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Langedijk et al. Nature Communications (2024)

Universal paramyxovirus vaccine design

Paramyxoviruses include some of the most medically significant RNA viruses, including measles, human respirovirus types 1 and 3 (RV1 and RV3), and zoonotic pathogens like Nipah and Hendra virus. Overcoming the instability of the Paramyxovirus fusion (F) protein is a critical step towards efficacious vaccines. Here we stabilized regions involved in the structural transformation to generate a preF protein vaccine antigen for RV3, with exceptional high expression and stability. Uniquely, by stabilizing the coiled-coil stem region, we prevented the need for a heterologous trimerization domain. Immunogenicity tests revealed strong neutralizing antibody responses in both naïve and pre-exposed animal models. Importantly, despite the evolutionary distance of these paramyxovirus F proteins, we successfully transferred the stabilizing substitutions to the distant preF proteins of RV1 and Nipah virus. This work presents a successful vaccine antigen design for RV3 and provides a toolbox for future paramyxovirus vaccine design and pandemic preparedness.

Milder ET AL. PNAS (2022)

Redesign of the influenza hemagglutinin pH switch regions

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing, stable, trimeric HAs. This repair-and-stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Juraszek ET AL. Nature Communications (2021)

Stabilizing the closed SARS-CoV-2 spike trimer

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to the intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct. Importantly, our design does not require a heterologous trimerization domain in contrast to previously published designs.

Our stabilized S protein is very stable, expresses to high yields, and importantly, the cryo-EM structure reveals a correctly folded, predominantly closed, pre-fusion conformation.

The here reported advancements will increase understanding of coronavirus S protein structure and will support vaccine development and serological diagnostics.

Bakkers et al. Nature Communications (2024)

AI-based engineering of a closed HMPV prefusion trimer

In order to stabilize the prefusion conformation of the human metapneumovirus fusion protein (hMPV Pre-F) we developed an AI convolutional classifier (‘ReCAP’) that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. Additionally, by molecular analysis of the virus’ processing pathway we uncovered a previously unnoticed processing event in the HMPV F protein. By combining additional processing, stabilization of interface regions and the membrane-proximal stem, we designed a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer.

Importantly, the protein induces high, cross-neutralizing, antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate.

Key publications

Human Respiratory Viruses

Efficacious human metapneumovirus vaccine based on AI-guided engineering of a closed prefusion trimer. Bakkers et al. Nat. Comm. 2024 [Read more]
Structural basis for potent neutralization of human respirovirus type 3 by protective single-domain camelid antibodies. Johnson et al. Nat. Comm. 2024 [Read more]
Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein. Langedijk et al. Nat. Comm. 2024 [Read more]
Design and Preclinical Evaluation of a Nanoparticle Vaccine against Respiratory Syncytial Virus Based on the Attachment Protein G. Voorzaat et al. Vaccines. 2024 [Read more]
Impact of SARS-CoV-2 spike stability and RBD exposure on antigenicity and immunogenicity. Rutten et al. Sci Rep. 2024 [Read more]
Enhancing breadth and durability of humoral immune responses in non-human primates with an adjuvanted group 1 influenza hemagglutinin stem antigen. Swart et al. NPJ Vaccines. 2023 [Read more]
Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike. Yu et al. PLoS Pathog. 2023 [Read more]
Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions. Milder et al. PNAS. 2022 [Read more]
Stabilizing the closed SARS-CoV-2 spike trimer. Juraszek et al. Nat. Comm. 2021 [Read more]
Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Mercado et al. Nature. 2020 [Read more]
Transient opening of trimeric prefusion RSV F proteins. Gilman et al. Nat. Comm. 2019 [Read more]
Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity. Bakkers et al. Cell Host Microbe. 2017 [Read more]
Coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation. Bakkers et al. PNAS. 2016 [Read more]
A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Krarup et al. Nat. Comm. 2015 [Read more]

