Articles Immunogenic dynamics and SARS-CoV-2 variant neutralisation of the heterologous ChAdOx1-S/ BNT162b2 vaccination: Secondary analysis of the randomised CombiVacS study Javier García-Perez,a,b,1 María Gonzalez-Perez,c,1 María Castillo de la Osa,d Alberto M. Borobia,e Luis Casta~ no,f María Jes n, Magdalena Campins, Antonio Portoles, David Lora, us Bertra g h i,j,s k,l,s,u Mercedes Bermejo, Patricia Conde,c a Lourdes Herna ndez-Gutierrez, Antonio Carcas, Eunate Arana-Arri, Marta Tortajada,m Inmaculada Fuentes,n Ana Ascaso,i d e f María Teresa García-Morales,k,s Humberto Erick de la Torre-Tarazona,a Jose-Ram on Arribas,b,o Natale Imaz-Ayo,f Eugenia Mellado-Pau,g Antonia Agustí,p,q Carla Perez-Ingidua,i Agustín Go mez de la Ca mara,k,l,r,s Jordi Ochando,c t Cristobal Belda-Iniesta, Jesus Frías, Jose Alcamí, * and Mayte Perez-Olmeda e a,b,2 b,d,2 **, on behalf of the CombiVacS study Group3 a Unidad de Inmunopatología del SIDA, Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), Madrid, Spain b Centro de Investigacion Biomedica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), Madrid, Spain c Laboratorio de Referencia en Inmunología, Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), Madrid, Spain d Laboratorio de Serología, Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), Madrid, Spain e Servicio de Farmacología Clínica, Departamento de Farmacología y Terap eutica, Facultad de Medicina, Hospital Universitario La Paz, IdiPAZ, Universidad Auto noma de Madrid, Madrid, Spain f Biocruces Bizkaia, Hospital Universitario Cruces, CIBERDEM, CIBERER, Endo-ERN, UPV-EHU, Barakaldo, Spain g Servicio de Medicina Preventiva y Epidemiologia, Hospital Clínic de Barcelona, Barcelona, Spain h Servicio de Medicina Preventiva y Epidemiología, Hospital Universitari Vall d'Hebron, Universitat Auto noma de Barcelona, Barcelona, Spain i Servicio de Farmacología Clínica, Hospital Clínico San Carlos, IdISSC, Madrid, Spain j Departamento de Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid (UCM), Madrid, Spain k Instituto de Investigaci on Sanitaria Hospital 12 de octubre (imas12), Facultad de Medicina, Universidad Complutense de Madrid (UCM) l Centro de Investigacion Biomedica en Red de Epidemiología y Salud P ublica (CIBERESP), Instituto de Salud Carlos III (ISCIII), Madrid, Spain m Servicio de Prevencion de Riesgos Laborales, Salud Laboral, Hospital Clínic de Barcelona, Barcelona, Spain n Unidad de Soporte a la Investigacio n Clínica, Vall d’Hebron Institut de Recerca, Barcelona, Spain o Servicio de Medicina Interna, Departamento de Medicina, Facultad de Medicina, Hospital Universitario La Paz, IdiPAZ, Universidad Auto noma de Madrid, Madrid, Spain p Servicio de Farmacología Clínica, Hospital Universitari Vall d’Hebron, Barcelona, Spain q Departament de Farmacologia, Terap noma de Barcelona, Bellaterra, Barcelona, Spain eutica i Toxicologia, Universitat Auto r Facultad de Medicina, Universidad Complutense de Madrid (UCM), Madrid, Spain s Spanish Clinical Research Network − SCReN − ISCIII, Madrid, Spain t Directorate Instituto de Salud Carlos III, Madrid, Spain u Facultad de Estudios Estadísticos, Universidad Complutense de Madrid (UCM), Madrid, Spain Summary Background The CombiVacS study was designed to assess immunogenicity and reactogenicity of the heterologous eClinicalMedicine ChAdOx1-S/BNT162b2 combination, and 14-day results showed a strong immune response. The present secondary 2022;50: 101529 analysis addresses the evolution of humoral and cellular response up to day 180. Published online xxx https://doi.org/10.1016/j. eclinm.2022.101529 *Corresponding author at: Unidad de Inmunopatologıa del SIDA Instituto de Salud Carlos III, 28220 Majadahonda, Spain. **Corresponding author at: Laboratorio de Serologıa, Instituto de salud Carlos III, 28220 Majadahonda, Spain. E-mail addresses: ppalcami@isciii.es (J. Alcamı), mayteperez@isciii.es (M. Perez-Olmeda). 1 Both authors contributed equally and should be considered as first authors. 2 Both authors contributed equally and should be considered joint senior authors. 3 Study group members are listed in Appendix 1. www.thelancet.com Vol 50 Month , 2022 1 Articles Methods Between April 24 and 30, 2021, 676 adults primed with ChAdOx1-S were enrolled in five hospitals in Spain, and randomised to receive BNT162b2 as second dose (interventional group [IG]) or no vaccine (control group [CG]). Individuals from CG received BNT162b2 as second dose and also on day 28, as planned based on favourable results on day 14. Humoral immunogenicity, measured by immunoassay for SARS-CoV-2 receptor binding domain (RBD), antibody functionality using pseudovirus neutralisation assays for the reference (G614), Alpha, Beta, Delta, and Omicron variants, as well as cellular immune response using interferon-g and IL-2 immunoassays were assessed at day 28 after BNT162b2 in both groups, at day 90 (planned only in the interventional group) and at day 180 (laboratory data cut-off on Nov 19, 2021). This study was registered with EudraCT (2021-001978-37) and Clini- calTrials.gov (NCT04860739). Findings In this secondary analysis, 664 individuals (441 from IG and 223 from CG) were included. At day 28 post vaccine, geometric mean titres (GMT) of RBD antibodies were 5616¢91 BAU/mL (95% CI 5296¢49−5956¢71) in the IG and 7298¢22 BAU/mL (6739¢41−7903¢37) in the CG (p < 0¢0001). RBD antibodies titres decreased at day 180 (1142¢0 BAU/mL [1048¢69−1243¢62] and 1836¢4 BAU/mL [1621¢62−2079¢62] in the IG and CG, respectively; p < 0¢0001). Neutralising antibodies also waned from day 28 to day 180 in both the IG (1429¢01 [1220¢37−1673¢33] and 198¢72 [161¢54−244¢47], respectively) and the CG (1503¢28 [1210¢71−1866¢54] and 295¢57 [209¢84−416¢33], respectively). The lowest variant-specific response was observed against Omicron-and Beta variants, with low propor- tion of individuals exhibiting specific neutralising antibody titres (NT50) >1:100 at day 180 (19% and 22%, respectively). Interpretation Titres of RBD antibodies decay over time, similar to homologous regimes. Our findings suggested that delaying administration of the second dose did not have a detrimental effect after vaccination and may have improved the response obtained. Lower neutralisation was observed against Omicron and Beta variants at day 180. Funding Funded by Instituto de Salud Carlos III (ISCIII). Copyright Ó 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Keywords: SARS-CoV-2; Heterologous vaccination; Neutralisation; Variants; Antibodies Implications of all the available evidence Research in context Antibody titres decay over time, but delay in second dose administration has no deleterious effect on the Evidence before this study immune response; on the contrary it resulted in better humoral responses. Functionally, all individuals exhib- We did not perform a systematic search of the literature because of the rapidly developing situation. In summer ited neutralising titres >1:100 against G614 reference 2021, the Delta variant of SARS-CoV-2 emerged and strain at day 28 after vaccine and a relevant proportion replaced the circulating Alpha strain. In November 2021 (76%) did so at day 180. However, variant-specific neu- tralisation was variable, with the lowest activity the Omicron variant was first described, and became predominant by March 2022 worldwide. In parallel, in observed at day 180 against Omicron variant followed summer 2021 different works pointed to a decrease by Beta and Delta (19%, 22% and 56% with in vaccine protection after six months of immunisation, NT50>1:100, respectively). These findings support the use of heterologous regimes, which is consistent with in particular against new variants, due to waning of neu- tralising antibody activity. Together, these observations that arising from homologous schemes, and a third open the debate on a third dose booster that was dose strategy in patients previously immunised with a adopted in many countries over the final months of combination of adenovirus- and mRNA-based vaccines. 2021. Added value of this study Introduction The present results provide additional evidence on late Early after the SARS-CoV-2 outbreak started − rapidly immunogenicity − up to 6 months − of the heterolo- evolving to a worldwide pandemic − active immuniza- gous ChAdOx1-S/BNT162b2 vaccination regime. Fur- tion emerged as the key priority of global healthcare pol- ther, SARS-CoV-2 variant-specific elicited immunity is icies. Most scientific efforts have focused in vaccines also reported here. development, which successfully resulted in three 2 www.thelancet.com Vol 50 Month , 2022 Articles homologous two-dose vaccines and one single-dose vac- effectiveness, as well as by cellular immunity, given the cine available for use in the European Union between relevant role of CD4+ and CD8+ responses found in late 2020 and early 2021. Notwithstanding, rare severe COVID-19 patients and cross-reactivity observed in thrombotic with thrombocytopenia events related to unexposed individuals.15,16 Also, as suggested for Omi- ChAdOx1-S vaccine (Vaxzevria, AstraZeneca) and short- cron variant, a decreased virulence could contribute to age in supplies had an impact on European vaccination lower the rates of hospital admissions and death.17,18 plans and drove an interest in heterologous regimes. In addition, decay of variant-specific antibody titres is The combination of ChAdOx1-S and the mRNA vaccine shifting focus towards the need of a third dose, espe- BNT162b2 (Comirnaty, BioNTech) has been the first cially in those with weakened immune systems such as heterologous scheme studied. Results on 14-day1 and older adults19 and immunosuppressed patients.20,21 28-day2 immunogenicity showed that robust humoral Considering the available evidence, the European Medi- and cellular immune responses were elicited. Accord- cines Agency (EMA) has issued recommendations for ingly, and as planned in the protocol, participants an extra dose with mRNA vaccines and is completing included in the control group were offered to receive conclusions on booster doses for people with a normal BNT162b2 as a second dose. immune system.22 Concurrently, public health plans to control the pan- Increasing evidence on immunity dynamics aiming demic faced a recurrent issue, namely the periodic out- to answer these questions derives mostly from homolo- break of new SARS-CoV-2 variants. To date, numerous gous vaccines, while heterologous regimes are still less variants have been identified, of which Beta (B.1.351), studied. Here we present additional results of the Com- Gamma (P.1), and Delta (B.1.617.2) are currently consid- biVacS study1 addressing a) total and neutralising anti- ered as variants of concern (VoC), the latter beginning body dynamics of heterologous ChAdOx1-S/BNT162b2 in summer 2021 and soon becoming dominant.3 More vaccines combination, and b) immune response against recently a new variant − Omicron (B.1.1.529) − was first different SARS-CoV-2 variants, including Delta and described in Botswana and South Africa. Because of its Omicron variants. This analysis also aims to provide high transmissibility, Omicron is displacing Delta as valuable data to the