VAX-seq: the future of mRNA vaccine analysis and quality assurance?

Published

2 years ago

September 22, 2023

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By Pooja Toshniwal Paharia Sep 22 2023 Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Nature Communications, researchers proposed a simplified approach for analyzing messenger ribonucleic acid (mRNA) vaccines using long-read sequencing. Study: mRNA vaccine quality analysis using RNA sequencing. Image Credit: Jo Panuwat D/Shutterstock.com Background

Messenger RNA vaccines demonstrated safety and efficacy during the COVID-19 pandemic, but extensive quality and purity testing is required to verify their efficacy and safety. Manufacturing advances have enabled billions of doses to be manufactured with acceptable quality and safety.

Various approaches are now utilized to assess mRNA vaccines; however, the efficacy of novel therapies depends on speedy and safe manufacture. Rigorous analytics are required at each stage of the production process to detect impurities and assure the safety of mRNA vaccines. About the study

In the present study, researchers investigated the VAX-seq method for quality analysis of messenger RNA vaccines.

The researchers developed VAX-seq, a simplified procedure for analyzing mRNA vaccines and therapeutics using long-read sequencing. This procedure compares VAX-seq to industry standards, including chromatography, capillary and agarose electrophoresis, and immunoblotting. The researchers employed a variety of methodologies, including Illumina plasmid DNA sequencing, ONT cDNA-PCR sequencing, and Oxford Nanopore direct ribonucleic acid sequencing.

Key messenger RNA quality features assessed by VAX-seq were sequence similarity, integrity, 3'-poly(A) nucleotide tail dimension, and RNA and DNA contamination. To assist VAX-seq, a software toolbox was created that provides thorough and automated reporting on mRNA quality. An enhanced green fluorescent protein (eGFP) messenger RNA was created and generated as a reference to demonstrate the application and validity of the methodology,

The plasmid template was amplified in Escherichia coli, isolated, purified, and linearized as the initial stage in the preparation process. The linearized pDNA template was then employed as a template for synthetic mRNA transcription in vitro. To examine the isolated mRNA, the program was combined with complementary deoxyribonucleic acid (cDNA) sequencing. VAX-seq attached a reverse transcriptase primer to the 3' terminus of the poly(A) nucleotide tail, allowing the length of the tail to be measured.

The researchers used the tailfindr program to normalize deletion mistakes and the read-specific nucleotide translocation rate. As part of the VAX-seq process, the complementary DNA library preparation introduced two flanking-type adaptors to the messenger RNA's 5' and 3' ends.

To identify complete-length molecules of mRNA from truncated messenger RNA, sequencing reads contained both flanking adaptors. Off-target RNA contaminants were identified using VAX-seq, which was used to assess fragmented and off-target RNA contaminants in cDNA libraries. Results

The analysis revealed that VAX-seq, a technique for sequencing mRNA vaccines, can detect sequence, length, integrity, and purity. It also enabled the examination of linearized plasmid DNA templates and the detection of impurities from plasmid amplification. VAX-seq easily established the length and similarity of mRNA vaccine sequences. The eGFP mRNA size profile revealed a major peak (77%) that was within 5.0% of the predicted length [1,153 nucleotide (nt)-long], as well as a varied spectrum of smaller, fragmented mRNAs. Short-read sequencing provided insufficient and inconsistent coverage, while heterogeneous alignment coverage was highly repeatable across replicates. Related StoriesDoes IQ influence COVID-19 vaccination decision-making?Inactivated poliovirus vaccine elicits persistent immunity for up to 10 yearsModernas adapted COVID-19 vaccine that targets Omicron XBB.1.5 approved by MHRA

Most sequences were aligned with the on-target messenger RNA product, and only a few reads revealed Escherichia coli contamination. The remaining seven percent of ribonucleic acid species were off-target RNA molecules, with 0.3% presumably originating from initiation sites of cryptic transcription. Direct ribonucleic acid sequencing libraries produce lower yields than comparable complementary DNA sequencing genetic libraries and cannot be multiplexed at the moment.

The researchers did, however, identify biases particular to direct ribonucleic acid sequencing, such as inferior-quality poly(A) nucleotide tail deletion. Direct ribonucleic acid sequencing found changed nucleosides in messenger RNA vaccines, demonstrating that including modified nucleosides in mRNA vaccines might lessen the innate immunological response while improving stability and translation.

