Category: 8. Health

Dublin, Aug. 11, 2025 (GLOBE NEWSWIRE) — The “Multi Cancer Early Detection Market and Forecasts 2025-2033” report has been added to ResearchAndMarkets.com’s offering.

The global multi cancer early detection market size was valued at USD 1 billion in 2024 and is likely to reach USD 4.3 billion by 2033, expanding at a CAGR 16.9% during the forecast period.

The multi cancer early detection market is experiencing significant growth as advancements in technology and changing healthcare paradigms evolve. Early detection of cancer can lead to improved treatment outcomes and potentially save lives, making this market increasingly important in the realm of healthcare.

One of the primary drivers of the multi cancer early detection market is the rapid advancement of diagnostic technologies. Innovations in genomics, artificial intelligence, and imaging techniques are enhancing the accuracy and efficiency of cancer detection. These cutting-edge technologies allow for the identification of multiple cancer types simultaneously, which is crucial for timely intervention.

Another key factor contributing to the market’s expansion is the growing public awareness regarding the importance of early cancer detection. More individuals are prioritizing preventive healthcare measures, leading to increased screenings and tests. Health campaigns and education initiatives are empowering patients to take charge of their health, thereby stimulating demand for multi-cancer early detection solutions.

Multi Cancer Early Detection Market Synopsis

This new 2025 market report presents an in-depth assessment of the global multi cancer early detection market dynamics, opportunities, future road map, competitive landscape and discusses major trends. The report offers the most up-to-date industry data on the actual market situation and future outlook in the global multi-cancer early detection market. The report also provides up-to-date historical market size data for the period 2020 – 2024 and an illustrative forecast to 2033 covering key market aspects like market value, share, analysis, and trends for the global multi cancer early detection market.

The report provides a detailed analysis of the current industry situation and market requirements, highlighting facts about the market size, market share, revenue for global multi cancer early detection market segments, and a vivid forecast to 2033.

It also provides a comprehensive analysis of the pricing landscape, policies and regulation, and reimbursement pattern by countries and regions. The report also offers analysis and information according to categories such as market segments, application, technology, geographies, companies and competitive landscape. The report also provides a detailed description of the porter’s five forces analysis, PESTEL analysis, SWOT analysis, funding, merger and acquisitions, pipeline, growth drivers and challenges of the global multi cancer early detection market.

The report concludes with the profiles of major market players in the global multi cancer early detection market. The key market players are evaluated on various parameters such as company overview, product landscape, recent developments in multi cancer early detection, funding & M&A, strategic outlook, challenges and risks of the global multi cancer early detection market.

Multi Cancer Early Detection Market by Key Players

• GRAIL, Inc. (an Illumina Company)
• Guardant Health
• Exact Sciences Corporation
• Illumina, Inc.
• Freenome Holdings, Inc.
• Burning Rock Biotech Limited
• Foundation Medicine, Inc.
• AnchorDx
• Beijing Lyman Juntai International Medical Technology Development Co.
• Genecast
• Singlera Genomics Inc
• Thrive Earlier Detection (acquired by Exact Sciences Corporation)

Key Features of the Report

• The global multi cancer early detection market provides granular level information about the market size, regional market share, historic market (2020-2024), and forecast (2025-2033).
• Annualized revenues and country level analysis for each market segment.
• Analysis of business strategies by identifying the key market segments positioned for strong growth in the future.
• Detailed analysis of geographical landscape with country level analysis of major regions.
• The report covers in-detail insights about the competitor’s overview, company share analysis, key market developments, and key strategies.
• The report outlines drivers, restraints, unmet needs, and trends that are currently affecting the market.
• The report tracks recent innovations, key developments, and start-up details that are actively working in the market.
• The report provides a plethora of information about market entry strategies, regulatory framework, and reimbursement scenarios.
• The report analyses the impact of the socio-political environment through SWOT analysis and competition through porter’s five force analysis and PESTEL analysis
• Through study of the key business strategies and recommendations on future market approach.
• Comprehensive analysis of the competitive structure of the market.
• Demand side and supply side analysis of the market.

Key Questions the Report Addresses

• How big is the global multi cancer early detection market?
• What is the current multi cancer early detection market size?
• What is the major driving factor for the multi cancer early detection market?
• Which factor is restraining the growth of the multi cancer early detection market?
• Who are the key players in the multi cancer early detection market?
• Which region has the biggest share in the multi cancer early detection market?
• Which is the fastest growing country in the multi cancer early detection market?