debate on extra booster doses. the dominant variant in most world countries in less than 1 month, representing a new challenge in the con- trol of pandemic.4 Not surprisingly, dynamics over time Methods of variant-specific immune response induced by vacci- nation is a hot matter of research. Several works have Study design and participants reported a deterioration of vaccine-induced antibody Data from the 12-month, phase 2, open-label, rando- response and a waning of protection against infection mised, controlled CombiVacS study are included in this with either homologous regimes.5,6 By variants, a 3¢5- secondary analysis. Full descriptions of the methods as fold to 14-fold reduction of serum neutralisation titres well as safety and initial immunogenicity analyses have against Beta variant from vaccinated individuals has been previously published in detail.1 Full study protocol been reported.7−9 Further, ChAdOx1-S vaccine results is provided in Appendix 1 (p 26). evidenced undetectable neutralisation activity against Healthy, or clinically stable, adults from 18 to Beta variant in 60% of vaccinated individuals and decay 59 years old with no history of SARS-CoV-2 infection by a factor of up to 31¢5% in the remaining 40%.10 Pre- who had been vaccinated with a single dose of ChA- liminary data from Omicron variant were even more dOx1-S between 8 and 12 weeks before screening were worrying as a 14 to 30-fold reduction in neutralisation enrolled in the CombiVacS study to evaluate immuno- susceptibility elicited by immunisation was reported.11,12 genicity and reactogenicity of a second dose of the Notwithstanding this, the impact of waning neutral- mRNA COVID-19 vaccine BNT162b2. ising antibodies on clinical efficacy is not clear. Some All participants provided written informed consent studies found that protection against hospitalization or before enrolment. The trial complies with the principles death persisted at a robust level,6 while others showed of the Declaration of Helsinki and Good Clinical Prac- that efficacy notably decreased, or even failed, against tice. This study was approved by the Spanish Agency of Beta10,13 and Delta6 variants. A consequent question is Medicines and Healthcare Products (AEMPS) and by whether this reduction makes the variant resistant to the Ethics Committee at University Hospital La Paz. vaccination. Some results on Beta variant comparing sera from naturally infected and vaccinated individuals have suggested that those vaccinated retain protective Randomisation and masking levels of humoral immunity,7 while others evidenced no Briefly, participants were randomly assigned (2:1) to efficacy in mild and moderate disease.10 It must be receive one intramuscular injection of BNT162b2 (inter- mentioned that effectiveness may be influenced by the ventional group, IG) or maintain observation (control interval between doses, with longer time associated to group, CG). Since the main immunogenicity objective an enhanced antibody response14 and higher was met, and reactogenicity was acceptable,1 www.thelancet.com Vol 50 Month , 2022 3 Articles participants included in the control group were offered compared to control without serum. Samples below the to receive BNT162b2 as a second dose at day 28, as detection threshold (1:32 serum dilution) were given planned in the protocol. A systematic randomisation 1:16 value. Positive and negative controls were included stratified by study site, gender and age (18-49 years, and in the assays and non-specific neutralisation was 50-59 years) was used. The randomization list was cen- assessed using a related pseudovirus expressing the trally generated with the SAS software for Windows vesicular stomatitis virus envelope (VSV-G). Cellular (version 9.4; SAS Institute Inc., Cary, NC, USA), and immune response was measured in participants from imported into the secure Research Electronic Data two pre-selected sites by quantification of interferon-g Capture platform (REDCap version 8.7.4; Vanderbilt (IFN-g) and interleukin-2 (IL-2) present in plasma on University, Nashville, TN, USA) used for the study elec- overnight stimulation of whole blood cultured with tronic case report form (eCRF). pools of SARS-CoV-2 spike peptides (2 mg/mL) or dimethyl sulfoxide control. Cytokines were quantified using the next-generation enzyme-linked immunosor- Procedures bent assay (ELISA) tool, Ella (ProteinSimple, San Jose, Study procedures have been described in full previously.1 CA, USA). Full details on the pseudo-virus neutralisa- In brief, at randomization clinical assessments were per- tion assays and cellular immunity quantification are formed and blood samples for safety and immunology provided in the Appendix 1 (pp 18-19). collected from all participants. Concurrently, participants in the interventional group were administered 0¢3 mL BNT162b2 dose as a single intramuscular injection (day Outcomes Outcomes included in the present secondary analysis 0), whilst individuals from control group were vaccinated were humoral immune response to vaccination as per on day 28 of study. Planned follow-up visits for safety antibodies titres and neutralising antibody titres at days and immunologic purposes were scheduled on days 7, 28, 90 and 180 after the BNT162b2 dose. Of note, in 14, 28, 90, 180 and 360. All vaccinated participants were the control group outcomes at day 28 post-vaccine corre- on-site monitored for safety for at least 15 minutes. Safety spond to day 56 of study, and outcomes at day 180 corre- procedures included both direct report from individuals spond to day 152 post