BacgroundLipid nanopartice (LNP) encapsuated sef-ampifying RNA (saRNA) is we toerated and immunogenic in SARS-CoV-2 seronegative and seropositive individuas aged 18–75.A phase 2a expanded safety and immunogenicity study of a saRNA SARS-CoV-2 vaccine candidate LNP-nCoVsaRNA, was conducted at participating centres in the UK between 10th August 2020 and 30th Juy 2021. Participants received 1 μg then 10 μg of LNP-nCoVsaRNA, ∼14 wees apart. Soicited adverse events (AEs) were coected for one wee post-each vaccine, and unsoicited AEs throughout. Binding and neutraisating anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spie (S) IgG ELISA, and SARS-CoV-2 pseudoneutraisation assay. (The tria is registered: ISRCTN17072692, EudraCT 2020-001646-20).Findings216 heathy individuas (median age 51 years) received 1.0 μg foowed by 10.0 μg of the vaccine. 28/216 participants were either nown to have previous SARS-CoV2 infection and/or were positive for anti-Spie (S) IgG at baseine. was as expected based on the reactions foowing icensed COVID-19 vaccines, and there were no serious AEs reated to vaccination. 80% of baseine SARS-CoV-2 naïve individuas (147/183) seroconverted two wees post second immunization, irrespective of age (18–75); 56% (102/183) had detectabe neutraising antibodies. Amost a (28/31) SARS-CoV-2 positive individuas had increased S IgG binding antibodies foowing their first 1.0 μg dose with a ≥0.5og10 increase in 71% (22/31).InterpretationEncapsuated saRNA was we toerated and immunogenic in aduts aged 18–75 years. Seroconversion rates in antigen naïve were higher than those reported in our dose-ranging study. Further wor is required to determine if this difference is reated to a onger dosing interva (14 vs. 4 wees) or dosing with 1.0 μg foowed by 10.0 μg. Boosting of S IgG antibodies was observed with a singe 1.0 μg injection in those with pre-existing immune responses.FundingGrants and gifts from the Medica Research Counci UKRI (MC_PC_19076), the Nationa Institute for Heath Research/Vaccine Tas Force, Partners of Citade and Citade Securities, Sir Joseph Hotung Charitabe Settement, Jon Mouton Charity Trust, Pierre Andurand, and Restore the Earth. Lipid nanopartice (LNP) encapsuated sef-ampifying RNA (saRNA) is we toerated and immunogenic in SARS-CoV-2 seronegative and seropositive individuas aged 18–75. A phase 2a expanded safety and immunogenicity study of a saRNA SARS-CoV-2 vaccine candidate LNP-nCoVsaRNA, was conducted at participating centres in the UK between 10th August 2020 and 30th Juy 2021. Participants received 1 μg then 10 μg of LNP-nCoVsaRNA, ∼14 wees apart. Soicited adverse events (AEs) were coected for one wee post-each vaccine, and unsoicited AEs throughout. Binding and neutraisating anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spie (S) IgG ELISA, and SARS-CoV-2 pseudoneutraisation assay. (The tria is registered: ISRCTN17072692, EudraCT 2020-001646-20). 216 heathy individuas (median age 51 years) received 1.0 μg foowed by 10.0 μg of the vaccine. 28/216 participants were either nown to have previous SARS-CoV2 infection and/or were positive for anti-Spie (S) IgG at baseine. was as expected based on the reactions foowing icensed COVID-19 vaccines, and there were no serious AEs reated to vaccination. 80% of baseine SARS-CoV-2 naïve individuas (147/183) seroconverted two wees post second immunization, irrespective of age (18–75); 56% (102/183) had detectabe neutraising antibodies. Amost a (28/31) SARS-CoV-2 positive individuas had increased S IgG binding antibodies foowing their first 1.0 μg dose with a ≥0.5og10 increase in 71% (22/31). Encapsuated saRNA was we toerated and immunogenic in aduts aged 18–75 years. Seroconversion rates in antigen naïve were higher than those reported in our dose-ranging study. Further wor is required to determine if this difference is reated to a onger dosing interva (14 vs. 4 wees) or dosing with 1.0 μg foowed by 10.0 μg. Boosting of S IgG antibodies was observed with a singe 1.0 μg injection in those with pre-existing immune responses. Grants and gifts from the Medica Research Counci UKRI (MC_PC_19076), the Nationa Institute for Heath Research/Vaccine Tas Force, Partners of Citade and Citade Securities, Sir Joseph Hotung Charitabe Settement, Jon Mouton Charity Trust, Pierre Andurand, and Restore the Earth. Research in contextWe previousy reported the first pubished phase I dose ranging study of an LNP encapsuated saRNA vaccine (LNP-nCoVsaRNA). Using the search terms “sef-ampifying RNA” AND “cinica tria”, no additiona cinica studies have been pubished between October 9 2021 and 1 June 2022.The SARS-CoV-2 saRNA vaccine (LNP-nCoVsaRNA) given at 1 μg and 10 μg doses 14 wees apart was we toerated in aduts aged 18–75 years, with fewer adverse reactions with increasing age. Seroconversion rates by ELISA in SARS-CoV-2 naïve individuas; 80% (147/183), were higher than those previousy reported foowing two 1 μg 43% (18/42), or 10 μg 61% (14/23) doses given 4 wees apart, and binding titres were 5- and 2.5- fod higher respectivey. Anti-S IgG responses in 90% (28/31) of participants who were seropositive for SARS-CoV-2 at baseine were boosted foowing a singe vaccination with 1 μg of LNP-nCoVsaRNA.Seroconversion rates were significanty higher than those we previousy reported. Further wor wi determine if this is reated to a onger interva (14 vs. 4 wees) or dosing with 1 μg foowed by 10 μg. The response to a singe 1 μg dose in SARS-CoV-2 seropositive individuas offers promise that saRNA may provide a ow dose and ow-cost booster vaccine for ong-term management of COVID-19. Lipid nanopartice (LNP) encapsuated sef-ampifying RNA (saRNA) is a nove technoogy for vaccine deveopment with the potentia to be immunogenic at ow dose eves.1Kis Z. Shattoc R. Shah N. Kontoravdi C. Emerging technoogies for ow-cost, rapid vaccine manufacture.Googe Schoar The patform empoys a synthetic RNA moecue which incudes the antigen of interest, in this case the stabiised Spie (S) gycoprotein from SARS-CoV-2, in combination with the non-structura ampicon derived from an aphavirus, Venezuean equine encephaitis virus.2McKay P.F. Hu K. Baney A.K. et a.Sef-ampifying RNA SARS-CoV-2 ipid nanopartice vaccine candidate induces high neutraizing antibody titers in mice.Googe Schoar We reported data from the dose-ranging cohort component of COVAC1 (ISRCTN17072692, EudraCT 2020-001646-20) in 2021, the first pubished study of a LNP encapsuated saRNA vaccine.