Key Attributes:

Report Attribute	Details
No. of Pages	150
Forecast Period	2024 – 2033
Estimated Market Value (USD) in 2024	$1 Billion
Forecasted Market Value (USD) by 2033	$4.3 Billion
Compound Annual Growth Rate	16.9%
Regions Covered	Global

Key Topics Covered:

1. Market Definition

2. Research and Methodology

3. Executive Summary

4. Global Multi Cancer Early Detection – Market Dynamics
4.1 Growth Drivers
4.2 Challengers
4.3 Funding and Merger & Acquisitions

5. Global Multi Cancer Early Detection Market – Industry Analysis
5.1 SWOT Analysis
5.2 Porter’s Analysis

6. Global Multi Cancer Early Detection (MCED) Market & Forecast

7. Global Multi Cancer Early Detection (MCED) Market Share & Forecast
7.1 By Types
7.2 By End Use
7.3 By Region
7.4 By Country

8. By Types – Global Multi Cancer Early Detection (MCED) Market & Forecast
8.1 Gene Panel, IDT & Others
8.2 Liquid Biopsy

9. By End Use – Global Multi Cancer Early Detection (MCED) Market & Forecast
9.1 Hospitals
9.2 Diagnostic Laboratories
9.3 Others

10. By Region – Global Multi Cancer Early Detection (MCED) Market & Forecast
10.1 North America
10.2 Europe
10.3 Asia Pacific
10.4 Latin America
10.5 Middle East & Africa

11. By Country – Global Multi Cancer Early Detection (MCED) Market & Forecast

12. Global Multi Cancer Early Detection Market – Key Players Profiles

• GRAIL, Inc. (an Illumina Company)
• Guardant Health
• Exact Sciences Corporation
• Illumina, Inc.
• Freenome Holdings, Inc.
• Burning Rock Biotech Limited
• FOUNDATION MEDICINE, INC.
• AnchorDx
• Beijing Lyman Juntai International Medical Technology Development Co.
• GENECAST
• Singlera Genomics Inc.
• Thrive Earlier Detection (acquired by Exact Sciences Corporation)

For more information about this report visit https://www.researchandmarkets.com/r/ovdma8

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• Multi Cancer Early Detection Market

Parents of children diagnosed with type 1 diabetes suffer an income drop in the years following the diagnosis. The impact is more pronounced in mothers, especially mothers of children diagnosed in preschool years. These novel findings from a study led by researchers at Uppsala University, Sweden, have now been published in Diabetologia, the Journal of the European Association for the Study of Diabetes (EASD).

Type 1 diabetes is a chronic autoimmune disease that requires daily insulin treatment and continual blood sugar monitoring. In Sweden, more than a thousand children are diagnosed with type 1 diabetes yearly. The parents of these children shoulder the main responsibility for treatment and monitoring, at home as well as in school settings. Previous research has shown that parents of children with type 1 diabetes are at increased risk of stress-related symptoms and may need to reduce their working hours.

“In our study, we observed reduced parental work-related incomes in the years following the child’s type 1 diabetes diagnosis. The drop was larger in mothers than in fathers. Since mothers earned significantly less than fathers in absolute terms, even before the child fell ill, the relative drop in mothers was 6.6% the year following diagnosis compared to 1.5% in fathers. We further note the greatest impact on work-related incomes in mothers of children diagnosed at preschool age,” says Beatrice Kennedy, physician at the Endocrine and Diabetes unit at Uppsala University Hospital and Associate Professor of Medical Epidemiology at Uppsala University, who led the study.

Builds on data from 26,000 parents of children with type 1 diabetes

The research project was an interdisciplinary collaboration across medical departments at Uppsala University, including the Centre for Health Economics Research, with the University of Gothenburg. It builds on data from national population and health registers and the Swedish Child Diabetes Register (Swediabkids). The study includes the parents of more than 13,000 children diagnosed with type 1 diabetes in Sweden in 1993−2014, as well as more than half a million parents in the general population who have children not diagnosed with diabetes.

The researchers observed that the maternal pension-qualifying incomes (a composite outcome including work-related income and societal benefits) initially increased after the child’s diagnosis. This was attributable to mothers applying for the parental care allowance from the Swedish Social Insurance Agency. The parental care allowance was intended to compensate for disease-related loss of work-related income and contribute toward disease-specific costs.

