🛡️ immunology

Isolation and characterisation of Nipah virus neutralising candidate therapeutic monoclonal antibodies from an mRNA-immunised pig

This study demonstrates the utility of an mRNA-immunised pig model for isolating novel Nipah virus neutralising monoclonal antibodies that target distinct epitopes on the G glycoprotein, with a specific candidate (mAb A2) showing enhanced therapeutic potential when combined with the existing standard mAb m102.4 to achieve complete protection against lethal viral challenge.

Original authors: Pedrera, M., Pipatpadungsin, N., Kobasa, D., Elrefaey, A. M. E., Holzer, B., McLean, R. K., Warner, B., Vendramelli, R., Thakur, N., Stass, R., Hayes, J. W. P., Medfai, L., Sealy, J. E., Crossley, S.

Published 2026-08-30

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CC BY 4.0

Original authors: Pedrera, M., Pipatpadungsin, N., Kobasa, D., Elrefaey, A. M. E., Holzer, B., McLean, R. K., Warner, B., Vendramelli, R., Thakur, N., Stass, R., Hayes, J. W. P., Medfai, L., Sealy, J. E., Crossley, S., Schwartz, J. C., Munir, D., Mwangi, W., Bailey, D., Truong, T., Tchilian, E., Pickering, B., Bowden, T. A., Graham, S. P.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Nipah virus is a dangerous pathogen that jumps from fruit bats to humans, often causing severe illness with a high fatality rate. It spreads through contact with infected secretions and can also move between people. While the virus is a known threat, there are currently no approved vaccines or medicines to treat an infection once it occurs. Scientists have long known that antibodies, the immune system's specialized proteins that hunt down invaders, can stop the virus. One such antibody, already in clinical trials, has shown promise, but relying on a single weapon is risky; viruses can mutate to evade it. To build a more robust defense, researchers need to find new antibodies that attack the virus in different ways, ideally combining them into a powerful cocktail that is harder for the virus to escape.

To find these new defenders, a team of researchers turned to an unlikely source: the pig. Pigs are not just a potential intermediate host for the virus; they are also excellent models for human immune responses. The scientists vaccinated a group of pigs with a modern mRNA vaccine designed to teach the immune system to recognize the Nipah virus. This vaccine contained instructions for making a specific part of the virus, a protein called the G glycoprotein, which sits on the virus's surface and acts like a key to unlock human cells. After the pigs were vaccinated, their immune systems produced a wide array of antibodies. The researchers then harvested blood cells from these animals to isolate the specific antibodies that were most effective at binding to the virus.

From this process, the team successfully isolated five distinct antibodies that bound tightly to the virus. One of these, named A2, was particularly special because it could also recognize a related virus called Hendra, suggesting it might have a broad range of protection. When tested in the lab, all five antibodies were able to neutralize a version of the Nipah virus found in Malaysia, but only antibody A2 could also stop the strain found in Bangladesh. This is a crucial distinction, as the two strains differ slightly, and a treatment needs to work against both to be truly effective. The researchers found that antibody A2 and another potent antibody, C1, worked in ways that did not clash with each other or with the existing leading candidate antibody. This meant they could potentially be used together without getting in each other's way.

To understand exactly how these new antibodies worked, the scientists used a powerful imaging technique called cryo-electron microscopy to take detailed three-dimensional pictures of the antibodies attached to the virus protein. They discovered that these antibodies did not block the specific spot where the virus normally grabs onto human cells. Instead, they latched onto different parts of the virus's surface. This finding suggests that these antibodies do not simply block the door; rather, they likely jam the mechanism that allows the virus to fuse with the cell or change shape to enter it. The images also revealed why one antibody worked against both virus strains while the other did not: the part of the virus targeted by the second antibody had small differences between the two strains, making it harder for that antibody to hold on to the Bangladesh version.

The final test took the research from the lab to a living model. The researchers gave the new antibody A2 to hamsters that were then exposed to the deadly Bangladesh strain of the virus. While the antibody alone saved 60 percent of the animals, it was not enough to save everyone. However, when the researchers combined the new antibody A2 with the existing leading candidate antibody, the result was complete protection; all the animals survived. This outcome demonstrated that mixing antibodies that target different parts of the virus creates a stronger shield than using either one alone. The study confirms that pigs can serve as a valuable source for discovering these therapeutic tools and highlights the potential of combining different antibodies to create a treatment that is both powerful and difficult for the virus to defeat.

Technical Summary: Isolation and Characterisation of Nipah Virus Neutralising Candidate Therapeutic Monoclonal Antibodies from an mRNA-Immunised Pig

Problem Statement
Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with significant epidemic potential, causing severe respiratory and neurologic disease in humans with case-fatality rates ranging from 40% to over 75%. Despite the threat, no licensed therapeutics or vaccines exist. While the monoclonal antibody (mAb) m102.4, which targets the receptor-binding domain (RBD) of the NiV attachment glycoprotein (G), has shown therapeutic promise and entered clinical trials, there is a critical need for additional mAbs. Combining mAbs targeting distinct epitopes is a recognized strategy to improve neutralising potency, broaden coverage against viral variants (such as the NiV Bangladesh strain), and reduce the risk of viral escape mutations. Furthermore, large animal models like pigs are essential for predicting human vaccine outcomes and serve as a source for isolating relevant therapeutic antibodies.