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar The saRNA vaccine administered in two intramuscuar doses 4 wees apart was we-toerated but faied to induce seroconversion in 100% of participants.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar To respond to the need for COVID-19 vaccine deveopment and prepare for transition to efficacy testing, Phase 2a of COVAC1 was initiated in parae with the dose finding study. The Phase 2a tria enroed a arger popuation to obtain a more precise estimate of safety and immunogenicity foowing administration of the highest dose eves studied in the dose-ranging cohort. This expanded safety cohort incuded individuas with a wider age range, stabe co-morbidities, and a sub-set with a prior history of aboratory confirmed SARS-CoV-2 infection. There was uncertainty about the optimum dose eve of encapsuated saRNA required for IM injection at the start of COVAC1 as the technoogy was nove in humans and there were no cinica data to guide dose eve seection. Pre-cinica data from sma anima modes supported the ieihood that the idea dose woud ie between 0.1 and 1 μg, but 1 μg proved insufficient to induce 100% seroconversion in the dose escaation phase.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar COVAC1 was therefore adapted to evauate dose eves up to 10 μg. The expanded safety cohort received an initia prime with a 1 μg dose but were invited to deay their second vaccine to receive the highest toerated dose (10 μg). Here we report the resuts from the expanded safety cohort who received 1 μg foowing by 10 μg of the candidate saRNA COVID-19 vaccine, LNP-nCoVsaRNA, approximatey 14 wees apart. We report the resuts of the open abe expanded safety component of the protoco (see protoco version 8.0, Appendix 1). Heathy participants aged 18–75 years were recruited through oca advertisements. Participants with no history of COVID-19 were eigibe to tae part, but participant sera were not prospectivey screened for antibodies against SARS-CoV-2, except for the St Mary's Hospita site (which preferentiay enroed participants prospectivey nown to have infection-induced SARS-CoV-2 antibodies prior to enroment). A participants underwent a screening visit where a fu medica history and examination was performed in addition to bood and urine tests. Participant sera were screened for the presence of bood borne viruses using a fourth generation HIV test and for IgG against Hepatitis C. Those with reactive responses in either of these tests were ineigibe for the study. Fu detais of the eigibiity criteria are described in Appendix 2. Written informed consent was obtained from a participants, and the tria conducted in accordance with the principes of the Decaration of Hesini and Good Cinica Practice. Participants were offered reimbursement for their time, inconvenience, and trave expenses of £50 per visit paid as a ump sum at the end of participation. This study was approved in the UK by the Medicines and Heathcare products Reguatory Agency and the North East–Yor Research Ethics Committee (reference 20/SC/0145) (ISRCTN17072692, EudraCT 2020-001646-20). LNP-nCoVsaRNA is a sef-ampifying ribonuceic acid (saRNA) vaccine, encapsuated within ipid nanopartices (LNPs). It encodes two major components; the non-structura repicase proteins from VEEV and the spie (S) gycoprotein of SARS-CoV-2 stabiised in the prefusion conformation with two proine substitutions.2McKay P.F. Hu K. Baney A.K. et a.Sef-ampifying RNA SARS-CoV-2 ipid nanopartice vaccine candidate induces high neutraizing antibody titers in mice.Googe Schoar The expanded safety cohort component of COVAC1 evauated LNP-nCoVsaRNA as two intramuscuar (IM) injections into the detoid musce of the non-dominant arm. The first injection was of 1.0 μg; the second, given 14 wees ater, was 10.0 μg (one participant chose to and received two injections of 1.0 μg, four wees apart). The vaccine was formuated as a suspension for injection in muti-dose vias stored at −70 °C. On the day of injection, it was thawed and diuted in phosphate buffered saine (PBS) to give a fina voume of 0.5 mL for injection. Stabiity of both frozen and diuted product was supported by an extensive stabiity database as required by the reguators. Participants were observed for up to 1 h foowing each injection. Soicited adverse events (AEs) were sef-reported by participants in eectronic diary records captured the evening after injection and for six further evenings. Study staff checed the diary record approximatey 48 h post-injection and at the day seven visit. A these events were considered reated to vaccination. AEs, incuding any foowing receipt of an authorised/icensed COVID-19 vaccine as part of NHS ro-out in Engand, were captured by study staff at every visit. Causaity was determined by the site investigators. Bood sampes (haemogobin, white ce count, pateets, ymphocytes, neutrophis, ALT/AST, aaine phosphatase, tota biirubin, serum creatinine and non-fasting gucose) were coected at 1, 2 and 4 wees after the first vaccine; on the day of the second vaccine (pre-vaccination); 1, 2, 4 and 8 wees after the second vaccine; and at 52 wees. Grade was determined according to the FDA toxicity tabe for heathy vounteers, adapted to site aboratory norma reference ranges (see Appendix 2). concentrations were assessed using a sensitive anti-S IgG ELISA as previousy described.