vaccine. In the control group, no during the post-vaccine observation period and online outcomes were planned at day 90, according to the pro- report using an electronic diary throughout the study fol- tocol. Alpha-, Beta-, Delta-, and Omicron-specific neu- low-up period. tralising antibody titres at days 28 and 180 post-dose To the present analysis, the commercial electroche- have been analysed in both study groups. Cellular miluminescence immunoassay (ECLIA) ElecsysÒ Anti- response defined as inflammatory IFN-g and IL-2 cyto- SARS-CoV-2 S assay (Roche Diagnostics GmbH, Man- kines production against SARS-CoV-2 spike peptide nheim, Germany) was used to detect antibodies (includ- pools at days 28 and 180 post-BNT162b2 dose were also ing IgG) specific to the SARS-CoV-2 spike protein assessed. receptor binding domain (RBD-S protein) on the Cobas e411 module (Roche Diagnostics GmbH, Mannheim, Germany),23 with a measuring range from 0¢4 to Statistical analysis 250 U/mL (up to 2,500 U/mL with onboard 1:10 dilu- To the present analysis, the immunogenicity population tion and up on 12,500 with onboard 1:50 dilution). Val- included all the participants who were randomly ues higher than 0¢8 BAU/mL were considered positive. assigned, completed all applicable visits, and for whom Measurement of neutralising antibodies titres in a serological samples were available at the baseline visit predefined subset of 198 participants was carried out by and on days 28, 56 (only applicable to control group), preincubation of diluted plasma samples with titrated 90 (only applicable to interventional group) and pseudoviruses (10 ng p24Gag per well) generated by co- 180. Laboratory data cut-off was on November 19, 2021. transfection of pNL4-3DenvRen and an expression vec- Day-28 variables (i.e. humoral immunogenicity by tor for the different viral spikes (pcDNA3.1-S-CoV2Δ19- RBD-specific IgG analysis, neutralising activity of G614, -Alpha, -Beta, -Delta, or -Omicron) and added to SARS-CoV-2-specific antibodies, and cellular immunity) Vero E6 cells in 96-well plates. Viral infectivity 48 hours were analysed in 658, 194 and 114 individuals, respec- post infection was assessed by measuring luciferase tively. Missing values from later visits were not imputed activity (Renilla Luciferase Assay, Promega, Madison, (Appendix 1 p 17). WI, USA) using a 96-well plate luminometer LB 960 Data were presented as geometric mean and 95% Centro XS3 (Berthold Technologies, Oak Ridge, TN, confidence interval (CI) or, for categorical variables, USA). The titre of neutralising antibodies was calcu- number, and percentage, unless otherwise stated. For lated as 50% inhibitory dose (neutralising titre 50, serological measurements, difference at each time point NT50), expressed as reciprocal of four-fold serial dilu- was evaluated using ratio of geometric means. Since the tion of heat-inactivated sera (range 1:32−1:131¢072) outcome variable, i.e. antibodies against SARS-CoV-2 resulting in a 50% reduction of pseudovirus infection spike protein RBD on day 28 post-dose, was restricted 4 www.thelancet.com Vol 50 Month , 2022 Articles by technical limitations, a truncated regression model group, mean (SD) interval between ChAdOx1-S and was used. The model incorporated right censoring val- BNT162b2 administration was 89¢03 days (5¢92). Nei- ues, raw data response with distribution lognormal ther differences between groups were found in demo- data, and treatment effect (interventional group versus graphic characteristics at day 180 (Appendix 1 p 15). control group) adjusted by sex, age, and time between Median time in collection of day-28 post- BNT162b2 vaccine doses. Additionally, reverse cumulative distribu- dose blood sample was similar in both study groups tion curve (RCDC) was plotted. A stratified analysis by (28 days [interventional] vs. 27 [control]), however vari- sex, age and interval between vaccine doses was done ability was higher in individuals from the control group for the humoral and cellular immunity endpoints. Labo- (range 21-38 days [interventional] vs. 16-42 [control]) ratory parameters with values below detection limit (Appendix 1 p 2). were replaced by a value equal to the lowest limit Results on immunogenicity dynamics in both divided by two. All analyses were carried out using the groups show a decay in titres of antibodies specific to statistical software SAS, version 9¢4. Sample size was the SARS-CoV-2 S-protein RBD over time (Appendix 1 calculated for primary efficacy endpoint − i.e. antibody pp 3-4). In the interventional group, geometric mean titres at day 14 −, and was also considered appropriate titres (GMT) of S-RBD antibodies decreased from to evaluate most of secondary endpoints (Appendix 1 p 7739¢21 BAU/mL (95% CI 7371¢53−8161¢96) at day 14 14). Sample size calculation methods as well as indepen- after BNT162b2 second dose (fully reported earlier)1 to dent data monitoring committee procedures have been 5616¢91 BAU/mL (95% CI 5296¢49−5956¢71) at day described previously.1 28, 2303¢28 BAU/mL (95% CI 2141¢66−2477¢1) at day Composition of the independent data monitoring 90 and 1142¢0 BAU/mL (1048¢69−1243¢62) at day 180. committee is provided in the Appendix 1 (p 23). This Of note, waning was slower from third to six month study was registered with EudraCT (2021-001978-37) (mean lognormal difference -0¢303 [95% CI -0¢324−(-) and ClinicalTrials.gov (NCT04860739). 