Need for increased support to parents of children with chronic disease

When the research team investigated long-term effects in mothers, they found that the pension-qualifying incomes gradually decreased after eight years, and had not recovered by the end of follow-up − 17 years after the children were diagnosed.

“In this study, we have focused on the income effects in parents of children diagnosed with type 1 diabetes. However, parenting children with other chronic childhood-onset conditions may entail similar income consequences. Our findings indicate that there is room for improvement in the societal targeted support to mothers of children with chronic conditions, to ensure that the financial impact of caring for a child with health concerns is alleviated,” says Tove Fall, Professor of Molecular Epidemiology at Uppsala University, who initiated the project.

New research, published in The Journal of Immunology, discovered that a parasitic worm suppresses neurons in the skin to evade detection. The researchers suggest that the worm likely evolved this mechanism to enhance its own survival, and that the discovery of the molecules responsible for the suppression could aid in the development of new painkillers.

Schistosomiasis is a parasitic infection caused by helminths, a type of worm. Infection occurs during contact with infested water through activities like swimming, washing clothes, and fishing, when larvae penetrate the skin. Surprisingly, the worm often evades detection by the immune system, unlike other bacteria or parasites that typically cause pain, itching, or rashes.

In this new study, researchers from Tulane School of Medicine aimed to find out why the parasitic worm Schistosoma mansoni doesn’t cause pain or itching when it penetrates the skin. Their findings show that S. mansoni causes a reduction in the activity of TRPV1+, a protein that sends signals the brain interprets as heat, pain, or itching. As part of pain-sensing in sensory neurons, TRPV1+ regulates immune responses in many scenarios such as infection, allergy, cancer, autoimmunity, and even hair growth.

The researchers found that S. mansoni produces molecules that suppress TRPV1+ to block signals from being sent to the brain, allowing S. mansoni to infect the skin largely undetected. It is likely S. mansoni evolved the molecules that block TRPV1+ to enhance its survival.

“If we identify and isolate the molecules used by helminths to block TRPV1+ activation, it may present a novel alternative to current opioid-based treatments for reducing pain,” said Dr. De’Broski R. Herbert, Professor of Immunology at Tulane School of Medicine, who led the study. “The molecules that block TRPV1+ could also be developed into therapeutics that reduce disease severity for individuals suffering from painful inflammatory conditions.”

The study also found that TRPV1+ is necessary for initiating host protection against S. mansoni. TRPV1+ activation leads to the rapid mobilization of immune cells, including gd T cells, monocytes, and neutrophils, that induce inflammation. This inflammation plays a crucial role in host resistance to the larval entry into the skin. These findings highlight the importance of neurons that sense pain and itching in successful immune responses

Identifying the molecules in S. mansoni that block TRPV1+ could inform preventive treatments for schistosomiasis. We envision a topical agent which activates TRPV1+ to prevent infection from contaminated water for individuals at risk of acquiring S. mansoni.“

Dr. De’Broski R. Herbert, Professor of Immunology, Tulane School of Medicine

In this study, mice were infected with S. mansoi and evaluated for their sensitivity to pain as well as the role of TRPV1+ in preventing infection. Researchers next plan to identify the nature of the secreted or surface-associated helminth molecules that are responsible for blocking TRPV1+ activity and specific gd T cell subsets that are responsible for immune responses. The researchers also seek to further understand the neurons that helminths have evolved to suppress.

In a discovery that could broaden access to next-generation biologic medicines and vaccines, researchers at the University of Chicago Pritzker School of Molecular Engineering (UChicago PME) have engineered polymer-based nanoparticles that form with a temperature change.

The new nanoparticles, described in Nature Biomedical Engineering, self-assemble in water at room temperature. Due to these gentle conditions, they can deliver proteins that are unstable in many existing nanoparticle formulations.

“What excites me about this platform is its simplicity and versatility. By simply warming a sample from fridge temperature to room temperature, we can reliably make nanoparticles that are ready to deliver a wide variety of biological drugs,” said co-senior author Stuart Rowan, the Barry L MacLean Professor for Molecular Engineering Innovation and Enterprise at UChicago PME.

