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Articles Immunogenicity and reactogenicity of BNT162b2 booster in ChAdOx1-S-primed participants (CombiVacS): a multicentre, open-label, randomised, controlled, phase 2 trial Alberto M Borobia*, Antonio J Carcas*, Mayte Pérez-Olmeda, Luis Castaño, María Jesús Bertran, Javier García-Pérez, Magdalena Campins, Antonio Portolés, María González-Pérez, María Teresa García Morales, Eunate Arana-Arri, Marta Aldea, Francisco Díez-Fuertes, Inmaculada Fuentes, Ana Ascaso, David Lora, Natale Imaz-Ayo, Lourdes E Barón-Mira, Antonia Agustí, Carla Pérez-Ingidua, Agustín Gómez de la Cámara, José Ramón Arribas, Jordi Ochando, José Alcamí, Cristóbal Belda-Iniesta†, Jesús Frías†, on behalf of the CombiVacS Study Group‡ Summary Background To date, no immunological data on COVID-19 heterologous vaccination schedules in humans have been Published Online reported. We assessed the immunogenicity and reactogenicity of BNT162b2 (Comirnaty, BioNTech, Mainz, Germany) June 25, 2021 https://doi.org/10.1016/ administered as second dose in participants primed with ChAdOx1-S (Vaxzevria, AstraZeneca, Oxford, UK). S0140-6736(21)01420-3 See Online/Comment Methods We did a phase 2, open-label, randomised, controlled trial on adults aged 18–60 years, vaccinated with a https://doi.org/10.1016/ single dose of ChAdOx1-S 8–12 weeks before screening, and no history of SARS-CoV-2 infection. Participants were S0140-6736(21)01442-2 randomly assigned (2:1) to receive either BNT162b2 (0·3 mL) via a single intramuscular injection (intervention group) For the French translation of the or continue observation (control group). The primary outcome was 14-day immunogenicity, measured by abstract see Online for appendix 1 immunoassays for SARS-CoV-2 trimeric spike protein and receptor binding domain (RBD). Antibody functionality For the Spanish translation of the was assessed using a pseudovirus neutralisation assay, and cellular immune response using an interferon-γ abstract see Online for appendix 2 immunoassay. The safety outcome was 7-day reactogenicity, measured as solicited local and systemic adverse events. *Contributed equally The primary analysis included all participants who received at least one dose of BNT162b2 and who had at least one †Contributed equally efficacy evaluation after baseline. The safety analysis included all participants who received BNT162b2. This study is ‡Study group members are listed in appendix 3 registered with EudraCT (2021-001978-37) and ClinicalTrials.gov (NCT04860739), and is ongoing. Servicio de Farmacología Clínica, Departamento de Findings Between April 24 and 30, 2021, 676 individuals were enrolled and randomly assigned to either the Farmacología y Terapéutica, intervention group (n=450) or control group (n=226) at five university hospitals in Spain (mean age 44 years Facultad de Medicina [SD 9]; 382 [57%] women and 294 [43%] men). 663 (98%) participants (n=441 intervention, n=222 control) (A M Borobia PhD, completed the study up to day 14. In the intervention group, geometric mean titres of RBD antibodies increased Prof A J Carcas PhD, Prof J Frías PhD), and Servicio de from 71·46 BAU/mL (95% CI 59·84–85·33) at baseline to 7756·68 BAU/mL (7371·53–8161·96) at day 14 Medicina Interna (p<0·0001). IgG against trimeric spike protein increased from 98·40 BAU/mL (95% CI 85·69–112·99) to (J R Arribas PhD), Hospital 3684·87 BAU/mL (3429·87–3958·83). The interventional:control ratio was 77·69 (95% CI 59·57–101·32) for RBD Universitario La Paz, IdiPAZ, Universidad Autónoma de protein and 36·41 (29·31–45·23) for trimeric spike protein IgG. Reactions were mild (n=1210 [68%]) or moderate Madrid, Madrid, Spain; (n=530 [30%]), with injection site pain (n=395 [88%]), induration (n=159 [35%]), headache (n=199 [44%]), and Laboratorio de Serología myalgia (n=194 [43%]) the most commonly reported adverse events. No serious adverse events were reported. (M Pérez-Olmeda PhD), Unidad de Inmunopatología del SIDA (J García-Pérez PhD, Interpretation BNT162b2 given as a second dose in individuals prime vaccinated with ChAdOx1-S induced a robust F Díez-Fuertes PhD, immune response, with an acceptable and manageable reactogenicity profile. J Alcamí PhD), Laboratorio de Referencia en Inmunología Funding Instituto de Salud Carlos III. (M González-Pérez MSc, J Ochando PhD), Centro Nacional de Microbiología, and