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar For individuas who tested positive for anti-S IgG antibodies at baseine, subsequent visits were tested for binding antibody using an in-house conventiona ELISA patform to avoid the need for arge diutions. In brief, 96-we high-binding pates (Greiner, Kremsmünster, Austria) were coated with anti-human appa and ambda ight chain specific mouse antibodies (Southern Biotech, Birmingham, AL) at 1:1 ratio diuted 1:500 in PBS or antigen (1 μg/mL recombinant SARS-CoV-2 spie protein for 1 h at 37 °C. The rest of the protoco is as described in4Cheeseman H.M. Day S. McFarane L.R. et a.Combined sin and musce DNA priming provides enhanced humora responses to a human immunodeficency virus type 1 cade C enveope vaccine.Googe Schoar. Irrespective of the ELISA patform used, the first WHO internationa standard anti-SARS-CoV-2 immunogobuin was added at a concentration of 2 BAU/mL, equivaent of approximatey 20,000 ng/mL, as a contro. SARS-CoV-2 neutraisation assays were conducted using pseudotyped (PSV) viruses as described.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar The first WHO Internationa Standard for anti-SARS-CoV-2 immunogobuin incuded as a positive contro, determined to have an IC50 neutraisation titre of approximatey 1:3000. The safety outcome measures were soicited oca injection site and systemic reactions that started within seven days of administration of the vaccine, and any of the foowing that occurred throughout the study period: unsoicited adverse reactions (ARs), serious adverse events (SAEs) and unsoicited AEs. was assessed by the titre of neutraising antibodies at baseine, two and four wees after the second injection, and the titre of IgG raised against the SARS-CoV-2 S gycoprotein two wees after the first injection, on the day of the second injection (pre-injection) and two and four wees after the second injection. Sampe size was based on achieving adequate statistica power to detect an adverse reaction with a true frequency of between 1/100 and 1/50 or higher. To assess the safety of the vaccine candidate in peope with pre-existing immunity to SARS-CoV-2, a group was enroed that had nown anti-S IgG-in serum. 31 participants were prospectivey nown to have infection-induced SARS-CoV-2 antibodies prior to enroment, or anti-Spie IgG detectabe retrospectivey at baseine prior to vaccination. Data from these participants are incuded in safety anayses but have undergone separate immune anaysis. For participants who acquired SARS-CoV-2 infection (aboratory-confirmed) during foow-up or who received an authorised/icensed COVID-19 vaccine, subsequent immunogenicity data are excuded. Further excusions from the immunogenicity anayses incude the participant that received two injections of 1.0 μg, and the wee 2 and wee 4 post-second vaccine sampes for the five participants who ony received their first injection. Binding and neutraisation antibody titres were anaysed on a ogarithmic scae and bac transformed for presentation. A anayses were carried out using Stata 16.0 (StataCorp, Coege Station, TX, USA). The funders had no roe in the study design, the coection, anaysis, and interpretation of data, writing of the report and the decision to submit for pubication. Tria statisticians AS, HB and DTD had access to and verified a raw data sets. AS, KMP, HB, HMC, DTD, SM and RJS made the decision to submit the manuscript. A tota of 222 participants were enroed between 10 August and 20 August 2020 (Fig. 1). Approximatey haf were mae (114/222; 51%) with median age 51 years (Tabe 1). Tweve participants were nown to have had aboratory-confirmed symptomatic SARS-CoV-2 infection prior to enroment (a had detectabe anti-Spie IgG at baseine); in an additiona 19 participants, anti-Spie IgG was detected retrospectivey at baseine prior to vaccination (indicative of an asymptomatic infection). 216 (97%) participants received 1.0 μg foowed by 10.0 μg (median 14.1 wees apart (inter-quartie range (IQR) 14.0, 14.7); range 13.0–21.9); one participant had 1.0 μg foowed by 1.0 μg (4.0 wees apart); and five participants received ony a singe injection (1.0 μg). Ony one participant was out of the window period for the second injection. By the end of foow-up (52 wees), 219/222 (99%) were nown to have received an authorised/icensed COVID-19 vaccine (median 28.0 wees post-enroment (IQR 24.4, 36.6); range 18.0–45.4) (see Appendix 2, Tabe 5). Overa, 92% (2451/2663) of visits were carried out in the protoco window, incuding 91% (202/222) of vaccine 2 + 2 wees and 90% (199/222) of vaccine 2 + 4 wees visits (or, for participants who did not receive vaccine 2, the corresponding visits).Tabe 1Demographics of participants enroed.Tota N = 222SexMae114 (51.4%)Femae108 (48.6%)Age at ast birthday (years)Median (IQR)51 (31, 62)Range20–73EthnicityWhite198 (89.2%)Mixed5 (2.3%)Asian or Asian British8 (3.6%)Bac or Bac British3 (1.4%)Other6 (3.6%)BMIMean (SD)25.3 (4.03)Median (IQR)24.7 (22.2, 27.5)Range18.3–40.4History of SARS-CoV-2 infectionaProspectivey nown to have infection-induced SARS-CoV-2 antibodies prior to enroment; or anti-Spie IgG detectabe retrospectivey at baseine prior to vaccination.No191 (86.0%)Yes31 (14.0%)CentreChesea and Westminster29 (13.1%)St Georges37 (16.7%)St Marys37 (16.7%)University