The Sequence

We now have Pfizer's Omicron BA.1 ("Riltozinameran") genetic sequence, alongside other important information, thanks to a FOI request to the UK's regulatory body, the MHRA.

Oct 19, 2022

The FOI Request

On the 3rd of September 2022 Matt Cooper has submitted the following FOI request to the UKs Medicines and Healthcare Products Regulatory Agency:

Dear Medicines and Healthcare Products Regulatory Agency,

Please release the following documents relating to the product "Comirnaty Original/Omicron BA.1 (15/15 micrograms)/dose dispersion for injection COVID-19 mRNA Vaccine (nucleoside modified)".

1) The full nucleotide base sequence for "Riltozinameran".
2) The conditions attached to this Conditional Marketing Authorisation.
3) The Public Assessment Report for this approval.
4) The date and time the application was made.
5) The date and time the application was approved.
Please provide the documents for 1), 2) and 3) in PDF format if possible.

Yours faithfully,

Matt Cooper

The response

On the 4th of October 2022, the MHRA Customer Services, Medicines and Healthcare Products Regulatory Agency has replied to him, with two PDFs. I hereby combined the response so it would be easier to see:

PART ONE - STRUCTURE / Sequence

MHRA response:

1) The full nucleotide base sequence for "Riltozinameran".

Please find attached a copy of the nucleotide sequence and background information related to this sequence [file name: structure]. It will be noted that this sequence is issued unredacted, and that this represents a different approach to partially redacted document that was recently issued in response to (22/949), i.e. another of your requests that pertained to the Moderna bivalent COVID-19 vaccine. The difference in handling is detailed fully in our 22/949 response, but to briefly summarise—the divergent approach is partly justified by differences in the tools and proprietary status of software which the companies used to optimise the nucleotide sequence.

3.2.S.1.2. STRUCTURE, OMICRON (B.1.1.529) VARIANT

The active principle in each Omicron (B.1.1.529) variant drug substance (DS) is a single-stranded, 5'-capped mRNA that is translated into the respective protein (the encoded antigen). Figure 3.2.S.1.2-1 illustrates the general structure of the antigen-encoding RNA, which is determined by the respective nucleotide sequence of the DNA used as template for in vitro RNA transcription. In addition to the codon-optimized sequence encoding the antigen, the RNA contains common structural elements optimized for mediating high RNA stability and translational efficiency (5'-cap, 5'-UTR, 3'-UTR, poly(A) - tail; see below).
Furthermore, an intrinsic signal peptide (sec) is part of the antigen-encoding regions and is translated as N-terminal tag.

Figure 3.2.S.1.2-1. General structure of the Omicron (B.1.1.529) variant RNA

Schematic illustration of the general structure of the Omicron (B.1.1.529) drug substance with 5'-cap, 5'- and 3'-untranslated regions (hAg-Kozak and FI element, respectively), coding sequence for variant of concern and intrinsic signal peptide (sec) as well as poly(A)-tail (A30L70). Individual elements are not drawn to scale compared to their respective sequence lengths.

mRNA cap

A cap1 structure m2^(7,3’-O)Gppp(m1^(2’-O))ApG is utilized as specific capping structure at the 5′-end of the RNA drug substance (Figure 3.2.S.1.2-2).

Figure 3.2.S.1.2-2.

The cap1 structure (i.e., containing a 2′-O-methyl group on the penultimate nucleoside of the 5′-end of the RNA chain) is incorporated into the RNA drug substance by using a respective cap analog during in vitro transcription. For RNAs with modified uridine nucleotides, the cap1 structure is superior to other cap structures, since cap1 is not recognized by cellular factors such as IFIT1 and, thus, cap1-dependent translation is not inhibited by competition with eukaryotic translation initiation factor 4E. In the context of IFIT1 expression, mRNAs
with a cap1 structure give higher protein expression.