Copyright © 2021 Elsevier Ltd. All rights reserved. Evaluation and Promotion of Research (C Belda-Iniesta PhD), Introduction (ie, sequential administration of the same vaccine).1 Instituto de Salud Carlos III, Madrid, Spain; Hospital Active immunisation is the cornerstone of global health- The ability to sequentially administer different COVID-19 Universitario de Cruces, care policies against COVID-19. To date, four COVID-19 vaccines—ie, a heterologous schedule—could be an Biocruces Bizkaia HRI, vaccines have been granted conditional marketing opportunity to make vaccination programmes more UPV/EHU, OSAKIDETZA, authorisation by the European Commission, namely the flexible and reliable in response to fluctuations in supply. CIBERDEM, CIBERER, Endo-ERN, Barakaldo-Bilbao, mRNA vaccines BNT162b2 (Comirnaty, BioNTech, Mainz, Additionally, these schemes are being studied for Spain (Prof L Castaño PhD, Germany) and CX-024414 (Moderna, Cambridge, MA, successive booster doses. E Arana-Arri PhD, USA), and the adenovirus vaccines ChAdOx1-S (Vaxzevria, Interest in a heterologous schedule for COVID-19 N Imaz-Ayo PharmG); Servicio AstraZeneca, Oxford, UK) and Ad26.Cov2.S (Janssen-Cilag vaccines came from the appearance of rare, but severe, de Medicina Preventiva y Epidemiología, Hospital Clínic International NV, Beerse, Belgium). thrombotic events with thrombocytopenia in people vac­ de Barcelona, Barcelona, Spain To date, the administration of both mRNA vaccines cinated with ChAdOx1-S.2 These uncommon side-effects (M J Bertran PhD, M Aldea MD, and ChAdOx1-S has followed a homologous schedule were more frequent in young people, resulting in the L E Barón-Mira MD); Servicio de www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 1 Articles Medicina Preventiva y Epidemiología Research in context (Prof M Campins PhD), Unidad de Soporte a la Investigación Evidence before this study strategy against SARS-CoV-2. Administration of a dose of Clínica, Vall d’Hebron Institut Heterologous regimens in COVID-19 have been proposed as BNT162b2 vaccine after a first dose of ChAdOx1-S provides a de Recerca (I Fuentes PhD), and an option to elicit combined antibody and cellular responses strong immune humoral and cellular response. Departmento de Farmacología, resulting in stronger, broader, or longer-lasting immunity. Terapéutica y Toxicología Implications of all the available evidence (A Agustí PhD), Servicio de However, no clinical evidence has been reported to date. This study confirms preclinical studies and suggestions Farmacología Clínica, Hospital We searched PubMed on April 15, 2021, for any article Universitari Vall d’Hebron, anticipating that a heterologous vaccination regimen could published from database inception until the date of search, Universitat Autònoma de elicit potent combined antibody and cellular responses, which without language restrictions, using the terms “heterologous” Barcelona, Barcelona, Spain; might lead to mix-and-match COVID-19 vaccine programmes. Servicio de Farmacología OR “heterologous vaccination” AND “vaccination” OR Trials directly comparing full homologous and heterologous Clínica, Hospital Clínico “vaccine” AND “COVID-19” OR “SARS-CoV-2”; however, San Carlos, IdISSC, vaccination strategies are warranted to confirm safety and no publications reporting reactogenicity and immune response Departamento de vaccine effectiveness of heterologous strategies. Farmacología y Toxicología after the use in humans of heterologous vaccination with (Prof A Portolés PhD, ChAdOx1-S and BNT162b2 in COVID-19 were returned. A Ascaso MD, C Pérez-Ingidua BSN), and Added value of this study Instituto de Investigación This is, to our knowledge, the first study evaluating the Sanitaria Hospital 12 de immune and cellular response to a heterologous vaccination Octubre, CIBER de Epidemiología y Salud Pública, Facultad de Medicina health authorities of several European countries3 and present reacto­genicity and immunogenicity 14 days after (M T García Morales MSc, Canada, among others, modifying their national vaccination. D Lora PhD, A Gómez de la Cámara PhD), immunisation strategies and reserving the ChAdOx1-S Universidad Complutense de vaccine for older people. Consequently, some countries, Methods Madrid, Madrid, Spain including Sweden, France, Germany, Norway, and Study design and participants Correspondence to: Denmark, advised