From problem to platform

Nanoparticles are key to protecting delicate drugs like RNA and proteins from being degraded in the body before they reach the right cells. For example, lipid nanoparticles (LNPs) made COVID-19 mRNA vaccines possible. However, LNPs rely on alcohol-based solvents and sensitive manufacturing steps – making them less suitable for protein delivery.

“We wanted to make a delivery system that could work for both RNA and protein therapies – because right now, most platforms are specialised for just one,” said first author Samir Hossainy, a UChicago PME graduate student. “We also wanted to make it scalable, without needing toxic solvents or complicated microfluidics.”

Hossainy theorised that polymer-based nanoparticles could offer a more robust and customisable alternative. He outlined that the immune system would only respond to particles with certain sizes, shapes and charges – and then used chemical tools to begin designing new nanoparticles from scratch.

After fine-tuning more than a dozen different materials, he found one that worked. In cold water, the polymer – and any desired protein – remained dissolved. But when heated to room temperature, the polymer self-assembled into uniformly sized nanoparticles surrounding the protein molecules.

“Our particle size and morphology are dictated only by the chemistry of the polymers that I designed from the bottom up,” explained Hossainy. “We don’t have to worry about different particle sizes forming, which is a challenge with a lot of today’s nanoparticles.”

Carrying versatile cargo

To put the new particles – known as polymersomes – to the test, Hossainy collaborated with colleagues in Rowan’s lab and with former UChicago PME Professor Jeffrey Hubbell. First, they showed that the particles can encapsulate more than 75 percent of protein and nearly 100 percent of short interfering RNA (siRNA) cargo. This is far higher than most current systems and they can be freeze-dried and stored without refrigeration.

In the context of vaccination, Hossainy and his collaborators found that the particles could effectively carry a protein. When injected into mice,  this triggered the animals’ immune systems to generate long-lasting antibodies against that protein. Another experiment showed that the nanoparticles could also carry proteins designed to prevent an immune response in the context of allergic asthma. A third demonstrated that injecting them into tumours could block cancer-related genes and suppress tumour growth in mice.

“The exciting thing is that we didn’t need to tailor a different system for each use case,” said Hossainy. “This one formulation worked for everything we tried – proteins, RNA, immune activation, immune suppression and direct tumour targeting.”

A scalable solution for worldwide vaccines

One of the biggest advantages of these new polymersomes over current LNPs is the potential for low-tech, decentralised production. Hossainy says he imagines being able to ship freeze-dried formulations of the nanoparticles to anywhere in the world. When they need to be used, they can be mixed in cold water, warmed up and will be ready to deliver to patients.

“Being able to store these dry drastically improves the stability of the RNA or protein,” said Hossainy.

The group is continuing to work on fine-tuning the particles to carry more types of cargo, including messenger RNA like that used in the COVID-19 vaccines, which are generally much larger than the siRNA used in the current trial. They also plan to collaborate on pre-clinical trials to apply the new nanoparticles to real-world vaccine and drug delivery challenges.

Air conditioning can feel heaven-sent on hot summer days. It keeps temperatures comfortable and controls humidity, making indoor environments tolerable even on the most brutally warm days.

But some people avoid using air conditioning (AC) no matter how hot it gets outside, out of fear that it will make them sick. While this may sound far-fetched to some, as a microbiologist I can say this fear isn’t altogether unfounded.

If an air conditioning system malfunctions or isn’t properly maintained, it can become contaminated with infectious microbes. This can turn your AC unit into a potential source of numerous airborne infections – ranging from the common cold to pneumonia.

Sick buildings

“Sick building syndrome” is the general name for symptoms that can develop after spending extended periods of time in air-conditioned environments. Symptoms can include headaches, dizziness, congested or runny nose, persistent cough or wheeze, skin irritation or rashes, trouble focusing on work and tiredness.

Related: Mysterious, Rapid Surge in Legionnaires’ Disease Linked to Cleaner Air

The condition tends to occur in people who work in office settings, but can happen to anyone who spends extended periods of time in air-conditioned buildings such as hospitals. The symptoms of sick building syndrome tend to get worse the longer you’re in a particular building, and are alleviated after you leave.

A 2023 study from India compared 200 healthy adults who worked at least six-to-eight hours per day in an air-conditioned office with 200 healthy adults who didn’t work in AC.

The AC group experienced more symptoms consistent with sick building syndrome over the two-year study period – particularly a higher prevalence of allergies. Importantly, clinical tests showed those who were exposed to AC had poorer lung function and were absent from work more often, compared with the non-AC group.