0¢283]) than from first to third (-0¢389 [95% CI -0¢405 −(-)0¢374]). The regression model for outcome variables to 180 days by treatment (interventional group versus Role of the funding source control group) and adjusted by covariables is showed in The funder − Institute of Health Carlos III (ISCIII), a the Appendix 1 p 16. Interestingly, immunogenic public research organization − designed the trial in response in the control group was significantly stronger cooperation with the Spanish Clinical Trials Platform at day 28 after second dose (7298¢22 BAU/mL [95%CI (SCReN), a public network of clinical trial units at the 6739¢41−7903¢37]) than in the interventional group Spanish National Health System funded by the ISCIII. (p < 0¢0001). Likewise, antibody levels remained higher Trial coordination, participant recruitment and data at day 180 in the control compared to interventional analysis were performed by SCReN. All immunological group (1836¢4 BAU/mL [95%CI 1621¢62−2079¢62]; procedures were performed at ISCIII. All authors p < 0¢0001) (Figure 2; Appendix 1 p 3). This effect was reviewed and approved the original draft. All authors also observed in stratified analyses by sex and age had full access to the full data in the study and accept (Appendix 1 pp 5−6). Adjusted differences in day-28 responsibility to submit for publication. and day-180 lognormal RBD values in the interventional vs. control group resulted from the regression model were -0¢0881 (95% CI -0¢1239−(-)0¢0523 [p < 0¢0001]) Results and -0¢1760 (95% CI -0¢2277−(-)0¢1242 [p < 0¢0001]), Of 676 participants enrolled and randomised between respectively. In addition, the effect of delayed vaccina- April 24th and 30th, 2021, 450 individuals were assigned tion in the control group is linear over time, resulting in to the interventional group, receiving BNT162b2 as sec- a difference of S-RBD antibody levels in the test vs. con- ond dose, and 226 were to the control group, maintain- trol group of -0¢1329 (95% CI -0¢1831−(-)0¢0826) over ing observation. Participants flow up to day 14 of study the follow-up period. has been fully described previously.1 441 participants Consistently with waning of antibody titres observed from the interventional group completed day 28 of in the interventional group, a decay in neutralising anti- study and 418 completed day 180. 223 individuals from bodies was also evidenced. At day 14, GMT of neutralis- control group received BNT162b2 vaccination at day 28; ing antibodies was 1905¢69 (95%CI 1625¢65−2233¢98) 212 and 199 of them completed day 56 of study (day 28 in the interventional group, which decreased to 1429¢01 post vaccination) and day 180 (day 152 post vaccine), (95%CI 1220¢37−1673¢33) at day 28, to 480¢68 (95%CI respectively (Figure 1). 398¢27−580¢13) at day 90 and to 198¢72 (95%CI 161¢54 Baseline and demographic characteristics of the pop- −244¢47) at day 180. In the control group, neutralising ulation are summarised in Table 1. 378 (57%) individu- antibody titres were similar to the interventional group als were women and 289 (43%) were men. 431 (65%) both 28 days after second dose (1503¢28 [95%CI 1210¢71 participants were aged 18−49 years, and the mean age −1866¢54]) and at day 180 (295¢57 [95%CI 209¢84 of both groups was 44¢03 years (SD 8¢82). In the control −416¢33]) (Figure 3; Appendix 1 p 7). RCDC for www.thelancet.com Vol 50 Month , 2022 5 Articles Figure 1. Trial profile. neutralising antibodies in both study groups are shown efficacy. Yet decreased, a relevant proportion of individ- in Appendix 1 (p 8). All patients from both interven- uals (76%) exhibited NT50 >1:100 at day 180 of study. tional and control group exhibited high neutralising (Appendix 1 p 9). activity (NT50 >1:100) against the reference variant Differences between variants and original G614 28 days post- BNT162b2 dose; a threshold that G614 strain at different time points were analysed has been recently described24 as associated with vaccine (Appendix 1, p 10). By SARS-CoV-2 variants, the poorest 6 www.thelancet.com Vol 50 Month , 2022 Articles Interventional group (n = 450) Control group (n = 226)d Overall (n = 676) Sex, n (%) Male 193 (43%) 101 (45%) 294 (43%) Female 257 (57%) 125 (55%) 382 (57%) Age (years), mean (SD) 43¢93 (8¢88) 44¢10 (8¢82) 43¢98 (8¢85) Age group, n (%) 18-49 years 293/450 (65%) 144/226 (64%) 437/676 (65%) Male 123/293 (42%) 65/144 (45%) 188/437 (43%) Female 170/293 (58%) 79/144 (55%) 249/437 (57%) 50-59 years 157/450 (35%) 82/226 (36%) 239/676 (35%) Male 70/157 (45%) 36/82 (44%) 106/239 (44%) Female 87/157 (55%) 46/82 (56%) 133/239 (56%) Days between vaccines, mean (SD) 61¢16 (5¢73) 89¢03 (5¢92) 70¢33 (14¢32) Table 1: Baseline characteristics of the population. d Based on 223 subjects (3 subjects withdrew consent before being immunized). neutralisation capability at day 28 post-vaccination was were evidenced for Alpha- and Delta-neutralising anti- found with Omicron variant in both interventional and body titres (Figure 4; Appendix 1 p 11). NT50 against control groups (GMT 144¢84 [95%CI 116¢65−179¢85] Delta variant on day 28 after second dose was 717¢13 and 204¢84 [95%CI 151¢99−276¢06]). A decrease in (95%CI 587¢27−876¢13) in the interventional group and NT50 was also observed for Beta variant in both inter- 837¢14 (95%CI 609¢7−1149¢41) in the control group. ventional and control groups (GMT 293¢21 [95%CI Overall, day-28 NT50 was above 1:100 in 94% to 100% 234¢8−366¢15] and 483¢89 [95%CI 352¢53−664¢2]). Of patients against all variants excepting Beta (88% note, titre of Omicron and Beta-neutralising antibodies patients) and Omicron (69%) (Appendix 1 p 12). at day 28 in the control group was higher than in the At day 180 Omicron-neutralising antibody titres interventional group (p = 0¢0641 and p = 0¢0102, decayed