that BNT162b2 should be administered CombiVacS is a phase 2, multicentre, open-label, ran­ Dr Cristóbal Belda-Iniesta, as the booster dose in people primed with ChAdOx1-S. domised, controlled trial done at five university hospitals Evaluation and Promotion of Research, Instituto de Salud This advice came without supporting data regarding in Spain (Hospital Universitario de Cruces, Vizcaya; Carlos III, Madrid 28029, Spain reactogenicity or immunogenicity of this schedule. Hospital Universitari Vall d’Hebron, Barcelona; Hospital cbelda@isciii.es Heterologous prime-boost strategies based on the Clínic de Barcelona, Barcelona; Hospital Clínico San or sequential administration of two gene expression Carlos, Madrid; and Hospital Universitario La Paz, Prof J Frías, Servicio de systems has been widely used for protection against Madrid). The trial complies with the principles of the Farmacología Clínica, different infectious diseases.1 Spencer and colleagues4 Declaration of Helsinki and Good Clinical Practice, and Departamento de Farmacología y Terapéutica, Facultad de had shown a combination of increased SARS-CoV-2 IgG- was approved by the Spanish Agency of Medicines and Medicina, Hospital Universitario specific titres with neutralisation ability and a robust Healthcare Products and by the ethics committee at La Paz, IdiPAZ, Universidad T-helper-1-type response using a heterologous regimen University Hospital La Paz. The study protocol and the Autónoma de Madrid, based on either ChAdOx1-S or BNT162b2 as prime or statistical analysis plan are provided in appendix 3 (p 28). Madrid 28046, Spain jesus.frias@uam.es booster doses in animal models,5 which is in agreement Our hypothesis was that immunogenicity after with the clinical efficacy (91%) shown by the heterologous BNT162b2 would be superior to no vaccination in See Online for appendix 3 Ad26 and Ad5 vaccine Gam-COVID-Vac (Sputnik V, ChAdOx1-S-primed participants. Participants were Gamaleya National Research Centre for Epidemiology healthy, or clinically stable, adults (aged 18–60 years) and Microbiology, Moscow, Russia).6 Shaw and who had received a prime ChAdOx1-S vaccination colleagues3 published initial data from the Com-COV between 8 weeks and 12 weeks (50–84 days) before the trial showing limited, short-lived reactogenicity when screening visit. Participants with documented RT-PCR- heterologous schedules were used in humans. confirmed COVID-19, or who had been vac­cinated with In many countries, at the beginning of April, 2021, any other vaccine since the prime dose were excluded. A many people vaccinated with a first dose of ChAdOx1-S SARS-CoV-2 RT-PCR test was done at the randomisation could not complete the vaccination scheme because visit, and blood samples were collected to determine health authorities had suspended administration of baseline SARS-CoV-2 serological status. Additional a second dose until risks were re-evaluated. This key exclusion criteria were the presence of clinically withdrawal left open the possibility of public health significant acute illness or temperature of at least 38°C authorities using heterologous vaccination schedules, within the 24 h before the planned dose of study vaccine, without data about immunogenicity outcomes in clinical manifestations compatible with COVID-19, humans. We designed a randomised, controlled, phase 2 and any condition contraindicating or discouraging trial to evaluate immuno­ genicity and reactogenicity BNT162b2 admin­ istration, including pregnancy. Full of a second dose of the mRNA vaccine BNT162b2 in eligibility criteria are given in the protocol (appendix 3 people prime vaccinated with ChAdOx1-S. Here, we p 28). 2 www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 Articles Study information was disseminated using social (grade 3), or life-threatening (grade 4). Causality of networking, and interested candidates contacted a study unsolicited adverse events was defined as related or site directly, at which time a personal interview was unrelated to study treatment based on reasonable booked for study personnel to explain the study and possibility, temporal relationship, and alter­ nate cause check selection criteria. All participants provided written criteria. Causality was also assessed in reported unsolicited informed consent before enrolment. adverse events. Safety definitions and a list of