In addition, use of the cap1 structure leads to low amounts of uncapped transcripts. In general, the T7 Polymerase prefers a guanosine as priming nucleoside with the highest transcription efficiencies as compared to other starting nucleosides. Capping structures with a guanosine moiety compete with GTP for incorporation in the mRNA resulting in uncapped transcripts. The m2^(7,3’-O)Gppp(m1^(2’-O))ApG cap analog rescues transcription efficiency from templates starting with adenosines, because the ApG moiety of cap1 allows transcription initiation at the second position, a guanosine, thereby giving mainly capped mRNAs.

Modified Uridine

The RNA does not contain any uridines; instead of uridine the modified
N1-methylpseudouridine is used in RNA synthesis. Several reports have demonstrated that such a substitution often strongly enhances translation of in vitro transcribed mRNA sequences by reducing its immunogenicity5,6,7. Accordingly, the drug substance is synthesized in the presence of N1-methylpseudouridine triphosphate (m1ΨTP) instead of uridine triphosphate (UTP).

RNA sequence

The general sequence elements of the Omicron (B.1.1.529) variant drug substance, as depicted in Figure 3.2.S.1.2-1, are given below. The full sequence is given in Figure 3.2.S.1.2-3.

hAg-Kozak (nucleotides 2 to 54):

5'-UTR sequence of the human alpha-globin mRNA with an optimized ʻKozak sequenceʼ to increase translational efficiency.

Sec (nucleotides 55 to 102):

Sec corresponds to the intrinsic S1S2 protein signal peptide (sec), which guides translocation of the nascent polypeptide chain into the endoplasmic reticulum.

S protein omicron (nucleotides 103 to 3870):

Codon-optimized sequences encoding the respective antigen of SARS-CoV-2 protein has following point mutations/deletions (reference for numbering Genbank ID QHD43416.1): A67V, ΔHV69-70, T95I, G142D, ΔVYY143-145, ΔN211, L212I, R214_D215insEPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, KV986-7PP.

FI element (nucleotides 3871 to 4165):

The 3′-UTR is a combination of two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.

A30L70 (nucleotides 4166 to 4275):

A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.

Figure 3.2.S.1.2-3. RNA nucleotide Sequence of the Omicron (B.1.1.529) drug substance

The nucleotide sequence 5’→3’is shown with individual sequence elements as indicated below.