Other studies have confirmed that AC office workers have a higher prevalence of sick building syndrome than those who do not work in an air-conditioned environment.

It’s suspected that one cause of sick building syndrome is malfunctioning air conditioners. When an AC unit isn’t working properly, it can release allergens, chemicals and airborne microorganisms into the air that it would normally have trapped.

Malfunctioning air conditioners can also release chemical vapours from AC cleaning products or refrigerants into the building’s air. Chemicals such as benzene, formaldehyde and toluene are toxic and can irritate the respiratory system.

Poorly maintained air conditioning systems can also harbour bacterial pathogens which can cause serious infections.

Legionella pneumophila is the bacteria that causes Legionnaires’ disease – a lung infection contracted from inhaling droplets of water containing these bacteria. They tend to grow in water-rich environments such as hot tubs or air conditioning systems.

A Legionella infection is most often caught in communal places such as hotels, hospitals or offices, where the bacteria have contaminated the water supply.

Symptoms of Legionnaires’ disease are similar to pneumonia, causing coughing, shortness of breath, chest discomfort, fever and general flu-like symptoms. Symptoms usually begin to show between two and 14 days after being exposed to Legionella.

Legionella infections can be life-threatening and often require hospitalisation. Recovery can take several weeks.

Fungal and viral infections

The accumulation of dust and moisture inside air conditioning systems can also create the right conditions for other infectious microbes to grow.

For instance, research on hospital AC systems has found that fungi such as Aspergillus, Penicillium, Cladosporium and Rhizopusspecies commonly accumulate within the water-rich areas of hospital ventilation systems.

These fungal infections can be serious in vulnerable patients such as those who are immunocompromised, have had an organ transplant or are on dialysis – as well as babies who were born premature. For example, Aspergillus causes pneumonia, abscesses of the lungs, brain, liver, spleen, kidneys and skin, and can also infect burns and wounds.

Symptoms of fungal infections are mostly respiratory and include persistent wheeze or cough, fever, shortness of breath, tiredness and unexplained loss of weight.

Viral infections can also be caught from air conditioning. One case study revealed that children in a Chinese kindergarten class were infected with the norovirus pathogen from their AC system. This caused 20 students to experience the stomach flu.

While norovirus is usually transmitted through close contact with an infected person or after touching a contaminated surface, in this instance it was confirmed, unusually, that the virus was spread through the air – originating from the air conditioning unit in a class restroom. Several other cases of norovirus being spread this way have been reported.

However, air conditioners can also help stop the spread of airborne viruses. Research shows AC units that are regularly maintained and sanitised can reduce circulating levels of common viruses, including COVID.

Another reason AC may increase your risk of catching an infection is due to the way air conditioners control humidity levels. This makes inside air drier than outside air.

Spending extended periods of time in low-humidity environments can dry out the mucus membranes in your nose and throat. This can affect how well they prevent bacteria and fungi from getting in your body – and can leave you more vulnerable to developing a deep-tissue infection of the sinuses.

Air conditioners are designed to filter air contaminants, fungal spores, bacteria and viruses, preventing them from entering the air we breathe indoors. But this protective shield can be compromised if a system’s filter is old or dirty, or if the system isn’t cleaned. Ensuring good AC maintenance is essential in preventing air-conditioner-acquired infections.

Primrose Freestone, Senior Lecturer in Clinical Microbiology, University of Leicester

This article is republished from The Conversation under a Creative Commons license. Read the original article.

Expression plasmids and transient transfections

To investigate the influence of glycosylation degree on the antigenicity and immunogenicity of the Marburg virus GPs, Angola-GP (KU978782) strain MARV GP ectodomains (including residues 1-637), containing the mucin-like domain (MARV GPΔTM) or deleting residues 264-425 to remove the mucin-like domain (MARV GPΔTM ΔMuc), were expressed in different cells. For Drosophila S2 cell expression, DNA encoding the glycoproteins was synthesized and codon-optimized for expression in insect cells at General Biol. The gene was cloned into the vector pMT/BiP/V5-His B(PMT-GP). For Expi293F cell expression, the gene was codon-optimized for expression in mammalian cells, and the gene of MARV GPΔTM was cloned into the vector pCAGGS (PCAGGS-GP); the other gene of MARV GPΔTM ΔMuc was cloned into the vector pcDNA3.4(pcDNA3.4-GP). All constructs contained a C-terminal Twin-Strep-tag for purification.