Modified nucleosides had minimal effect on messenger RNA quality features and complementary DNA sequencing errors between messenger RNAs, including native N1-methylpseudouridine and uridine, but direct ribonucleic acid sequencing had a larger error rate.

Complementary DNA and direct ribonucleic acid sequencing revealed that modified messenger RNA vaccines had more truncated-type transcripts, with 41% complete-length and 54% truncated messenger RNA molecules, especially those less than 500 nt in length. Direct ribonucleic acid sequencing discovered nucleosides of N1-methylpseudouridine with a distinctive base-calling mistake that miscategorized N1-methylpseudouridine into cytosines, skewing the messenger RNA length profile. Conclusions

Overall, the study findings showed that VAX-seq was a procedure based on sequencing long reads that assessed essential mRNA quality characteristics such as integrity, contamination, and sequence identity. This technique can potentially become important to developing and producing mRNA medicines, offering a thorough and integrated evaluation at various manufacturing stages. VAX-seq employed full-length complementary DNA sequencing using Nanopore chemistry, which allowed for accurate assessment of the poly(A) molecular tail length as well as various off-target readings.

The approach offered a sensitive and quantitative assessment of mRNA characteristics, making it a more efficient alternative to conventional analytical techniques. VAX-seq enabled real-time identification of antisense RNA and messenger RNA integrity, allowing for swift testing lasting a few hours post-manufacture.

It might also identify complicated off-target ribonucleic acid contaminants created during transcription in vitro, as well as the degradation or sharing of messenger RNA vaccines during manufacturing, storage, and transportation. VAX-seq needed only a small quantity of messenger RNA as input and may be integrated to allow for large-scale and low-cost validation of vaccination batches. Journal reference: Gunter, H.M., Idrisoglu, S., Singh, S. et al. mRNA vaccine quality analysis using RNA sequencing. Nat Commun 14, 5663 (2023). doi: https://doi.org/10.1038/s41467-023-41354-y https://www.nature.com/articles/s41467-023-41354-y

Entertainment

Taylor Swift announces 12th studio album

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1 hour ago

August 12, 2025

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Taylor Swift announces 12th studio album

Taylor Swift has announced her 12th studio album during an appearance on her boyfriend Travis Kelce’s podcast.

The pop megastar, appearing on New Heights, did not say when the record, titled The Life Of A Showgirl, will be released.

Fans can pre-order the album in various formats now and Swift’s website says physical copies will be shipped by 13 October.

On Monday, Taylor Nation – an official branch of the singer’s marketing team – teased the release on TikTok with a slideshow of 12 images alongside the caption: “Thinking about when she said ‘See you next era…'”

Swift is seen wearing orange in every picture.

A special limited vinyl edition of the album will be released in “Portofino orange glitter”, according to a pre-order page on her site. A special cassette edition is also available for pre-order.

A brief clip from the New Heights podcast, hosted by Swift’s NFL star boyfriend, Travis Kelce and his brother Jason, a former NFL player, was posted on Instagram early on Tuesday.

The video showed Swift pulling a copy of the album from a briefcase with the cover blurred.

The full podcast episode will be released at 11pm on Tuesday, UK time.

Swift is living up to her reputation as pop’s hardest-working star

Gemma Peplow

Culture and entertainment reporter

@gemmapeplow

You might think that after pulling off the highest-grossing tour in history, all while writing and releasing an unexpected record-breaking double album at the same time, Taylor Swift would be happy to take a little break.

But no. The singer-songwriter has announced her 12th album, her sixth in six years.

Since her self-titled debut in 2006, the longest period Swifties have had to wait is just three years, between 2014’s 1989 and 2017’s Reputation; the period in which the star took time out following her public feud with Kim Kardashian and Kanye West.

Over the past few years, Swift has also re-recorded and re-released four of her early albums in a (now resolved) battle over the rights to her master recordings.

With the new announcement, she’s living up to her reputation as the hardest-working star in pop.

Album number 12 is titled The Life Of A Showgirl, hinting at inspiration drawn from spending the best part of two years on the road – and perhaps a return to pop after embracing folk and her more gothic side.

Fans are now eagerly waiting to find out what Swift’s new era will bring.

New Heights had previously teased Swift’s appearance by posting an orange image on social media with a mysterious silhouette, which many correctly identified as the pop star.