to 34¢46 (95%CI 27¢72−42¢85) and 61¢52 respectively), whilst no differences between groups (95%CI 43¢66−86¢71) in the interventional and control Figure 2. RBD (anti-spike) antibody titres measured in both interventional (red) and control (blue) groups over time. Interventional group was immunised at day 0 and control group at day 28. Accordingly day 180 corresponds to day 152 post vaccine in the control group. www.thelancet.com Vol 50 Month , 2022 7 Articles Figure 3. Neutralising antibodies titres (NT50) measured in both interventional (red) and control (blue) groups over time. Interven- tional group was immunised at day 0 and control group at day 28. Accordingly, day 180 corresponds to day 152 post vaccine in the control group. groups, respectively (p = 0¢0038). Beta-neutralising anti- Discussion body titres decayed to 37¢08 (95%CI 29¢76−46¢2) and Our results provide evidence that humoral immune 76¢24 (95%CI 53¢99−107¢67) in the interventional and response of patients vaccinated with the heterologous control groups, respectively (p = 0¢0004). Delta NT50 ChAdOx1-S/BNT162b2 regime decays over time after decayed to 94¢57 (95%CI 71¢83−124¢52) and 192¢89 peaking at day 14 post- BNT162b2 dose. A decline rang- (95%CI 126¢93−293¢12) at day 180 in the interventional ing 25%-27% in total RBD and neutralising antibody and control groups, respectively (p = 0¢0043) (Figure 4; levels was observed after 28 days, which increased up to Appendix 1 p 11). 70%-75% on day 90 and 86%-90% on day 180. This Regarding dynamics of functional spike-specific waning of immunogenicity was expected consistently T-cell response, an increase in levels of both IFN-g with previous reports from COVID-19 mRNA and IL-2 after vaccination is followed by a progres- vaccines5,25,26 that reported antibody half-life of 28- sive waning over time. In the interventional group, 33 days.26 A similar decline of about 80% in antibody maximum IFN-g production was observed at day 14 levels was found with homologous BNT162b2 regime at post-dose. Levels decreased to 380¢93 pg/mL (95% 90 days after the second dose5 as well as with homolo- CI 309¢07−469¢5) at day 28 and 223¢8 pg/mL gous mRNA-1273 regime, albeit the latter to a lesser (166¢25−301¢28) at day 180. In the control group, extent (decline around 60%).25 With homologous vacci- IFN-g levels 28 days after BNT162b2 dose were nation with ChAdOx1, antibodies are induced at lower 485¢32 pg/mL (343¢51−685¢68), and decreased to levels than with homologous RNA regimens or heterol- 171¢23 pg/mL (120¢15−244¢02) at day 180. Similarly, ogous vaccination of ChAdOx1 with BNT162b227 or IL-2 concentrations were maximum at day 28 post- mRNA-1273,28 although it has been reported that anti- vaccination in both the interventional and control bodies decay with a slower kinetics.29 Furthermore, groups (244¢07 pg/mL [95% CI 204¢89−290¢74] these results are consistent with known kinetics of and 299¢2 [217¢81−411¢01], respectively) and pro- humoral immune response against acute viral infec- gressively decayed until day 180 (171¢54 [133¢08 tions, in which extrafollicular short-lived plasmablasts −221¢12] and 170¢25 [122¢24−237¢11], respectively). contribute to early antibody production − IgM, IgG, Of note, day-180 levels of both IFN-g and IL-2 were IgA −, while a secondary increasing contribution of ger- higher than those present at baseline (Figure 5; minal centre-derived plasma cells − with longer life- Appendix 1 p 13). span and larger secretory capacity − leads to secretion 8 www.thelancet.com Vol 50 Month , 2022 Articles Figure 4. Neutralising antibodies titres (NT50) against SARS-CoV-2 variants measured in both interventional (red) and control (blue) groups at days 28 and 180 after BNT162b2 administration. Interventional group was immunised at day 0 and control group at day 28. Accordingly, day 180 corresponds to day 152 post vaccine in the control group. Dashes and circles inside boxes indicated the median and arithmetic mean, respectively. Box limits indicate the interquartile range (IQR). Whiskers are adjusted to maximal and minimal values if lower than 1.5 times the IQR. Further outliers are indicated as circles. of class-switched antibodies, mainly IgG. Considering and CG at day 28 after the second dose of the heterolo- that half-life of IgM is substantially shorter than IgG, a gous ChAdOx1-S/BNT162b2 scheme. decay in antibody titres is common once the extrafollicu- Regarding differences found at day 180 between con- lar response is resolved. However they will rapidly rise if trol and intervention group it must be noted that this memory B cells are re-exposed to viral antigens in the measurement carried over the 28-day delay in future.30 In this regard, we observed a slowing-down in BNT162b2 administration to the control group (mea- SARS-CoV-2 antibody decay from month 3 to month 6, sured at day 152 − instead of 180 − after dose). A second consistent with previous reports.26 explanation for these differences could be related with BNT162b2 administration to the control group vaccination delay itself, supporting an apparent 28 days later than the interventional one did not result benefit for longer intervals between doses as found in worse or weaker antibody responses 28 days after previously.14,31 Indeed, results from regression models immunization. Actually, S-RBD antibodies and all vari- pointed in this direction. Such delay could favour the ant-specific neutralising titres were higher − S-RBD maturation process of memory B cells from germinal and Beta-specific titres significantly higher− in the con- centres (GC), over