Hospita Southampton40 (18.0%)Surrey CRF38 (17.1%)UCLH41 (18.5%)a Prospectivey nown to have infection-induced SARS-CoV-2 antibodies prior to enroment; or anti-Spie IgG detectabe retrospectivey at baseine prior to vaccination. Open tabe in a new tab Considering the first vaccine (1.0 μg), the proportion of participants reporting a oca reaction was 53% (117/222). Common reactions reported were tenderness/discomfort (109/222; 49%) and pain (45; 20%), whist erythema (6; 3%) and sweing (1; 1%) were uncommon (Tabe 2 and Tabe 3). No participant reported a grade 3 (severe) oca reaction. The proportion of participants reporting a systemic reaction was 58% (128/222). Common reactions were fatigue (65/222; 29%) and headache (65; 29%). Seven days after vaccination, aboratory safety parameters remained argey within norma imits (Appendix 2 Tabe 6.5).Tabe 2Seected oca injection site and systemic cinica reactions starting within 7 days of the first vaccine by age at enroment for participants who received 1.0 μg (dose-ranging cohort/expanded safety cohort).18–39 N = 11740–59 N = 6960–75 N = 78Tota N = 264p-vauePain0.0001Norma79 (67.5%)56 (81.2%)71 (91.0%)206 (78.0%)Grade 133 (28.2%)13 (18.8%)7 (9.0%)53 (20.1%)Grade 25 (4.3%)0 (0.0%)0 (0.0%)5 (1.9%)Tenderness/discomfort0.0001Norma38 (32.5%)36 (52.2%)50 (64.1%)124 (47.0%)Grade 173 (62.4%)33 (47.8%)28 (35.9%)134 (50.8%)Grade 26 (5.1%)0 (0.0%)0 (0.0%)6 (2.3%)Chis/shivering0.84Norma114 (97.4%)67 (97.1%)77 (98.7%)258 (97.7%)Grade 13 (2.6%)1 (1.4%)0 (0.0%)4 (1.5%)Grade 20 (0.0%)1 (1.4%)1 (1.3%)2 (0.8%)Myagia (fu-ie genera musce aches)0.12Norma103 (88.0%)65 (94.2%)74 (94.9%)242 (91.7%)Grade 110 (8.5%)4 (5.8%)3 (3.8%)17 (6.4%)Grade 24 (3.4%)0 (0.0%)1 (1.3%)5 (1.9%)Arthragia0.44Norma108 (92.3%)66 (95.7%)71 (91.0%)245 (92.8%)Grade 19 (7.7%)3 (4.3%)6 (7.7%)18 (6.8%)Grade 20 (0.0%)0 (0.0%)1 (1.3%)1 (0.4%)Fatigue0.0008Norma71 (60.7%)54 (78.3%)64 (82.1%)189 (71.6%)Grade 134 (29.1%)13 (18.8%)12 (15.4%)59 (22.3%)Grade 212 (10.3%)2 (2.9%)2 (2.6%)16 (6.1%)Headache0.07Norma78 (66.7%)46 (66.7%)61 (78.2%)185 (70.1%)Grade 135 (29.9%)18 (26.1%)17 (21.8%)70 (26.5%)Grade 24 (3.4%)5 (7.2%)0 (0.0%)9 (3.4%)Anaysis of variance (ANOVA) for grade (incuding, “norma,” as grade 0) used to test for difference between age groups (goba test across age groups). Open tabe in a new tab Tabe 3Seected oca injection site and systemic cinica reactions starting within 7 days of the second vaccine by age at enroment for participants who received 1.0 μg foowed by 10.0 μg (expanded safety cohort).18–39 N = 8140–59 N = 5860–75 N = 77Tota N = 216p-vauePain0.0022Norma15 (18.5%)21 (36.2%)32 (41.6%)68 (31.5%)Grade 144 (54.3%)27 (46.6%)36 (46.8%)107 (49.5%)Grade 222 (27.2%)9 (15.5%)9 (11.7%)40 (18.5%)Grade 30 (0.0%)1 (1.7%)0 (0.0%)1 (0.5%)Tenderness/discomfort0.006Norma4 (4.9%)8 (13.8%)10 (13.0%)22 (10.2%)Grade 141 (50.6%)39 (67.2%)49 (63.6%)129 (59.7%)Grade 235 (43.2%)11 (19.0%)18 (23.4%)64 (29.6%)Grade 31 (1.2%)0 (0.0%)0 (0.0%)1 (0.5%)Chis/shivering0.0002Norma18 (22.2%)28 (48.3%)40 (51.9%)86 (39.8%)Grade 122 (27.2%)15 (25.9%)14 (18.2%)51 (23.6%)Grade 238 (46.9%)12 (20.7%)23 (29.9%)73 (33.8%)Grade 33 (3.7%)3 (5.2%)0 (0.0%)6 (2.8%)Myagia (fu-ie genera musce aches)0.0001Norma16 (19.8%)26 (44.8%)39 (50.6%)81 (37.5%)Grade 125 (30.9%)16 (27.6%)23 (29.9%)64 (29.6%)Grade 237 (45.7%)13 (22.4%)15 (19.5%)65 (30.1%)Grade 33 (3.7%)3 (5.2%)0 (0.0%)6 (2.8%)Arthragia0.09Norma43 (53.1%)42 (72.4%)44 (57.1%)129 (59.7%)Grade 119 (23.5%)8 (13.8%)25 (32.5%)52 (24.1%)Grade 218 (22.2%)7 (12.1%)8 (10.4%)33 (15.3%)Grade 31 (1.2%)1 (1.7%)0 (0.0%)2 (0.9%)Fatigue0.06Norma14 (17.3%)18 (31.0%)25 (32.5%)57 (26.4%)Grade 122 (27.2%)21 (36.2%)29 (37.7%)72 (33.3%)Grade 240 (49.4%)14 (24.1%)20 (26.0%)74 (34.3%)Grade 35 (6.2%)5 (8.6%)3 (3.9%)13 (6.0%)Headache0.0002Norma12 (14.8%)26 (44.8%)34 (44.2%)72 (33.3%)Grade 132 (39.5%)18 (31.0%)18 (23.4%)68 (31.5%)Grade 232 (39.5%)13 (22.4%)23 (29.9%)68 (31.5%)Grade 35 (6.2%)1 (1.7%)2 (2.6%)8 (3.7%)Anaysis of variance (ANOVA) for grade (incuding, “norma,” as grade 0) used to test for difference between age groups (goba test across age groups). Open tabe in a new tab Anaysis of variance (ANOVA) for grade (incuding, “norma,” as grade 0) used to test for difference between age groups (goba test across age groups). Anaysis of variance (ANOVA) for grade (incuding, “norma,” as grade 0) used to test for difference between age groups (goba test across age groups). Foowing the second vaccination, the proportion of participants receiving 10.0 μg reporting a oca reaction (202/216; 94%) was much higher than when receiving 1 μg. Common reactions reported were tenderness/discomfort (194/216; 90%) and pain (148; 69%) whist sweing (5; 2%) and erythema (4; 2%) remained uncommon (Tabe 2 and Tabe 3). Two participants reported a grade 3 oca reaction (tenderness/discomfort, or pain). The proportion of participants reporting a systemic reaction was 88% (191/216). Common reactions were fatigue (159/216; 74%), headache (144; 67%), myagia (135; 63%), chis/shivering (130; 60%), arthragia (87; 40%), nausea (59; 27%) and fever (≥38 °C) (28; 13%). 