solicited adverse events are provided in appendix 3 (p 28). Randomisation and masking Antigen-specific humoral immune response was Participants were randomly assigned (2:1) to receive one analysed using two commercial immunoassays and intramuscular injection of BNT162b2 (intervention one pseudovirus neutralisation assay. The Elecsys Anti- group) or maintain observation (control group). Partici­ SARS-CoV-2 S assay (Roche Diagnostics, Mannheim, pants assigned to the intervention group were vaccinated Germany) is an electrochemiluminescence immuno­assay by health-care personnel who were aware of group used to detect antibodies (including IgG) to the allocation, but were not otherwise involved in trial SARS-CoV-2 spike protein receptor-binding domain procedures or data collection. If the main immunogenicity (RBD) on the Cobas e411 module (Roche Diagnostics, objective was met, and always under the perspective of Mannheim, Germany),7 with a measuring range from acceptable reactogenicity, participants included in the 0·4 U/mL to 250 U/mL (up to 2500 U/mL with onboard control group would be offered BNT162b2 as a second 1:10 dilution, and up to 12 500 U/mL with onboard dose at day 28. Alternatively, ChAdOx1-S might be used 1:50 dilution). Values higher than 0·8 BAU/mL were as a second dose in the control group if requested by the considered positive. Correlation between U/mL and participant or established by local health authorities. The BAU/mL was, U is 0·972 BAU. The LIAISON SARS-CoV-2 randomisation list was centrally generated using SAS, TrimericS IgG assay (DiaSorin, Stillwater, MN, USA) is a version 9.4; systematic randomisation stratified by study chemiluminescence immunoassay used to detect IgG site, sex, and age (18–49 years and 50–59 years) was used antibodies to the anti-trimeric spike glycoprotein of to achieve balanced randomisation in the two study SARS-CoV-2 in human serum or plasma samples on groups. The randomisation list was imported into the the LIAISON XL (DiaSorin, Saluggia, Italy),8 with a secure Research Electronic Data Capture platform measuring range from 4·81 BAU/mL to 2080·00 BAU/mL. (REDCap, version 8.7.4) used for the study electronic case As per the manufacturer’s instructions, values more report form. than 2080·00 BAU/mL were diluted 1:20 and values higher than 33·8 BAU/mL were considered positive.9 Procedures To measure neutralising antibody titres, diluted plasma BNT162b2 was administered at the approved dose samples were preincubated with pseudoviruses generated of 0·3 mL as a single intramuscular injection. All by cotransfection of the plasmid pNL4-3ΔenvRen and participants were clinically assessed and had blood an expression vector for the viral spike (pcDNA3·1-S- samples drawn for safety and immunology at day 0 CoV2∆19-G614) and added at a concentration of 10 ng (randomisation and BNT162b2 dose administration). p24Gag per well to Vero E6 cells in 96-well plates. At 48 h Follow-up visits on days 7 and 14 were scheduled to post infection, viral infectivity was assessed by measuring measure vital signs, review any adverse events, update luciferase activity (Renilla Luciferase Assay, Promega, medical and medication records, and collect blood Madison, WI, USA) using a 96-well plate luminometer samples. The trial is ongoing and further follow-up data LB 960 Centro XS³ (Berthold Technologies, Oak Ridge, will be reported in future publications. TN, USA). The titre of neutralising antibodies was Participants in the intervention group were observed on calculated as 50% inhibitory dose (neutralising titre 50, site for at least 15 min after BNT162b2 vaccination for safety NT50), expressed as the reciprocal of four-fold serial monitoring. Any adverse events occurred up to the end of dilution of heat-inactivated sera (range 1:32–1:131·072), the observation period were recorded. All participants were resulting in a 50% reduction of pseudovirus infection asked to record any adverse events using an electronic compared with control without serum. Samples below diary throughout the 14-day follow-up period. Participant- the detection threshold (1:32 serum dilution) were given recorded events were accessible to the study team online 1:16 value. Positive and negative controls were included in through the electronic diary, which emailed an automatic the assay and non-specific neutralisation was assessed alert