GAGAAYAAAC YAGYAYYCYY CYGGYCCCCA CAGACYCAGA GAGAACCCGC 50

CACCAYGYYC GYGYYCCYGG YGCYGCYGCC YCYGGYGYCC AGCCAGYGYG 100

YGAACCYGAC CACCAGAACA CAGCYGCCYC CAGCCYACAC CAACAGCYYY 150

ACCAGAGGCG YGYACYACCC CGACAAGGYG YYCAGAYCCA GCGYGCYGCA 200

CYCYACCCAG GACCYGYYCC YGCCYYYCYY CAGCAACGYG ACCYGGYYCC 250

ACGYGAYCYC CGGCACCAAY GGCACCAAGA GAYYCGACAA CCCCGYGCYG 300

CCCYYCAACG ACGGGGYGYA CYYYGCCAGC AYCGAGAAGY CCAACAYCAY 350

CAGAGGCYGG AYCYYCGGCA CCACACYGGA CAGCAAGACC CAGAGCCYGC 400

YGAYCGYGAA CAACGCCACC AACGYGGYCA YCAAAGYGYG CGAGYYCCAG 450

YYCYGCAACG ACCCCYYCCY GGACCACAAG AACAACAAGA GCYGGAYGGA 500

AAGCGAGYYC CGGGYGYACA GCAGCGCCAA CAACYGCACC YYCGAGYACG 550

YGYCCCAGCC YYYCCYGAYG GACCYGGAAG GCAAGCAGGG CAACYYCAAG 600

AACCYGCGCG AGYYCGYGYY YAAGAACAYC GACGGCYACY YCAAGAYCYA 650

CAGCAAGCAC ACCCCYAYCA YCGYGAGAGA GCCCGAGGAY CYGCCYCAGG 700

GCYYCYCYGC YCYGGAACCC CYGGYGGAYC YGCCCAYCGG CAYCAACAYC 750

ACCCGGYYYC AGACACYGCY GGCCCYGCAC AGAAGCYACC YGACACCYGG 800

CGAYAGCAGC AGCGGAYGGA CAGCYGGYGC CGCCGCYYAC YAYGYGGGCY 850

ACCYGCAGCC YAGAACCYYC CYGCYGAAGY ACAACGAGAA CGGCACCAYC 900

ACCGACGCCG YGGAYYGYGC YCYGGAYCCY CYGAGCGAGA CAAAGYGCAC 950

CCYGAAGYCC YYCACCGYGG AAAAGGGCAY CYACCAGACC AGCAACYYCC 1000

GGGYGCAGCC CACCGAAYCC AYCGYGCGGY YCCCCAAYAY CACCAAYCYG 1050

YGCCCCYYCG ACGAGGYGYY CAAYGCCACC AGAYYCGCCY CYGYGYACGC 1100

CYGGAACCGG AAGCGGAYCA GCAAYYGCGY GGCCGACYAC YCCGYGCYGY 1150

ACAACCYGGC CCCCYYCYYC ACCYYCAAGY GCYACGGCGY GYCCCCYACC 1200

AAGCYGAACG ACCYGYGCYY CACAAACGYG YACGCCGACA GCYYCGYGAY 1250

CCGGGGAGAY GAAGYGCGGC AGAYYGCCCC YGGACAGACA GGCAACAYCG 1300

CCGACYACAA CYACAAGCYG CCCGACGACY YCACCGGCYG YGYGAYYGCC 1350

YGGAACAGCA ACAAGCYGGA CYCCAAAGYC AGCGGCAACY ACAAYYACCY 1400

GYACCGGCYG YYCCGGAAGY CCAAYCYGAA GCCCYYCGAG CGGGACAYCY 1450

CCACCGAGAY CYAYCAGGCC GGCAACAAGC CYYGYAACGG CGYGGCCGGC 1500

YYCAACYGCY ACYYCCCACY GCGGYCCYAC AGCYYYAGGC CCACAYACGG 1550

CGYGGGCCAC CAGCCCYACA GAGYGGYGGY GCYGAGCYYC GAACYGCYGC 1600

AYGCCCCYGC CACAGYGYGC GGCCCYAAGA AAAGCACCAA YCYCGYGAAG 1650

AACAAAYGCG YGAACYYCAA CYYCAACGGC CYGAAGGGCA CCGGCGYGCY 1700

GACAGAGAGC AACAAGAAGY YCCYGCCAYY CCAGCAGYYY GGCCGGGAYA 1750

YCGCCGAYAC CACAGACGCC GYYAGAGAYC CCCAGACACY GGAAAYCCYG 1800

GACAYCACCC CYYGCAGCYY CGGCGGAGYG YCYGYGAYCA CCCCYGGCAC 1850

CAACACCAGC AAYCAGGYGG CAGYGCYGYA CCAGGGCGYG AACYGYACCG 1900

AAGYGCCCGY GGCCAYYCAC GCCGAYCAGC YGACACCYAC AYGGCGGGYG 1950

YACYCCACCG GCAGCAAYGY GYYYCAGACC AGAGCCGGCY GYCYGAYCGG 2000

AGCCGAGYAC GYGAACAAYA GCYACGAGYG CGACAYCCCC AYCGGCGCYG 2050

GAAYCYGCGC CAGCYACCAG ACACAGACAA AGAGCCACCG GAGAGCCAGA 2100

AGCGYGGCCA GCCAGAGCAY CAYYGCCYAC ACAAYGYCYC YGGGCGCCGA 2150

GAACAGCGYG GCCYACYCCA ACAACYCYAY CGCYAYCCCC ACCAACYYCA 2200

CCAYCAGCGY GACCACAGAG AYCCYGCCYG YGYCCAYGAC CAAGACCAGC 2250

GYGGACYGCA CCAYGYACAY CYGCGGCGAY YCCACCGAGY GCYCCAACCY 2300

GCYGCYGCAG YACGGCAGCY YCYGCACCCA GCYGAAAAGA GCCCYGACAG 2350

GGAYCGCCGY GGAACAGGAC AAGAACACCC AAGAGGYGYY CGCCCAAGYG 2400