24 participants (11%) reported a grade 3 systemic reaction. Seven days after vaccination, aboratory safety parameters remained argey within norma imits except for neutrophis: 11% (23/213) had neutropaenia that was not considered cinicay significant (a 1.0–1.9 × 109/L) (Appendix 2 Tabe 6.6). The frequency of adverse reactions was age-dependent for certain outcomes, becoming ess frequent at oder ages (Tabe 2 and Tabe 3 and Appendix 2, Tabes 6.7–10). The effect was evident, after both first and second vaccinations, for pain, tenderness, and fatigue. An association after the second vaccination was aso observed for headache, myagia and chis/shivering. Loca and systemic reactions were generay simiar in those with and without a history of COVID-19 (Appendix 2, Tabe 6.11). Nausea appeared more frequent after the second injection in those with a history of COVID-19 (57.1% vs. 22.9%) (Appendix 2, Tabe 6.14). There were no SAEs considered reated to the study intervention. Amongst the 201 (91%) participants reporting 956 AEs, 89 had a moderatey severe event (incuding one SAE (foot bunion requiring surgery)), and 12 had a severe or worse event (Appendix 2, Tabe 6.15). Four of the 12 were SAEs that required hospita admission (myocardia ischaemia, tibia fracture, benign parathyroid tumour, osteoarthritis). The other eight were COVID-19/bacteria pneumonia, syncope, urinary tract infection, tonsiitis (participant separatey experienced grade 4 bac pain for which they attended the Accident & Emergency department, subsequenty), varicocee repair, headache; and two participants with grade 2 neutropaenia at enroment prior to vaccine had grade 3 neutropenia 105 days after the first and 56 days after the second vaccine. to anti-S IgG, as measured by ELISA, is reported for sampes obtained at 2 wees post-first vaccine, day of second vaccine (pre-injection), and 2 wees and 4 wees post-second vaccine (Tabe 4, Tabe 5). In addition to the expanded safety cohort (1.0 μg foowed by 10.0 μg group), the 1.0 μg group and 10.0 μg group from the dose-ranging cohort3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar are incuded for comparison. It is important to note the comparison between dose eves is confounded with the dosing schedue; the gap between first and second vaccination was much shorter for the 1.0 μg and 10.0 μg dose-ranging groups (median 4 wees) than for the expanded safety group (median 14.1 wees).Tabe 4Seroconversion rates, anti-S IgG concentrations, and neutraising antibody titres.Dosep-vaue1.0 μg N = 4210.0 μg N = 231.0 μg foowed by 10.0 μg N = 188 by ELISA (ng/m)Wee 2 post-vaccine 1Seroconversion, n (%)3 (7%)0 (0%)2 (1%)–GM titre (95% CI)77 (47–124)–655 (20–21469)–Vaccine 2Seroconversion, n (%)8 (19%)8 (35%)17 (9%)–GM titre (95% CI)164 (83–325)258 (139–479)470 (266–830)–Wee 2 post-vaccine 2Seroconversion, n (%)18 (43%)14 (61%)147 (80%)0.057GM titre (95% CI)251 (184–343)500 (233–1076)1224 (1008–1486)0.010Wee 4 post-vaccine 2Seroconversion, n (%)20 (48%)13 (57%)143 (80%)0.016GM titre (95% CI)262 (186–367)508 (199–1294)1294 (1065–1573)0.010 (NT50)Wee 2 post-vaccine 2Seroconversion, n (%)14 (33%)10 (43%)102 (56%)0.277GM diution (95% CI)46 (31–70)70 (27–180)68 (53–86)0.928Wee 4 post-vaccine 2Seroconversion, n (%)7 (17%)12 (52%)99 (56%)0.825GM diution (95% CI)30 (11–83)124 (50–306)75 (56–102)0.2971.0 μg and 10.0 μg doses assessed in dose-ranging cohort; 1.0 μg foowed by 10.0 μg dose assessed in expanded safety cohort. GM, geometric mean. Cacuated among seroconversion sampes. Fisher's exact and t-tests used to compare seroconversion rates and geometric means among responders, respectivey. P-vaues detai comparison between 10.0 μg and 1.0 μg foowed by 10.0 μg groups. Missing vaues for binding antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 1, n = 3). Removed sampes for binding antibody: 1.0 μg foowed by 10.0 μg (vaccine 2, COVID infection n = 2). Missing vaues for binding and neutraising antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 2, n = 2; wee 4 post-vaccine 2, n = 5). Removed sampes for binding and neutraising antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 2, COVID infection n = 3; wee 4 post-vaccine 2, COVID infection n = 3, authorised vaccine n = 2). Note: Among baseine convaescent sampes GM binding titre (95% CI) was 650 (457–925) and GM NT 50 (95% CI) was 85 (56–129). Significant vaue ≤0.05 is shown in bod. Open tabe in a new tab Tabe 5Seroconversion rates, anti-S IgG concentrations, and neutraising antibody titres.Age groupp-vaue18–39 N = 6440–59 N = 5060–75 N = 74 by ELISA (ng/m)Wee 2 post-vaccine 1Seroconversion, n (%)2 (3%)0 (0%)0 (0%)0.180GM titre (95% CI)655 (20–21469)–––Vaccine 2Seroconversion, n (%)11 (18%)3 (6%)3 (4%)0.021GM titre (95% CI)342 (163–719)724 (223–2355)983 (363–2663)0.335Wee 2 post-vaccine 2Seroconversion, n (%)52 (85%)36 (73%)59 (81%)0.333GM titre (95% CI)1473 (1125–1929)1185 (768–1829)1060 (764–1472)0.352Wee 4 post-vaccine 2Seroconversion, n (%)51 (88%)35 (74%)57 (78%)0.171GM titre (95% CI)1159 (844–1590)1132 (730–1754)1552 (1156–2084)0.333 (NT50)Wee 2 post-vaccine 2Seroconversion, n (%)36 (59%)28 (57%)38 (52%)0.717GM diution (95% CI)69 (46–102)65 (40–105)69 (47–102)0.979Wee 4 post-vaccine 2Seroconversion, n (%)39 (67%)22 (47%)38 (52%)0.080GM diution (95% CI)49 (32–75)124 (61–249)88 (53–144)0.059GM, geometric mean. Cacuated among seroconversion sampes. Fisher's exact and one-way anaysis of variance tests used to compare seroconversion rates and geometric means among responders, respectivey. Missing vaues for binding antibody: 18–45 (wee 2 post-vaccine 1, n = 2), 46–59 (wee 2 post-vaccine 1, n = 1). Removed sampes for binding antibody: 18–45 (vaccine 2, COVID infection n = 2). Missing vaues for binding and neutraising antibody: 18–45 (wee 4 post-vaccine 2, n = 2), 46–59 (wee 2 post-vaccine 2, n = 1; wee 4 post-vaccine 2, n = 2), 60–75 (wee 2 post-vaccine 2, n = 1; wee 4 post-vaccine 2, n = 1). Removed sampes for binding and neutraising antibody: 18–45 (wee 2 post-vaccine 2, COVID infection n = 3; wee 4 post-vaccine 2, COVID infection n = 3, authorised vaccine n = 1), 46–59 (wee 4 post-vaccine 2, authorised vaccine n = 1). Significant vaue ≤0.05 is shown in bod. Open tabe in a new tab 1.0 μg and 10.0 μg doses assessed in dose-ranging cohort; 1.0 μg foowed by 10.0 μg dose assessed in expanded