which B cells accumulate somatic trol group four weeks after vaccination suggesting a mutations in their variable region leading to selection of benefit of second dose delay. However, lack of antibody those with higher affinity for a given viral antigen.30 A determination 14 days after immunization − when top recent study has demonstrated that antigen-driven acti- levels of antibodies are reached − does not allow to per- vation of memory B cells persisted and matured up to 6 form a parallel kinetics of S-RBD and neutralizing anti- months after SARS-CoV-2 infection.32 The observation bodies between CG and IG to fully demonstrate that that at day 28 neutralisation activity in the control group delayed administration of BNT162b2 results in better against Beta was higher − and trended to higher against antibody responses. Omicron − while no differences were observed between Importantly, our results suggest that high levels of groups for G614 reference strain, Alpha and Delta var- protection against Delta-variant persisted in both IG iants suggests that the 4-week delay in vaccination www.thelancet.com Vol 50 Month , 2022 9 Articles Figure 5. IFN-g (a) and IL-2 (b) measured in both interventional (red) and control (blue) groups over time. Interventional group was immunised at day 0 and control group at day 28. Accordingly, day 180 corresponds to day 152 post vaccine in the control group. window for the control group in our study might have found in both groups at different time points which is contributed to a better affinity maturation against consistent with the generation of memory T lympho- ‘difficult’ variants such as Beta and Omicron. Regarding cytes in which maturation process and selection of Tc cellular responses, similar decay of IFN-g and IL-2 were receptor affinity is not dependent on somatic mutation. 10 www.thelancet.com Vol 50 Month , 2022 Articles Notwithstanding this, the sharp decay in RBD anti- Contributors bodies and neutralisation titres observed at day 180 as Trial conceptualisation was done by C.B.-I., J.A., M.P.- compared to day 14 and 28 support the use of a third O., A.M.B., A.J.C., J.F., J.R.A. and M.C. A.J.C., J.F., and immunization to reach higher protection levels, particu- A.Ag. developed the study methods. J.A., M.P.-O., A.M. larly considering the high infectivity potential of the B., J.F., L.C., M.J.B., J.G.-P., M.C., A.P., M.G.-P., E.A.- Omicron variant.33 Actually, despite the persistence of A., M.T., A.As., N.I.-A., E.M., C.P.-I., J.O., M.C.-O., M. immune memory, antibody decay increases the risk of B., P.C., L.H.-G., I.F., H.E.D.T. and J.R.A. were study SARS-CoV-2 infection and a third dose becomes neces- investigators. M.T.G.M., D.L., J.G.-P. and A.G.C. sary to achieve protection against asymptomatic and ensured data verification. J.G.-P., D.L., J.A., M.P.-O., M. symptomatic infections, particularly in aged groups T.G.M., J.O., A.M.B. and A.J.C. were responsible for the above 60 and patients with risk factors for developing present secondary statistical analysis. C.B.-I., J.A., M.P.- severe COVID-19 as immune suppression.19,20 It has O., J.G.-P., M.G.-P., A.M.B., L.C., M.C., M.J.B., A.P., J. been described that boosting with a third dose of mRNA O., J.F., and J.R.A. supervised the study. C.B.-I. was vaccines generate potent neutralization of Omicron, responsible for funding acquisition. J.A., M.P.O., J.O., J. shortening the difference in neutralization titres with G.-P. and A.M.B. wrote the original draft of this Article. other variants.34 Thus, increased protection of a booster All authors reviewed and edited the manuscript, and dose would be related not only to higher levels of neu- approved the manuscript for submission. All authors tralizing antibodies but to antibody maturation leading reviewed and approved the original draft. All authors to the generation of antibodies with increased affinity to had full access to the full data in the study and accept their targets. These qualitative changes are particularly responsibility to submit for publication. important against escape variants as Omicron and rep- resent an added value for a third dose. Unfortunately, very recently it has been described a waning effective- Data sharing statement ness of a third dose of BNT162b2 against hospital Individual participant data will be made available when admission after 3 months due to the Omicron variant.35 the trial is complete, on request to the corresponding As previously described,1 the main limitation of the authors. After approval of a proposal, data will be shared CombiVacS study is the absence of a control group com- through a secure online platform. pleting the homologous ChAdOx1-S scheme to compare with the heterologous ChAdOx1-S/BNT162b2 regimen. This arm would had been very useful to compare anti- Declaration of interests body waning and neutralization activity in individuals JA has received fees for educational programs from vaccinated with homologous or heterologous vaccine Gilead, MSD, GSK and Janssen outside of the submit- regimes. Besides, the abovementioned 4-week delay ted work. MC has participated in advisory boards and between both study groups in BNT162b2 administra- has received research funding from GSK, Sanofy Pas- tion led to capture 5-month, rather than 6-month, post teur, Pfizer, Novavax and Janssen.CB-I is the deputy dose data in the control group. Although this limitation general manager of the Instituto de Salud Carlos III. may have influenced some differences observed at day JRA has received fees from Janssen, outside of the sub- 180, overall results are