Emerging and neglected viruses

Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein Trimers. Rutten et al. Cell Rep. 2020 [Read more]
CD164 is a host factor for lymphocytic choriomeningitis virus entry. Bakkers et al. PNAS. 2022 [Read more]

Human Immunodeficiency virus (HIV)

CoPoP liposomes displaying stabilized clade C HIV-1 Env elicit tier 2 multiclade neutralization in rabbits. Koornneef et al. Nat. Commun. 2024 [Read more]
Automated Design by Structure-Based Stabilization and Consensus Repair to Achieve Prefusion-Closed Envelope Trimers in a Wide Variety of HIV Strains. Rawi et al. Cell. Rep. 2020 [Read more]
A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers. Rutten et. al. Cell Rep. 2018 [Read more]
HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike. Eggink et al. J. Virol. 2016 [Read more]
A sweet surprise for HIV broadly neutralizing antibodies. Langedijk et al. Nature Medicine. 2012 [Read more]

Filed Patents

Vaccine design and others

STABILIZED PRE-FUSION HMPV FUSION PROTEINS (WO2023110618A1)
STABILIZED PRE-FUSION HMPV FUSION PROTEINS (WO2023217988A1)
PRE-FUSION HUMAN PIV1 F PROTEINS (WO2024061757A1)
STABILIZED PRE-FUSION PIV3 F PROTEINS (WO2024074584A1)
RSV IMMUNOGENS (WO2024017682A1)
STABILIZED PRE-FUSION RSV FB ANTIGENS (US-20240132548-A1)
STABILIZED VACCINE COMPOSITIONS (US-20230310573-A1)
RECOMBINANT INFLUENZA ANTIGENS (US-20220204567-A1)
COMPOSITIONS AND METHODS FOR PREVENTING AND TREATING CORONAVIRUS INFECTION – SARS-COV-2 VACCINES (US-20210388032-A1)
RNA REPLICON ENCODING A STABILIZED CORONA VIRUS SPIKE PROTEIN (US-20210347828-A1)
SARS-CoV-2 VACCINES (US-20210346492-A1)
STABILIZED FILOVIRUS GLYCOPROTEIN TRIMERS (US-20210292371-A1)
COMPOSITIONS AND VACCINE COMBINATIONS CONTAINING SYNTHETIC HUMAN IMMUNODEFICIENCY VIRUS (HIV) ENVELOPE ANTIGEN, AND METHODS OF USE THEREOF (US-20210220468-A1)
INFLUENZA VIRUS VACCINES AND USES THEREOF (US-20200377555-A1)
POXVIRUS VECTORS ENCODING HIV ANTIGENS, AND METHODS OF USE THEREOF (US-20200138938-A1)
VACCINE AGAINST RSV (US-20200061183-A1)
VACCINE AGAINST RSV (US-20190125854-A1)
STABILIZED SOLUBLE PRE-FUSION RSV F PROTEINS (US-20190119330-A1)
TRIMER STABILIZING HIV ENVELOPE PROTEIN MUTATIONS (US-20190023744-A1)
STABILIZED VIRAL CLASS I FUSION PROTEINS (US-20180346521-A1)
VACCINE AGAINST RSV (US-20180200360-A1)
STABILIZED SOLUBLE PRE-FUSION RSV F POLYPEPTIDES (US-20180194808-A1)
TRIMER STABILIZING HIV ENVELOPE PROTEIN MUTATIONS (US-20180072777-A1)
HUMAN IMMUNODEFICIENCY VIRUS ANTIGENS, VECTORS, COMPOSITIONS, AND METHODS OF USE THEREOF (US-20170165355-A1)
STABILIZED SOLUBLE PRE-FUSION RSV F POLYPEPTIDES (US-20160176932-A1)
HUMAN ANTIBODIES BINDING TO RSV G PROTEIN (US-20160145321-A1)
HUMAN ANTIBODIES BINDING TO RSV G PROTEINS (US-20160145322-A1)
STABILIZED SOLUBLE PREFUSION RSV F POLYPEPTIDES (US-20160102123-A1)