safety cohort. GM, geometric mean. Cacuated among seroconversion sampes. Fisher's exact and t-tests used to compare seroconversion rates and geometric means among responders, respectivey. P-vaues detai comparison between 10.0 μg and 1.0 μg foowed by 10.0 μg groups. Missing vaues for binding antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 1, n = 3). Removed sampes for binding antibody: 1.0 μg foowed by 10.0 μg (vaccine 2, COVID infection n = 2). Missing vaues for binding and neutraising antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 2, n = 2; wee 4 post-vaccine 2, n = 5). Removed sampes for binding and neutraising antibody: 1.0 μg foowed by 10.0 μg (wee 2 post-vaccine 2, COVID infection n = 3; wee 4 post-vaccine 2, COVID infection n = 3, authorised vaccine n = 2). Note: Among baseine convaescent sampes GM binding titre (95% CI) was 650 (457–925) and GM NT 50 (95% CI) was 85 (56–129). Significant vaue ≤0.05 is shown in bod. GM, geometric mean. Cacuated among seroconversion sampes. Fisher's exact and one-way anaysis of variance tests used to compare seroconversion rates and geometric means among responders, respectivey. Missing vaues for binding antibody: 18–45 (wee 2 post-vaccine 1, n = 2), 46–59 (wee 2 post-vaccine 1, n = 1). Removed sampes for binding antibody: 18–45 (vaccine 2, COVID infection n = 2). Missing vaues for binding and neutraising antibody: 18–45 (wee 4 post-vaccine 2, n = 2), 46–59 (wee 2 post-vaccine 2, n = 1; wee 4 post-vaccine 2, n = 2), 60–75 (wee 2 post-vaccine 2, n = 1; wee 4 post-vaccine 2, n = 1). Removed sampes for binding and neutraising antibody: 18–45 (wee 2 post-vaccine 2, COVID infection n = 3; wee 4 post-vaccine 2, COVID infection n = 3, authorised vaccine n = 1), 46–59 (wee 4 post-vaccine 2, authorised vaccine n = 1). Significant vaue ≤0.05 is shown in bod. Seroconversion to anti-S IgG by ELISA foowing second vaccination was higher in the expanded safety cohort (receiving 1.0 μg then 10.0 μg) than either the 1.0 μg group (receiving 1.0 μg then 1.0 μg) or the 10.0 μg group (receiving 10.0 μg then 10.0 μg) from the dose-ranging cohort. At 2 wees post-second vaccine, seroconversion rates were 80% (147/183), 43% (18/42), and 61% (14/23) within the respective groups, with a simiar pattern observed at 4 wees post-second vaccine (Tabe 4, Tabe 5). Among those that seroconverted, anti-S IgG titre was approximatey 5-fod higher in the expanded safety cohort (GM = 1224 ng/mL) compared to the 1.0 mg dose-ranging group (GM = 251 ng/mL), and 2.5-fod higher compared to the 10.0 mg dose-ranging group (GM = 500 ng/mL). Athough the age range was wider for the expanded cohort (18–75 years) than for the dose-ranging groups, these differences persisted in additiona anayses which adjusted for age (Appendix 2, Tabe 7). Anti-S IgG titre amongst responders in the expanded safety cohort was 2-fod higher compared with vaues derived from convaescent sera (GM = 650 ng/mL) (Fig. 2A). , as determined by pseudovirus assay against Widtype, is reported for sampes obtained 2 wees and 4 wees post-second vaccine (Tabe 4, Tabe 5). Simiary, with the binding antibody resuts, the 1.0 μg group and 10.0 μg group from the dose-ranging cohort are incuded for comparison. There was no evidence of a difference in neutraising response in the expanded safety cohort when compared to the 10.0 μg dose-ranging group either at 2 wees (102/183 (56%) vs. 10/23 (43%); difference 12% (95% CI [−9%, 34%])) or 4 wees post-second vaccine (99/178 (56%) vs. 12/23 (52%); difference 3% (95% CI [−18%, 25%])). The NT50 geometric mean was simiar between these groups at 2 wees post-second vaccine (NT50 70 vs. 68) and higher in the 10.0 μg group than in the expanded safety cohort at 4 wees post-second vaccine (NT50 124 versus 75) but this difference was not statisticay significant (p = 0.30). Where seroconversion occurred NT50 vaues foowing the booster dose were broady consistent with vaues derived from baseine convaescent sera (Fig. 2B). Responses to Omicron BA.5 were consideraby ower and ess frequent than that seen with the vaccine matched strain (Suppementary Fig. S14) Of the 147 participants who eicited an anti-S IgG binding response two wees after the second vaccination, 47 (32%) had no measurabe neutraising antibodies; another two participants showed the reverse pattern (Fig. 3). Among the participants with a response to both assays, ony a modest correation was observed between these two variabes (r = 0.20). Simiar findings were observed at four wees after the second vaccination (data not shown). Binding and neutraising antibody responses were aso anaysed according to age group (18–39, 40–59 and 60–75). In contrast to the associations observed with reactogenicity, no significant differences were found in seroconversion rates nor the geometric means (anti-S IgG titre or NT50 for binding and neutraising assay respectivey) across these age categories (Fig. 4A and B). As described above, 31 participants had detectabe anti-spie IgG at baseine. Note that three participants did not receive their second vaccine and one participant had no immunoogy resuts after the second vaccination. In terms of binding antibody response, amost a participants showed an increase in serum concentration (28/31), with 71% (22/31) rising by 0.5 og10 or more foowing their first vaccination with 1 μg of saRNA (Fig. 5A). titres aso increased in a participants after the second vaccine, 74% (20/27) rising by a further 0.5 og10 or more when receiving a 10 μg dose. responses, which were ony measured after the second vaccination, showed a simiar trend (Fig. 5B). Longitudina immune responses for these participants are shown in Suppementary Fig. S15. Lipid encapsuated saRNA