To generate GP glycoprotein from insect cells, S2 cells cultured in Schneider’s Drosophila Medium (Gibco, 21720024) containing 10% heat-inactivated FBS (Gibco, 10099141-C), 0.1% Pluronic F-68 (Gibco, 24040032) and 50 U/mL Penicillin-Streptomycin (Gibco, 15140122) were co-transfected with pMT-GP using calcium phosphate transfection (YEASEN, 40803ES70). Prepare cultured cells for transfection by seeding 1 × 10⁶ S2 cells in a 6-well plate in 2 mL complete Schneider’s Drosophila Medium. After growing for 16 h at 27 °C until cells reach a density of 2 × 10⁶-4 × 10⁶ cells/mL, 6 µg pMT-GP by calcium phosphate transfection was co-transfected for each well, and the medium was changed after 24 h of incubation. Copper sulfate was added to the medium to a final concentration of 500 µM 2 days after transfection. Six days after transfection, the culture medium was collected and then clarified by centrifugation at 800 × g for 15 min, 4000 × g for 15 min, and filtered using a 0.45 μm filter (Millipore, SLHVR33RB). For Expi293F, PCAGGS-GP (MARV GPΔTM) or pcDNA3.4-GP (MARV GPΔTM ΔMuc) constructs were transiently expressed in Expi293F cells with the ExpiFectamine™ 293 transfection kit (Gibco, A14524) at 37 °C at 100 rpm with 5% CO₂ according to the manufacturer’s protocol. The supernatant was collected at 120 h post-transfection.

The supernatant was purified through a StrepTactin affinity purification strepTrap(Cytiva,29401322) purification column according to the manufacturer’s instructions. Then, the elution was exchanged with 0.01 M phosphate-buffered saline (PBS, pH 7.2) and concentrated using a 30 kD Centrifugal Filter Unit (Millipore, UFC903096). Proteins were further purified by SEC on a Superdex 200 Increase 10/300 GL column (Cytiva, 28990944). Then the fractions were harvested and analyzed by SDS-PAGE. The resulting purified Marburg GPs glycoprotein was used to ELISA, western blotting, and immunization experiments.

Antibody production and purification

The antibody sequences of MR78, MR191, MR228 were used as reported [22, 23] and codon optimized for mammalian cells. The heavy and light chains were cloned into the pcDNA3.4 expression vector, and then the constructs were transfected into Expi293F cells using the ExpiFectamine™ 293 Transfection Kit (Gibco, A14524) according to the manufacturer’s specifications. Antibodies were purified from culture supernatants using Protein A column (Cytiva, 17040201) followed by running buffer (PBS). Bound proteins were eluted using elution buffer (0.1 M glycine, pH 2.7). This fraction was concentrated by 50 kD  Amicon Ultra concentrators (Millipore, UFC905096) and was identified using SDS-PAGE. The purified protein was aliquoted and stored at -80 °C until further use.

SDS-PAGE, Native-PAGE and Western blotting

For SDS-PAGE, 5 µg of reduced or non-reduced protein was loaded onto a SurePAGE™ Plus, Bis-Tris, 4-12% gel (GenScript, M00653). After electrophoresis, the gel was stained with Coomassie brilliant blue G-250. For western blotting, gels were transferred onto nitrocellulose membranes (Cytiva, 10600001) using the trans-Blot Turbo transfer system (GenScript). Membranes were then blocked with 5% skim milk for 1 h. After washing three times with wash buffer containing PBS and 0.2% Tween 20 (PBST), the membranes were incubated with MR78 (1 µg/mL) or anti-strep-tag II antibody (1:2000 dilution; Abcam, ab307676) for 1 h. The membranes were washed again and then incubated with HRP-conjugated Goat Anti-Human IgG Fc (1:5000 dilution; Abcam, ab97225) or Goat Anti-Rabbit IgG Secondary Antibody (HRP) (0.01 µg/mL; Sino Biological, SSA004) for 1 h. Membranes were washed again and then incubated with Chemiluminescent Substrate Kit (Millipore, WBKLS0100). Images were acquired using an iBright FL1500 imaging system (Invitrogen).