to the investigator when the adverse event was using a non-related pseudovirus expressing the vesicular reported as severe by the participant. In all severe cases, stomatitis virus envelope. Cellular immune response the investigator contacted the participant to assess was measured by quantification of interferon-γ (IFN-γ) seriousness according to the adverse events severity scale. present in plasma on overnight stimulation of whole At 14 days, participants were asked about both solicited and blood with pools of SARS-CoV-2 spike peptides (2 μg/mL) unsolicited adverse events up to day 7, as well as unsolicited or dimethyl sulfoxide control in whole blood culture, adverse events up to day 14. Intensity of adverse events requiring only 1 mL of blood.10,11 Cytokines were quantified was graded as mild (grade 1), moderate (grade 2), severe using the next-generation ELISA tool, Ella (ProteinSimple, www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 3 Articles San Jose, CA, USA). Neutralising anti­ bodies were analyses were carried out using the statistical software analysed in a subset of 198 participants randomly selected SAS, version 9·4. The reactogenicity analysis population and stratified by centre, while cellular immune response included all the participants who had received at least was analysed in participants from the two study sites in one dose of BNT162b2 in the inter­ ventional group Madrid. Full details on the pseudovirus neutralising assay regardless of the availability of data for primary endpoint and cellular immunity quantification are provided in analysis. Reactogenicity analyses were presented as appendix 3 (pp 20–21). numbers and percentages of participants who had suffered local and systemic adverse events during Outcomes 7 consecutive days after each vaccination. Sample size The primary outcome was the assessment of the calculation for a log-transformed outcome measure12 humoral immune response to vaccination as per was done to assess the humoral immune response antibodies against SARS-CoV-2 spike protein titres against SARS-CoV-2 14 days after the dose of BNT162b2 measured by immuno­assay 14 days after the BNT162b2 in participants that received a previous single dose of dose. A secondary immuno­genicity outcome measure ChAdOx1-S, as compared with no dosing. A sample size was neutralising antibody titres measured by virus of 600 participants (n=400 in the interventional group) neutralisation assay at day 14. 1-year safety was also was required to identify a 35% increase in antibody titres planned to be assessed. The exploratory outcomes were in participants who received BNT162b2 (denoted G[Y1]) the relationship between neutralising antibodies and compared with participants who did not receive it antibodies against SARS-CoV-2 spike protein measured (denoted G[Y2]) at 14 days, assuming a coefficient of by immunoassay, and cellular response to vaccination variation equal to 1·2 or 1·0 and similar between groups, (defined as inflammatory IFN-γ cytokine production at least 80% power and a one-sided 1% significance level against SARS-CoV-2 spike peptide pools at day 14). Other (H1 G[Y1]/G[Y2]>1). The sample size was increased secondary and exploratory immunogenicity and efficacy by 15% due to possible non-participation. outcomes—planned at day 28, 90, 180, or 360—are not The primary analysis included all participants who applicable to the present analysis, but are detailed in the received at least one dose of BNT162b2 and who had protocol (appendix 3 p 28). at least one efficacy evaluation after baseline. The safety analysis included all participants who received Statistical analysis BNT162b2. The immunogenicity analysis population included all An independent data monitoring committee consisting the participants who were randomly assigned, completed of independent scientists not otherwise involved in the all visits, and for whom serological samples were study was appointed and reviewed, and will continue to available both on day 14 and at the baseline visit. review the data regularly during the study for safety and 663 individuals were included in the immunogenicity scientific integrity. The committee will make recom­ analysis for RBD-specific and trimeric spike protein- mendations to the funder regarding the stopping of specific IgG analysis to explore main objective of the an intervention for harm or