The Life Of A Showgirl follows Swift’s The Tortured Poets Department, released last year during her record-breaking Eras tour, which generated more than $2.2bn (£1.6bn) across two years and five continents, making it the highest-grossing tour of all time.

It marks her first release since she took back control over her entire back catalogue from private equity firm Shamrock Capital for an undisclosed amount.

In an effort to regain control over her music in recent years, Swift has been re-recording and releasing her first six albums. The move was prompted by Hybe America CEO Scooter Braun’s purchase and sale of her early catalogue.

Some of the ‘Taylor’s Version’ releases have included new songs as well as Easter eggs and visuals to offer a deeper understanding of her work.

The four re-recorded albums released so far have been massive commercial and cultural successes, each one entering the Billboard 200 US album chart at number one, helping her become the woman with the most number one albums in history.

UK

Thames Water crisis: Ministers line up administrator for utility giant

Published

1 hour ago

August 12, 2025

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Ministers have lined up insolvency practitioners to prepare for the potential collapse of Thames Water, Britain’s biggest water utility.

Sky News can exclusively reveal that Steve Reed, the environment secretary, has signed off the appointment of FTI Consulting to advise on contingency plans for Thames Water to be placed into a Special Administration regime (SAR).

Sources said on Tuesday that the advisory role established FTI Consulting as the frontrunner to act as the company’s administrator if it fails to secure a private sector bailout – although approval of such an appointment would be decided in court.

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Thames Water, its largest group of creditors and Ofwat, the industry regulator, have been locked in talks for months about a deal that would see its lenders injecting about £5bn of new capital and writing off roughly £12bn of value across its capital structure.

The discussions are said to be progressing constructively, although they appear to rely in part on the prospect of the company being granted forbearance on hundreds of millions of pounds of regulatory fines.

Responding to an enquiry from Sky News on Tuesday, a government spokesperson said: “The government will always act in the national interest on these issues.

“The company remains financially stable, but we have stepped up our preparations and stand ready for all eventualities, including applying for a Special Administration Regime if that were to become necessary.”

Insiders stressed that FTI Consulting’s engagement by the Department for the Environment, Food and Rural Affairs (DEFRA) did not signal that Thames Water was about to collapse into insolvency proceedings.

A SAR would ensure that customers would continue to receive water and sewage services if Thames Water collapsed, while putting taxpayers on the hook for billions of pounds in bailout costs – a scenario the chancellor, Rachel Reeves, is keen to avoid at a time when the public finances are already severely constrained.

The SAR process can only be instigated in the event that a company becomes insolvent, can no longer fulfil its statutory duties or breaches an enforcement order, according to insiders.

Mr Reed has repeatedly stressed the government’s desire to avoid taking Thames Water into temporary public ownership, but that it was ready to deal with “all eventualities”.

“Thames Water must meet its statutory and regulatory obligations to its customers and to the environment–it is only right that the company is subject to the same consequences as any other water company.

The company remains financially stable, but we have stepped up our preparations and stand ready for all eventualities,” he told the House of Commons in June.

Thames Water, which has about 16m customers, serves about a quarter of the UK’s population.

It is drowning under close to £20bn of debt, and was previously owned by Macquarie, the Australian infrastructure and banking behemoth.

Its most recent consortium of shareholders, which included the Universities Superannuation Scheme and an Abu Dhabi sovereign wealth fund, have written off the value of their investments in the company.

The government’s SAR process has only been tested once before, when the energy retailer Bulb failed in 2021.

Bulb was ultimately sold to Octopus Energy with the taxpayer funding used to save and run the company since having been repaid.

Thames Water is racing to secure a rescue plan involving funds such as Elliott Management and Silver Point Capital, with a deadline of late October to appeal to the Competition and Markets Authority against Ofwat’s final determination on its next five-year spending plan.

Ofwat has ruled that Thames Water can spend £20.5bn during the period from 2026, with the company arguing that it requires a further sum of approximately £4bn.

Mike McTighe, a veteran corporate troubleshooter who chairs BT Group’s Openreach division, has been parachuted in to work with the funds.

The company said in its accounts last month that there was “material uncertainty” over whether it could be solvently recapitalised.

Earlier this year, Thames Water was fined a record £123m over sewage leaks and the payment of dividends, with Ofwat lambasting the company over its performance and governance.