Methodology
The study employed a multi-disciplinary approach combining immunology, structural biology, and in vivo challenge models:

Immunisation and Cell Sorting: Pigs were immunised with lipid nanoparticle-formulated mRNA encoding the soluble NiV Malaysia (NiV-M) G glycoprotein. Peripheral blood mononuclear cells (PBMCs) were collected 7 days post-boost. NiV G-specific IgG+ B cells were identified via flow cytometry using NiV G tetramers and sorted individually.
Antibody Generation: Single-cell RT-PCR was used to amplify heavy and light chain variable regions, which were cloned into porcine expression vectors. Recombinant porcine mAbs were expressed in Expi293F or ExpiCHO cells.
In Vitro Characterisation:
- Binding and Kinetics: Indirect ELISA and Biolayer Interferometry (BLI) assessed binding to NiV-M and Hendra virus (HeV) G proteins and determined affinity constants ( $K_d$ ).
- Neutralisation: Pseudovirus neutralisation assays (NiV-M and NiV-B strains) and cell-cell fusion inhibition assays measured neutralising potency ( $IC_{50}$ ).
- Epitope Binning: Competitive ELISA determined epitope overlap between the new mAbs and the reference mAb m102.4.
Structural Analysis: Single-particle cryogenic electron microscopy (cryo-EM) was performed on the NiV-M G RBD complexed with Fab fragments of the lead candidates (mAb A2 and mAb C1) to resolve structures at 3.19 Å resolution.
In Vivo Efficacy: Syrian golden hamsters were challenged with lethal NiV-B. Groups received prophylactic administration of mAb A2, mAb m102.4, or a combination of both (0.5 mg of each). Survival rates and viral shedding were monitored.

Key Results

Isolation and Binding: Five high-affinity porcine mAbs (A2, C1, D8, F4, G9) were isolated. All bound NiV-M sG with sub-nanomolar affinity ( $K_d < 0.1$ nM). Only mAb A2 cross-reacted with HeV sG.
Neutralisation Breadth: All five mAbs neutralised NiV-M pseudovirus. However, only mAb A2 and the reference mAb m102.4 neutralised the NiV-B pseudovirus. In fusion inhibition assays, mAb C1 was the most potent against NiV-M, while only mAb A2 neutralised NiV-B fusion.
Epitope Mapping: Competitive ELISA and cryo-EM revealed that mAbs A2 and C1 recognise distinct, non-overlapping epitopes that do not overlap with the receptor-binding site targeted by m102.4.
- mAb A2: Binds to blades $\beta$ 1 and $\beta$ 6 of the G RBD, occluding the dimerisation interface. It cross-neutralises NiV-M and NiV-B because its epitope residues are fully conserved between strains.
- mAb C1: Binds to blades $\beta$ 2 and $\beta$ 3. It fails to neutralise NiV-B due to five amino acid sequence differences in its epitope region between NiV-M and NiV-B.
- Structural Conformation: Both A2 and C1 bind to conformations of the G RBD that closely resemble the ephrinB2-bound state rather than the apo state, suggesting they may trap the protein in a fusion-incompetent conformation or sterically impede receptor engagement.
In Vivo Protection: In hamsters challenged with NiV-B:
- mAb m102.4 provided 100% protection.
- mAb A2 alone provided 60% protection.
- A combined dose of mAb A2 (0.5 mg) and mAb m102.4 (0.5 mg) provided 100% protection, equivalent to m102.4 alone.
- Viral shedding was significantly reduced in treated groups compared to controls.

Significance and Claims
The paper claims that the porcine model is a viable and effective source for isolating therapeutic candidate mAbs against henipaviruses. The study successfully characterised two novel neutralising epitopes on the NiV G glycoprotein that are distinct from the canonical receptor-binding site.

The authors state that these findings contribute to:

Understanding the Antigenic Landscape: Defining less commonly targeted regions on the NiV G surface (specifically the dimerisation interface and the $\beta$ 2/ $\beta$ 3 blade region).
Therapeutic Development: Demonstrating the potential for mAb combinations. While mAb A2 alone showed partial efficacy against NiV-B, its combination with m102.4 achieved complete protection, supporting the strategy of using complementary mechanisms of neutralisation.
Mechanism of Action: Providing structural evidence that neutralisation can occur via steric impedance or interference with fusion activation downstream of receptor engagement, rather than direct competition with receptor binding.

The paper concludes that vaccination of pigs with mRNA-encoded NiV G elicited robust B cell responses suitable for isolating high-affinity mAbs, validating the pig as a model for both therapeutic antibody discovery and vaccine evaluation.

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