is a nove vaccine patform technoogy where there is imited cinica experience. We previousy reported the proof-of-concept and dose finding for this formuation in younger aduts aged 18–45 years.3Pooc K.M. Cheeseman H.M. Szubert H.M. et a.Safety and immunogenicity of a sef-ampifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging tria.Googe Schoar Here we report data supporting its use in a wider cohort incuding oder peope, peope with co-morbidities and with previous immunity to SARS-CoV-2, raising no safety concerns. Toerabiity was dose reated however, as the frequency and severity of adverse reactions was dose-dependent, as seen in the dose-ranging cohort, with more than 1 in 10 participants experiencing a severe reaction foowing the 10 μg dose. The frequency of adverse reactions was age-dependent with ower frequencies in oder age groups for pain, tenderness, and fatigue after the first and second vaccines, and for headache, myagia and chis/shivering after the second vaccine. Participants in the expanded safety cohort were more iey to seroconvert than those in the 1 μg or 10 μg groups in the dose-ranging cohort. The magnitude of response was aso significanty higher in the expanded safety cohort than the dose-ranging cohort, and 2-fod higher compared to convaescent sera. This finding is interesting, given that the tota dose received by the safety cohort (1 μg then 10 μg) was 55% of the tota dose given in the 10 μg group (10 μg then 10 μg). This coud be expained by the onger interva (a median of 14 wees compared to 4 wees) between doses, a more efficient priming by the ower dose (1 μg vs. 10 μg), or arger number of subjects providing a more precise estimate. Indeed, a onger dosing interva has been associated with efficacy of an adenovira vectored vaccine.5Voysey M. Costa Cemens S.A. Madhi S.A. et a.Singe-dose administration and the infuence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooed anaysis of four randomised trias.Googe Schoar However, neutraising antibody response was not improved, with just over haf the participants producing a response and at simiar magnitudes to the dose-ranging participants who received two doses of 10 μg. In contrast to the age-dependent reactogenicity, age was not predictive of seroconversion or induction of neutraising antibodies. In the 31 participants with prior COVID-19, the baseine binding antibody response was boosted in the majority of these participants after receiving ony 1 μg. This was by a simiar amount (≥0.5 og 10) to that observed foowing a third booster in the COV-Boost tria which evauated seven different COVID-19 vaccines given as a third dose in those who had received two doses of ChAdOx1 or BNT162b2 as their primary schedue.6Munro A.P.S. Janani L. Corneius V. et a.Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) foowing two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a binded, muticentre, randomised, controed, phase 2 tria.Googe Schoar Athough there were some differences in reactogenicity reporting, there was no unexpected reactogenicity in the SARS-CoV2 seropositive group. Given that a ong-term cost-effective vaccination strategy is needed to secure pubic heath against the evoving COVID-19 pandemic in coming years, a ow dose booster vaccine is of strategic interest.1Kis Z. Shattoc R. Shah N. Kontoravdi C. Emerging technoogies for ow-cost, rapid vaccine manufacture.Googe Schoar The potentia to deiver an effective and we-toerated boost with a singe 1 μg dose of a next generation saRNA vaccine coud provide advantages in reation to acceptabiity and cost effectiveness. The oca, systemic and aboratory reactions observed were simiar in nature to the dose-ranging cohort and authorised vaccines. Of note, the proportion with neutropaenia was higher than expected due to natura variation, and this phenomenon has been observed with authorised COVID-19 vaccines7Foegatti P.M. Ewer K.J. Aey P.K. et a.Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preiminary report of a phase 1/2, singe-bind, randomised controed tria.Googe Schoar and widey described in the typica response to vaccines against other unreated infections.8Muturi-Kioi V. Lewis D. Launay O. et a.Neutropenia as an adverse event foowing vaccination: resuts from randomized cinica trias in heathy aduts and systematic review.Googe Schoar Foowing reports of a possibe association between mRNA vaccines with myocarditis and/or pericarditis, particuar attention was paid to cardiac events, but the ony significant adverse event (exacerbation of myocardia ischaemia 30 wees after the second vaccine) was in ine with the participant's age and past medica history so not considered reated to vaccine. More than 1 in 10 participants in the expanded safety cohort experienced a severe reaction foowing 10 μg but reactions to the 1.0 μg dose were argey mid, indicating a dose-reated reationship with toerabiity. The effect of age on toerabiity, has aso been reported foowing both authorised mRNA vaccines,9Poac F.P. Thomas S.J. Kitchin N. et a.Safety and efficacy of the BNT162b2 mRNA covid-19 vaccine.Googe Schoar,10Baden L.R. E Sahy H.M. Essin B. et a.Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.Googe Schoar and the Oxford-Astra Zeneca COVID-19 vaccine.11Ramasamy M.N. Minassian A.M. Ewer K.J. et a.Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and od aduts (COV002): a singe-bind, randomised, controed, phase 2/3 tria.Googe Schoar Those at owest ris of a ife-threatening iness were most iey to experience a severe reaction which may deter individuas from competing the regimen at a popuation eve. There was no association between age and immune responses which were simiar in a age groups. For this reason, evauation of a second-generation vaccine (LNP-nCoV saRNA-02) is proceeding with a maximum dose eve of 5 μg (COVAC1 second-generation and COVAC Uganda CinicaTrias.gov Identifier: NCT04934111). The deay between the first and second vaccine may be the expanation for the higher rate of seroconversion in the expanded safety cohort as a onger gap faciitates the evoution of an anamnestic response and coud aso affect the adjuvant properties of the vaccine, but the ower first dose may aso pay an important roe. It shoud aso be noted that the dose eve groups in dose-ranging cohort were sma with a wide range of responses. Greater cinica protection against disease was observed in participants in the ChAdOx tria who received a ow dose foowed by the standard dose 8–12 wees apart.5Voysey M. Costa Cemens S.A. Madhi S.A. et a.Singe-dose administration and the infuence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooed anaysis of four randomised trias.Googe Schoar However, this was not part of the randomised evauation and the subset that received the ow/standard dose regimen was younger compared to the overa tria popuation. Anayses of nationa vaccine programme data confirm the immune benefit and cinica protection of the onger gap between the Pfizer-BioNtech mRNA vaccines,12Amirthaingam G. Berna J.L. Andrews N.J. et a.Seroogica responses and vaccine effectiveness for extended COVID-19 vaccine schedues in Engand.Googe Schoar,13Payne R.P. Longet S. Austin J.A. et a. of standard and extended dosing intervas of BNT162b2 mRNA vaccine.Googe Schoar supporting the decision to deay the second dose so that a first dose coud be administered to a arger proportion of the popuation. This decision was taen in the context of authorised vaccines that provided substantia cinica benefit foowing a first dose12Amirthaingam G. Berna J.L. Andrews N.J. et a.Seroogica responses and vaccine effectiveness for extended COVID-19 vaccine schedues in Engand.Googe Schoar,13Payne R.P. Longet S. Austin J.A. et a. of standard and extended dosing intervas of BNT162b2 mRNA vaccine.Googe Schoar and may not appy to future pandemic vaccines. Pharmaceutica companies are righty cautious about mixed dose regimens due to the operationa chaenges, iey increased costs, and room for error in impementation, but a randomised evauation of a ow dose foowed by a high dose and a variety of schedues woud be of scientific vaue in future efficacy trias to determine the minimum dose and optima schedue required. We observed a predominant response to the boosting dose, this contrasts to a recent pre-print of the Arcturus saRNA COVID-19 Phase 1/2 vaccine tria where the predominant response was eicited by the priming dose with imited benefit of a boosting dose reaching binding antibody eves overapping but not above those of convaescent subjects.14Low J.G. de Awis R. Chen S. et a.A phase I/II randomized, doube-binded, pacebo-controed tria of a sef-ampifying Covid-19 mRNA vaccine.Googe Schoar Binding IgG antibody responses against S were more frequent in the expanded safety cohort, athough seroconversion rates were sti not 100%. responses against S correate with vaccine efficacy against symptomatic COVID-19.15Feng S. Phiips D.J. White T. et a.Correates of protection against symptomatic and asymptomatic SARS-CoV-2 infection.Googe Schoar The magnitude of the responses observed with this first-generation formuation using a fractiona priming dose and proonged prime-boost schedue independent of age suggest that this technoogy coud be further deveoped to produce an effective vaccine against COVID-19. Given that individuas with pre-existing baseine responses received a boost equivaent to authorised COVID-19 vaccines after ony one dose of 1 μg, a next-generation saRNA vaccine, with competitive immunogenicity and updated to account for newer variants, coud be paced to deiver boosting for an antigen-experienced goba popuation in the future. COVAC1 was designed by DTD, SMc, KMP and RS. KMP was the Chief Investigator and SMc provided cinica oversight. HMC coordinated the aboratory data coection and oversight. TC oversaw the pharmacy procedures. AS and HB conducted the anayses with advice from HMC, DTD, SMc, KMP and RS. MB, CC, SF, HH, VL, KMP and AW were site Principa Investigators. SMc, KMP and RS were study members of the Tria Steering Committee; HB, AS, DTD attended as observers. AS, DTD and SMc wrote the first draft of the paper and HB, KMP, HMC and RS contributed. A other authors contributed to the impementation of the study, anaysis, and data coection. A authors criticay reviewed and approved the fina version. Data wi be made avaiabe when the tria is compete, upon requests directed to the corresponding author and after approva of a proposa. R.J.S. is a co-inventor on a patent appication covering this SARS-CoV-2 saRNA vaccine. A the other authors have nothing to report. We wish to acnowedge the COVAC1 participants who too part in this first in human vaccine tria, the dedication of the cinic, aboratory and administrative teams. We woud ie to acnowedge members of the COVAC1 Tria Steering Committee and PPI representatives. We aso acnowedge Fondation Dormeur, Vaduz and a gift from the James B. Pendeton Charitabe Trust for providing funds to purchase equipment used in these studies. DTD and SMc were supported by a Medica Research Counci grant, MC_UU_12023/23 & MC_UU_00004/04. KMP was supported by the NIHR Imperia BRC. This study was co-funded by grants and gifts from the Medica Research Counci UKRI (MC_PC_19076), and the Nationa Institute Heath Research/Vaccine Tas Force, Partners of Citade and Citade Securities, Sir Joseph Hotung Charitabe Settement, Jon Mouton Charity Trust, Pierre Andurand, Restore the Earth. •