For native-PAGE, purified protein samples were mixed with 3× loading buffer (Thermo Scientific, BN2008) and then loaded onto 4-15% Native-Page Tris-Gly gradient gels (Beyotime, P0465S) as described by the manufacturer. Proteins were electrophoresed at 150 V for 60 min. After electrophoresis, the protein was also stained with Coomassie brilliant blue G-250.

High-performance liquid chromatography (HPLC)

All high-purity GPs were analyzed on a HPLC system (Waters) using a TSK Gel G5000PWXL 7.8 × 300 mm column (TOSOH, 0008023), which was pre-equilibrated with PBS, pH 7.4 (Gibco, C10010500BT). Samples were loaded at a flow rate of 0.4 mL/min, and eluted proteins were detected at 280 nm.

Deglycosylation and glycan staining

Glycans were removed from purified GPs using peptide-N-glycosidase F (PNGase F) (New England BioLabs, P0704S) and Endoglycosidase (Endo H) (New England BioLabs, P0702S). In brief, 20 µg of GP protein was denatured at 100 °C for 10 min in 1× glycoprotein denaturing buffer, then digested with 1 µL PNGase F or endo-N-acetylglucosaminidase H (Endo H) at 37 °C for 1 h according to the manufacturer’s instructions. The deglycosylated protein was analyzed by Western blot with anti-Strep antibody as the detection antibody. For MARV GPΔTM ΔMuc, the detection method is as described above. For MARV GPΔTM, the membranes were incubated with StrepMAB-Classic HRP (1:3000 dilution; iba, 2-1509-001). The glycan staining was performed using periodic acid schiff (PAS) staining (Thermo Scientific, 24562) for glycoproteins as described by the manufacturer.

Enzyme-linked immunosorbent assay (ELISA)

To determine the antigenicity of Marburg virus GP, Costar™ 96-well assay plates (Costar, 9018) were coated overnight at 4 °C with serially diluted Marburg GP. Then washed three times with wash buffer containing PBS and 0.2% Tween 20 (PBST). Each well was then coated with 100 µl of blocking buffer containing PBST with 2% BSA for 1 h at 37 °C and then washed three times with wash buffer. Marburg mAbs MR78 and MR191 were then separately added at a concentration of 10 µg/mL, MR228 was added at a concentration of 1 µg/mL, followed by incubation for 1 h at 37 °C. Then, after washing three times with wash buffer, Goat Anti-Human IgG Fc (HRP) (1:10000 dilution; Abcam, ab97225) was added and incubated for 1 h. Finally, after washing three times with wash buffer, the wells were developed with 100 µL of TMB (Solarbio, PR1200) for 3 min at 37 °C, then the reaction was stopped with 50 µL of ELISA Stop Solution (Solarbio, C1058). The absorbance was measured at 450 nm (reference: 630 nm). Data was analyzed by four-parameter nonlinear regression using GraphPad Prism 8.4.2 software.

Mice immunization

Specific pathogen-free (SPF) female BALB/c mice (6-8 weeks) were randomly divided into 6 groups with 8 mice per group, used for intramuscular immunization. Each mouse was injected with 100 µL of vaccine sample including 5 µg purified Marburg GP glycoprotein antigens, 200 µg aluminum adjuvant (InvivoGen, vac-alu-50), or PBS solution. The five purified Marburg GP glycoprotein antigens included Expi293F-derived GPΔTM, S2-derived GPΔTM, Expi293F-derived GPΔTM ΔMuc, S2-derived GPΔTM ΔMuc peak1, and S2-derived GPΔTM ΔMuc peak2. The control mice were vaccinated with PBS. The mice were immunized at weeks 0 and 3, and the blood samples were collected from the tail vein and processed by centrifugation on days 0, 14, 21 and 35. Then the processed serum was heated and inactivated at 56 °C for 30 min, aliquoted, and then stored at -80 °C until analysis was performed.

Antibody measurement

To measure GP-specific antibody responses in serum samples by ELISA, purified GP was coated into the wells of 96-well microtiter plates (Costar, 9018) at 10 µg/mL (100 µL) and incubated at 4 °C overnight. After blocking, Sera at 1:100 were three-fold serially diluted and added to the wells (100 µL) as primary antibodies, followed by HRP-conjugated anti-mouse IgG antibody (1:10000 dilution; Abcam, ab97265). After color development, the absorbance was read at 450 nm and 630 nm on a microplate reader (TECAN). For the given serum sample, the cutoff value is defined as 2.1 times the reading of the blank control (without serum added). The endpoint titers were defined as the dilution of the cutoff value.