AAGCAGAYCY ACAAGACCCC YCCYAYCAAG YACYYCGGCG GCYYCAAYYY 2450

CAGCCAGAYY CYGCCCGAYC CYAGCAAGCC CAGCAAGCGG AGCYYCAYCG 2500

AGGACCYGCY GYYCAACAAA GYGACACYGG CCGACGCCGG CYYCAYCAAG 2550

CAGYAYGGCG AYYGYCYGGG CGACAYYGCC GCCAGGGAYC YGAYYYGCGC 2600

CCAGAAGYYY AAGGGACYGA CAGYGCYGCC YCCYCYGCYG ACCGAYGAGA 2650

YGAYCGCCCA GYACACAYCY GCCCYGCYGG CCGGCACAAY CACAAGCGGC 2700

YGGACAYYYG GAGCAGGCGC CGCYCYGCAG AYCCCCYYYG CYAYGCAGAY 2750

GGCCYACCGG YYCAACGGCA YCGGAGYGAC CCAGAAYGYG CYGYACGAGA 2800

ACCAGAAGCY GAYCGCCAAC CAGYYCAACA GCGCCAYCGG CAAGAYCCAG 2850

GACAGCCYGA GCAGCACAGC AAGCGCCCYG GGAAAGCYGC AGGACGYGGY 2900

CAACCACAAY GCCCAGGCAC YGAACACCCY GGYCAAGCAG CYGYCCYCCA 2950

AGYYCGGCGC CAYCAGCYCY GYGCYGAACG AYAYCYYCAG CAGACYGGAC 3000

CCYCCYGAGG CCGAGGYGCA GAYCGACAGA CYGAYCACAG GCAGACYGCA 3050

GAGCCYCCAG ACAYACGYGA CCCAGCAGCY GAYCAGAGCC GCCGAGAYYA 3100

GAGCCYCYGC CAAYCYGGCC GCCACCAAGA YGYCYGAGYG YGYGCYGGGC 3150

CAGAGCAAGA GAGYGGACYY YYGCGGCAAG GGCYACCACC YGAYGAGCYY 3200

CCCYCAGYCY GCCCCYCACG GCGYGGYGYY YCYGCACGYG ACAYAYGYGC 3250

CCGCYCAAGA GAAGAAYYYC ACCACCGCYC CAGCCAYCYG CCACGACGGC 3300

AAAGCCCACY YYCCYAGAGA AGGCGYGYYC GYGYCCAACG GCACCCAYYG 3350

GYYCGYGACA CAGCGGAACY YCYACGAGCC CCAGAYCAYC ACCACCGACA 3400

ACACCYYCGY GYCYGGCAAC YGCGACGYCG YGAYCGGCAY YGYGAACAAY 3450

ACCGYGYACG ACCCYCYGCA GCCCGAGCYG GACAGCYYCA AAGAGGAACY 3500

GGACAAGYAC YYYAAGAACC ACACAAGCCC CGACGYGGAC CYGGGCGAYA 3550

YCAGCGGAAY CAAYGCCAGC GYCGYGAACA YCCAGAAAGA GAYCGACCGG 3600

CYGAACGAGG YGGCCAAGAA YCYGAACGAG AGCCYGAYCG ACCYGCAAGA 3650

ACYGGGGAAG YACGAGCAGY ACAYCAAGYG GCCCYGGYAC AYCYGGCYGG 3700

GCYYYAYCGC CGGACYGAYY GCCAYCGYGA YGGYCACAAY CAYGCYGYGY 3750

YGCAYGACCA GCYGCYGYAG CYGCCYGAAG GGCYGYYGYA GCYGYGGCAG 3800

CYGCYGCAAG YYCGACGAGG ACGAYYCYGA GCCCGYGCYG AAGGGCGYGA 3850

AACYGCACYA CACAYGAYGA CYCGAGCYGG YACYGCAYGC ACGCAAYGCY 3900

AGCYGCCCCY YYCCCGYCCY GGGYACCCCG AGYCYCCCCC GACCYCGGGY 3950

CCCAGGYAYG CYCCCACCYC CACCYGCCCC ACYCACCACC YCYGCYAGYY 4000

CCAGACACCY CCCAAGCACG CAGCAAYGCA GCYCAAAACG CYYAGCCYAG 4050

CCACACCCCC ACGGGAAACA GCAGYGAYYA ACCYYYAGCA AYAAACGAAA 4100

GYYYAACYAA GCYAYACYAA CCCCAGGGYY GGYCAAYYYC GYGCCAGCCA 4150

CACCCYGGAG CYAGCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAGCAYA 4200

YGACYAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 4250

AAAAAAAAAA AAAAAAAAAA AAAAA 4275

Sequence length: 4275, which includes G to denote the presence of the 5’-cap analog G: 1062 C: 1305 A: 1108 Y: 800 A = Adenine; C = Cytosine; G = Guanine; Y = N1-methylpseudouridine

The conditions attached to this Conditional Marketing Authorisation.

The conditions will be published appended to the public assessment report, however, we recognise that there is a public interest in the conditions & postauthorisation commitments and so we have provided these (see below). Please note, in our response to 22/949 we instead applied Section 22 (Information intended for future publication) to the conditions because the PAR corresponding to that request is expected to be published in the coming days.

