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

World

Overnight attacks in Russia and Ukraine as Zelenskyy eyes talks with Trump over peace plan

Published

3 hours ago

November 25, 2025

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Overnight attacks in Russia and Ukraine as Zelenskyy eyes talks with Trump over peace plan

Donald Trump and Volodymyr Zelenskyy are set to hold talks over the Ukraine peace plan.

US and Ukrainian officials have held discussions in Geneva about a controversial 28-point proposal drawn up by America and Russia, which has since been countered by an amended deal drawn up by Kyiv’s European allies.

The White House said there were still a “couple of points of disagreement” as of Monday night, but spokeswoman Karoline Leavitt said there was a “sense of urgency” to strike an agreement.

“The president wants to see this deal come together, and to see this war end,” she added.

Mr Zelenskyy echoed that message, saying “there is still work for all of us to do to finalise the document”.

“We must do everything with dignity,” he said in his nightly video address, adding: “The sensitive issues, the most delicate points, I will discuss with President Trump.”

It comes after Mr Trump, who had accused Ukraine of not being grateful enough for US military support while the Geneva talks were under way, suggested the process could be moving in the right direction.

He had earlier given Kyiv until Thursday to agree to the plan, but US Secretary of State Marco Rubio downplayed the deadline, saying officials could keep negotiating.

Moscow, however, has already signalled its opposition to the European version of the peace plan.

It would halt fighting at present front lines, leaving discussions of territory for later, and also include a NATO-style US security guarantee for Ukraine.

The talks in Geneva, Switzerland, had begun with Mr Rubio denying the original plan was written by Russia.

It appeared to include a number of longstanding Kremlin demands that have proved impossible for Kyiv, including sacrificing territory Russian forces have not even seized since the war began.

Ms Leavitt has also insisted the US is not favouring the Russians.

Starmer to lead talks of Ukraine’s allies

Ukraine’s allies in the so-called “coalition of the willing” will hold a virtual meeting today, chaired by Sir Keir Starmer.

The British prime minister said the alliance was focused on achieving a “just and lasting peace”.

It “matters for all of us, because the conflict in Ukraine has had a direct impact here in the UK”, he added.

Russia and Ukraine report overnight attacks

The talks will begin hours after the governor of Russia’s Rostov region reported three people had been killed and 10 more injured in a Ukrainian attack overnight.

The Russian defence ministry said 249 Ukrainian drones were downed over Russian regions in total.

Meanwhile, Russian drone strikes in Kyiv left at least two dead and triggered fires on residential buildings – forcing evacuations, and leaving several people injured.

The war was also a topic of discussion in a call between Mr Trump and China’s Xi Jinping on Monday.

Mr Xi urged “all parties” in the conflict to “reduce differences”, according to Chinese state news agency Xinhua.

He reiterated that China supported all efforts conducive to peace.

China has remained a consistent ally of Russia throughout its invasion of Ukraine, and is the top buyer of Russian oil, along with India.

World

Return to China ‘not an option’, Taiwan warns – as Xi mentions issue in phone call with Trump

Published

4 hours ago

November 25, 2025

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Return to China 'not an option', Taiwan warns - as Xi mentions issue in phone call with Trump

Taiwan’s prime minister has warned that a “return” to China is not an option.

Cho Jung-tai’s remarks came hours after President Xi Jinping mentioned the issue on a phone call with his US counterpart Donald Trump.

Mr Xi had described Taiwan’s return to mainland China as “an integral part of the post-war international order”.

But the island’s democratically elected government has rejected this – describing Taiwan as a “fully sovereign and independent country”.

“For the 23 million people of our nation, ‘return’ is not an option – this is very clear,” Mr Cho added.

China has offered Taiwan a “one country, two systems” model, but this position is not supported by any mainstream political party.

Meanwhile, relations between Beijing and Tokyo recently plunged to fresh lows after Japan’s prime minister suggested any attack on Taiwan would spark a military response.

Sanae Takaichi’s remarks were swiftly denounced by China’s foreign ministry, which said Japan had “crossed a red line that should not have been touched”.

Sun Yun from the Stimson Centre thinktank said: “My best guess is China is worried about the escalation with Japan.

“The reference to Taiwan and the post-World War II order directly points to the spat with Japan over Taiwan.”

The US has not taken a side when it comes to Taiwan’s sovereignty but is opposed to any use of force to seize the territory.

Mr Trump’s administration has also been ambiguous over whether it would deploy US troops if war broke out in the Taiwan Strait.

Yesterday, Taiwan’s defence ministry spotted a single balloon from China flying over the strait and claims this is part of a campaign of harassment by Beijing.

Chinese officials have previously dismissed these complaints, arguing the balloons are for meteorological purposes.

In other developments, Mr Trump has confirmed that he will visit Beijing in April – with Mr Xi set to travel to Washington for a state visit later in 2026.

Relations have improved in recent weeks after the pair met face to face in South Korea, with the US president moving to cut tariffs.

On Truth Social, he wrote: “Our relationship with China is extremely strong!”

Politics

SEC issues ‘rare’ no-action letter for Solana DePIN project token FUSE

Published

5 hours ago

November 25, 2025

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SEC issues ‘rare’ no-action letter for Solana DePIN project token FUSE

The SEC has just issued its second “no-action letter” toward a decentralized physical infrastructure network (DePIN) crypto project in recent months, giving its native token “regulatory cover” from enforcement.

The no-action letter was sent to the Solana DePIN project Fuse, which issues a network token, FUSE, as a reward to those actively maintaining the network.

Fuse initially submitted a letter to the SEC’s Division of Corporation Finance on Nov. 19, asking for official confirmation that it would not recommend the SEC take enforcement action if the project continues to offer and sell FUSE tokens.

Fuse also outlined in its letter that FUSE is designed for network utility and consumptive purposes, not for speculation. They can only be redeemed for an average market price via third parties.

“Based on the facts presented, the Division will not recommend enforcement action to the Commission if, in reliance on your opinion as counsel, Fuse offers and sells the Tokens in the manner and under the circumstances described in your letter,” the Division of Corporation Finance’s deputy chief counsel, Jonathan Ingram, wrote on Monday.

*SEC’s no-action letter to Fuse Crypto. Source:* *SEC*

The latest SEC no-action letter comes just a few months after the SEC issued a similar “highly coveted” letter to Double Zero, which was seen as a result of a new, more crypto-friendly leadership at the SEC.

At the time, DoubleZero co-founder Austin Federa said such letters are common in TradFi but are “very rare” in the crypto space.

“It was a months long process, but we found the SEC to be quite receptive, we found them to be quite professional, quite diligent, there was no crypto animosity.”

The SEC was put under new leadership in April, after Paul Atkins was sworn in as the 34th chairman, and the agency has since been seen taking a more balanced approach to crypto. As part of the leadership, crypto-friendly Hester Peirce also heads up the agency’s crypto task force.

SEC no-action letters are a form of regulatory clarity

Adding to the discussion on X, Rebecca Rettig, a legal representative of Solana MEV infrastructure platform Jito Labs, said that no-action letters are sought after by many crypto projects.