consistent between groups. As mitted work. AMB is principal investigator of clinical mentioned, lack of antibody determination at day 14 in trials sponsored by GlaxoSmithKline, Daiichi-Sankyo, the CG limits the interpretation of the results. Also, we Janssen, and Farmalider, outside of the submitted have found a low proportion of high-responder outliers, work. All other authors declare no competing interests. in particular before immunization with the second vac- cine dose. We cannot rule out asymptomatic SARS- CoV-2 infection between both vaccine doses leading to a Acknowledgements “booster-like” response after first immunization. Funded by Instituto de Salud Carlos III (ISCIII). AMB, In conclusion, follow-up of individuals included in AJC, JO, and JF are members of the VACCELERATE the CombiVacS trial that were immunised with heterol- (European Corona Vaccine Trial Accelerator Platform) ogous ChAdOx1-S/BNT162b2 confirm waning of Network, which aims to facilitate and accelerate the humoral and cellular responses over time, nevertheless design and implementation of COVID-19 phase 2 and 3 a relevant proportion of individuals exhibited neutralis- vaccine trials. JO is a member of the INsTRuCT (Inno- ing activity > 1:100 six months after full vaccination vative Training in Myeloid Regulatory Cell Therapy) excepting against Beta and Omicron variants. These Consortium, a network of European scientists from aca- results support the use of a third dose six months after demia and industry focused on developing innovative regular vaccination to enhance immune response, par- immunotherapies. This work is funded by Instituto de ticularly against new VoCs, as Omicron. Further studies Salud Carlos III, a Spanish public body assigned to the addressing immunogenicity using different heterolo- Ministry of Science and Innovation that manages and gous vaccination schemes are warranted. promotes public clinical research related to public www.thelancet.com Vol 50 Month , 2022 11 Articles health. The Spanish Clinical Trials Platform is a public FIST NAME SURNAME network funded by the Instituto de Salud Carlos III (grant numbers PTC20/00018 and PT17/0017), the Lucía Martínez de Soto State Plan for Research, Development, and Innovation Amelia Rodríguez Mariblanca 2013−16, the State Plan for Scientific and Technical Lucía Díaz García Research and Innovation 2017−20, and the Subdirec- Elena Ramírez García torate General for Evaluation and Promotion of Enrique Seco Meseguer Research, Instituto de Salud Carlos III, cofinanced with Stefan Mark Stewart Balbas FEDER funds. CombiVacS was designed under the Alicia Marín Cand on umbrella of the VACCELERATE project. VACCELER- Irene García García ATE and INsTRuCT received funding from the EU’s Mikel Urroz Elizalde Horizon 2020 Research and Innovation Programme Jaime Monserrat Villatoro (grant agreement numbers 101037867 and 860003). Paula de la Rosa The Instituto de Salud Carlos III is the Spanish partner Marta Sanz García in the VACCELERATE project. This work is partially Cristina L opez Crespo funded by Institute of Health Carlos III (Instituto de Vega Maule on Martínez Salud Carlos III − ISCIII −), (grants PI19CIII/00004 Raquel de Madariaga Castell to JA and PI21CIII/00025 to MPO and JGP), and Laura Vit on Vara COVID-19 FUND (grants COV20/00679 and COV20/ Julio García Rodríguez 00072 to MPO and JA) and CIBERINFEC, co-financed Antonio Bu~ no by the European Regional Development Fund (FEDER) Eduardo L opez Granados “A way to make Europe”. The authors thank all trial par- Carmen Camara ticipants, the international data safety monitoring board Esther Rey Cuevas (Appendix 1 p 23), and the trial steering committee Pilar Ayllon García (Appendix 1 pp 24−25). The authors thank Esther Prieto María Jimenez Gonzalez for editorial assistance and writing support (employed Victoria Hernandez Rubio by Hospital Universitario La Paz; funded by the Insti- Paloma Moraga Alapont tuto de Salud Carlos III, grant number PCT20/00018) Amparo Sanchez and Marıa Castillo-de la Osa (PEJ2018-004557-A) for Rocío Prieto excellent technical assistance. Silvia Llorente G omez Cristina Miragall Roig Marina Aparicio Marlasca Fernando de la Calle Supplementary materials Marta Arsuaga Supplementary material associated with this article can Blanca Duque be found in the online version at doi:10.1016/j. Susana Meijide eclinm.2022.101529. Aitor García de Vicu~ na Ana San Exp osito Appendix 1. CombiVacS Vaccine Trial Group Mikel Gallego Dolores García-Vazquez FIST NAME SURNAME Ana Belen de la Hoz Isabel Jado Gustavo Perez-Nanclares Concepci on Perea Olaia Velasco Giovanni Fedele Josu Aurrekoetxea Javier Hernandez Maria Angeles Lazaro María Jes us Llamas Alejandro García Casta~ no Francisco Díez-Fuertes Ines Urrutia Esther Calonge Rosa Martínez Almudena Cascajero Bego~ na Calvo Paloma Jim enez-Santana Laura Saso Jana Baranda Sara G omez Isabel Cervera Marta Aldea Marcos Berges-Buxeda Lourdes E Bar on-Mira Pilar Portol es  M’ Angeles Marcos (Continued) (Continued) 12 www.thelancet.com Vol 50 Month , 2022 Articles FIST NAME SURNAME FIST NAME SURNAME Laura Gran es Angel Hernandez-Bartolome Sulayman Lazaar Daniel Lozano-Martín Sara Herranz Ver onica Alvarez-Morales Montserrat Malet Oliver Astasio Sebastiana Quesada Natalia Perez-Macías Anna Vilella María Aranzazu Urrutia-de-la-Plaza Anna Llupia Carmen Sanz Victoria Oliv e Agustín Molina Antoni Trilla Bego~ na G omez Elisenda Gonzalez Sheila Romero Francisco Javier Gamez Cristina Casals References 1 Borobia AM, Carcas AJ, Perez-Olmeda M, et al. 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