Cell-surface binding using fluorescence activated cell sorting (FACS)

For the cell surface-displayed GP, 4 µg of plasmid encoding full-length GP was transfected into HEK293T cells cultured in T75 flasks using VirusGEN transfection reagent (Mirus, MIR 6700). After 24 h, cells were detached with 2% (v/v) FBS in PBS and transferred at a total of 5 × 10⁵ cells to each flow tube, then stained with polyclonal sera (1:100) obtained from mice vaccinated with different forms of GP. Cells were subsequently stained with anti-mouse IgG antibody conjugated APC (BioLegend, 405308) followed by flow cytometry analysis on a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software, using the following gating strategy: size & granularity > single cells > GP+ (Ab positive). All results were expressed as mean fluorescence intensity (MFI) or cell percentage.

Pseudovirus-based neutralization assays

For pseudovirus packaging, the gene encoding the full-length GP of Angola was human codon-optimized and inserted into the pcDNA3.1 vector to construct GP protein-expressing plasmids. HEK293T cells were inoculated into T75 flasks (Corning, 430641) and cultured at 37 °C and 5% CO₂ to 70-90% confluence for transfection. 4 µg of pcDNA3.1-Marburg GP were co-transfected with 32 µg of HIV backbone vector pNL4-3.Luc.R-E- into HEK293T cells using the VirusGEN transfection reagent (Mirus, MIR 6700). Supernatants containing pseudovirus particles were collected at 48 h post-transfection by centrifugation at 800 × g for 5 min. The harvested pseudovirus solution was stored in aliquots at − 80 °C after filtering through a 0.45 μm filter.

The titers of neutralizing antibodies in these sera were detected by HIV-based pseudovirus-type neutralization assay. For neutralization testing, serum samples were diluted at 1:20 using Dulbecco’s Modified Eagle’s Medium (Gibco, C11995500BT) with 10% fetal bovine serum at 50 uL per well. The serum samples were mixed with HIV-based pseudovirus-type virus with an equal volume (50 µL) and then incubated at 37 °C for 1 h before adding to HEK293T cells. After 48 h at 37 °C, the luciferase activity was measured by a microplate reader (TECAN) using the Bright-Lite Luciferase Assay System (Vazyme, DD1204). The formula for calculating the percentage of neutralization is “100% – (sample signals – blank control signals) / (virus control signals – blank control signals) × 100%”. The neutralization percent of each sample was performed using the GraphPad Prism 8.4.2 software.

Competitive ELISA

To compare the antibody epitope-specific responses, competitive binding enzyme-linked immunosorbent assay (ELISA) was performed. For the competitive ELISA, the steps were the same as the ELISA method described previously. First, Costar™ 96-well assay plates (Costar, 9018) were coated overnight at 4 °C with 1 µg/mL of Marburg GP. Wash buffer was used to wash the 96-well plate three times; mouse antisera, which was serially diluted from 1:10, was added to the wells and incubated at 37 °C for 1 h. MR228 antibodies were conjugated with HRP by using the EZ-Link Plus Activated Peroxidase kit (Solarbio, EX7000) according to the instructions. The plates were washed 3 times prior to the addition of HRP-conjugated MR228 (0.03 µg/mL) to the wells and incubated at 37 °C for 1 h. Finally, the cleaning step was repeated, and the wells were developed with 100 µL of TMB (Solarbio, PR1200) for 3 min at 37 °C, then the reaction was stopped with 50 µL of termination buffer (Solarbio, C1058). After color development, colorimetric analysis was performed at 450 nm and 630 nm in a microplate reader (TECAN). The formula for calculating the inhibition rate is “(blank control signals – sample signals) / blank control signals × 100%.”

Statistical analysis

Where applicable, results are expressed as the mean ± SD of representative results. Comparisons of GP-specific antibody titers (Fig. 1B) and cell-surface binding results (Fig. 1C) were performed using Kruskal-Wallis one-way ANOVA. Comparisons of neutralization assays (Fig. 1D) and inhibition rates (Fig. 1F) between groups were performed using one-way ANOVA. Differences were considered statistically significant at a P value of 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). All statistical analyses were performed with GraphPad Prism 8.4.2 software.