In recent weeks, Thames Water has been engulfed in a row over the legitimacy of bonuses paid to chief executive Chris Weston and other bosses, even as it attempts to secure its survival.

Under new laws, Thames Water is among half a dozen water companies which have been barred from paying bonuses this year because of their poor environmental records.

The creditor group was effectively left as the sole bidder for Thames Water after the private equity firm KKR withdrew from the process, citing political and reputational risks.

The Hong Kong-based investor CK Infrastructure Holdings (CKI), which already owns Northumbrian Water, has sought to re-engage in talks about a rescue deal but has gained little traction in doing so.

News of FTI Consulting’s appointment also comes on the same day as a “nationally significant” water shortfall was declared across swathes of the country.

Last week, Sky News revealed that David Black, the Ofwat chief executive, was to step down following the publication of a government-commissioned review which recommended the regulator’s abolition.

He has been replaced by Chris Walters, another Ofwat executive, on an interim basis.

Technology

Why a new UK internet safety law is causing an outcry on both sides of the Atlantic

Published

1 hour ago

August 12, 2025

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Why a new UK internet safety law is causing an outcry on both sides of the Atlantic

As of July 25, porn sites are required to implement effective age verification methods for U.K. users.

Jack Taylor | Getty Images

It was well intentioned but a U.K. law mandating age verification on adult sites and a number of other platforms has sparked a backlash from both internet users in the country, and U.S. politicians and tech giants.

Last month, new provisions in the Online Safety Act requiring large online platforms to implement age checks to prevent children from accessing pornographic and appropriate material came into force.

The measures have led PornHub, RedTube and other porn sites to force U.K. visitors to sign up and verify their age to gain access to their services.

What is the Online Safety Act?

Broadly, the Online Safety Act is a law that imposes a duty of care on social media firms and other user-generated content sites to ensure they take responsibility for harmful content uploaded and spread on their platforms.

In particular, the legislation aims to prevent children from being exposed to pornographic content and material that promotes suicide, self-harm, eating disorders or abusive and hateful behaviour.

The regulation has been years in the making and faced numerous delays in its development — not least due to concerns that it may infringe internet users’ right to privacy and result in censorship.

Why has it led to backlash?

The latest measures have been imposed with the aim of ensuring children aren’t able to view harmful and inappropriate content.

However, they have led to complaints from internet users due to the requirement of having to share personal information such as their ID, credit card details and selfies — in some cases for platforms that don’t even qualify as porn sites.

Spotify, Reddit, X and a number of other platforms have introduced their own respective age verification systems to stop users under the age of 18 from consuming explicit content.

These moves have subsequently led to providers of virtual private networks (VPNs) to report that their services, which allow users to mask their location, are surging in the U.K.

Meanwhile, on Monday, Wikipedia was dealt a legal blow in the U.K. as a High Court judge ruled the platform should be treated as a “category one” service, which would subject to certain user verification requirements.

The Online Safety Act requires category one platforms to offer users the ability to verify their identity and access tools that reduce their exposure to content from non-verified users.

Wikimedia, the parent company of Wikipedia, has said previously that it could limit visitor numbers from the U.K. in order to exempt it from category one status.

U.S. politicians weigh in

A number of U.S. politicians have blasted the new rules in recent days. Last week, Vice President JD Vance — who has previously criticized the U.K.’s internet safety rules — again raised concerns with the law, fearing it could unfairly restrict American tech companies.

“I just don’t want other countries to follow us down what I think was a very dark path under the Biden administration,” Vance told reporters during a trip to the country last week.

House Judiciary Chairman Jim Jordan, R-Ohio, who also visited the U.K. recently, said in a statement after his return that sweeping online safety laws in Europe are having “a serious chilling effect on free expression and threaten the First Amendment rights of American citizens and companies.”

There has been speculation over whether the U.S. may press Britain to relax the regulations during trade talks — however, U.K. officials say the issue is not open to debate.

Could other countries follow suit?

Other countries are already adopting their own respective internet age verification laws.

Australia and Ireland have both passed similar age verification measures, while Denmark, Greece, Spain, France and Italy have started testing a common age verification app to protect users online.

In the U.S., Louisiana passed a law in 2022 requiring age verification on websites where at least a third of the content is of an adult nature, while several other states are seeking to pass similar legislation.