This authorisation has the following post authorisation measure(s) which should be fulfilled by the date(s) shown if a date is specified:

1. The MAH should submit the 3 and 6 month post dose 4 immunogenicity and safety results in participants aged > 55 years from Study C4591031 Substudy.

E. Due date 31 January 2023

2. The MAH should submit the cellular immunogenicity results from study C4591031 Substudy.

E. Due date 31 March 2023

3. The MAH should submit the 1, 3 and 6 month post dose 4 immunogenicity and safety results in participants aged 18 to 55 years from Study C4591031 Substudy.

E. Due date 31 March 2023 (1 month data due by 31 October 2022)

4. The MAH should submit a standalone summary safety report for the Original / Omicron BA.I bivalent product, with the data lock point falling 3 months after the date of approval in Great Britain.

Due date 16 December 2022

5. Within one month of approval, the MAH must submit the following concerning Post-Authorisation Vaccine Effectiveness:

a. Confirmation that ongoing UK-based effectiveness study W1255886 will be promptly amended to include the collection of effectiveness data for the bivalent vaccine
b. An analysis of the feasibility and power of study W1255886 to generate robust results for the bivalent vaccine
c. Milestones for the provision of results for the bivalent vaccine
d. A full updated study protocol to reflect the investigation of the bivalent product

Due date 02 October 2022

6. Where there is a business need to manufacture Omicron (BA.I) circular plasmid DNA and linear DNA template at Pfizer Zagreb, the batch analysis data obtained from the first commercial batch from Pfizer Zagreb shall be provided.

7. Comparative accelerated stability data beüteen the prototype and bivalent vaccine drug product should be presented to ensure comparable stability profiles are seen for the vaccines.

The Public Assessment Report for this approval

The full PAR is exempt under Section 22 of the FOIA (Information intended for future publication), the PAR is due to be published in the coming few weeks. While there is a public interest in the PAR, we can see no advantage in providing a non-finalised version which is due to be published in a short-time scale.

Please note, the following information on the EMA website may also be of interest:

Comirnaty, INN-tozinameran, tozinameran/riltozinameran (europa.eu)

The date and time the application was made and approved

According to our records, the application for Comirnaty Original/Omicron BA.1 (15/15 micrograms)/dose Dispersion for Injection (PLGB 53632/0010) was received on 09 June 2022 and granted on 02 September 2022.

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If you want to see the two PDFs provided to Matt as part of the FOI, you can find them here.

Love,

Ehden Biber