for futility (appendix 3 trial. Secondary objectives to explore functionality of pp 25, 50). SARS-CoV-2-specific antibodies included 198 individuals This study is registered with EudraCT (2021-001978-37) randomly selected from both groups and, after a protocol and ClinicalTrials.gov (NCT04860739). amendment, 151 individuals from Madrid sites for cellular immunity analysis. Data were presented as Role of the funding source geometric mean and 95% CI or, for categorical variables, The funder, Institute of Health Carlos III, designed the number, and percentage, unless otherwise stated. For trial in cooperation with the Spanish Clinical Trials serological measurements, dif­ference at each time— Platform. Trial coordination, participant recruitment, basal, 7 days, and 14 days—was evaluated using ratio of and data analysis were done by the Spanish Clinical geometric means. Antibody titres against SARS-CoV-2 Research Network. All immunological procedures were spike protein at 14 days was the response variable, and done at Instituto de Salud Carlos III. treatment effect was evaluated comparing the inter­ ventional group titre and control group titre. Additional Results post-treatment ANCOVA adjust­ing for pretreatment was Between April 24 and 30, 2021, 676 participants were done, with baseline immunity value, age, and sex as enrolled and randomly assigned to receive BNT162b2 covariables. Additionally, reverse cumulative distribution vaccine (intervention group n=450) or no vaccine (control curve (RCDC) was plotted. A subgroup analysis by sex group n=226). Two individuals in the intervention group and age groups was done at baseline and 14 days for the and one individual in the control group withdrew consent primary and secondary endpoints. Missing values were before vaccination. 663 participants (intervention group not imputed (appendix 3 p 19). Laboratory parameters n=441, control group n=222) were included in the with values below detection limit were replaced by a immunogenicity analyses, after seven participants from value equal to the lowest limit divided by two. All the interventional group and three from the control group 4 www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 Articles 678 participants assessed for eligibility 2 ineligible because they did not meet inclusion criteria 676 enrolled and randomised 450 assigned to receive BNT162b2 226 assigned to receive control 2 withdrew consent 1 withdrew consent 448 completed day 14 225 completed day 14 7 excluded 3 excluded 4 withdrew consent 2 withdrew consent 2 visits out of window 1 absence of blood sample 1 absence of blood sample 441 included in the immunogenicity analysis 448 included in the reactogenicity analysis 222 included in the immunogenicity analysis 441 included in receptor-binding 222 included in receptor-binding domain-specific IgG analysis domain-specific IgG analysis 441 included in trimeric spike 222 included in trimeric spike protein-specific IgG analysis protein-specific IgG analysis 129 included in antibody functionality 69 included in antibody functionality analysis analysis 99 included in cellular immunity analysis 52 included in cellular immunity analysis Figure 1: Trial profile 663 individuals were included in the immunogenicity analysis for receptor-binding domain-specific and trimeric spike protein-specific IgG analysis to explore the primary objective of the trial. Secondary objectives to explore functionality of SARS-CoV-2-specific antibodies included 198 individuals randomly selected from both groups and, after a protocol amendment, 151 individuals from Madrid sites for cellular immunity analysis. were excluded (figure 1). 448 participants who received the second dose were included in the reactogenicity population, including one from the control group who Intervention group Control group Overall was erroneously vaccinated. One individual was lost to (n=450) (n=226) (n=676) follow-up after receiving the BNT162b2 dose and was Sex excluded. Male 193 (43%) 101 (45%) 294 (43%) Demographics and baseline characteristics (table; Female 257 (57%) 125 (55%) 382 (57%) appendix 3 p 2) were balanced between groups. No race Age, years 43·93 (8·88) 44·10 (8·82) 43·98 (8·85) or ethnicity data were collected. 382 (57%) participants Age group were women and 294 (43%) were men. 437 (65%) par­ 18–49 years 293/450 (65%) 144/226 (64%) 437/676 (65%) ticipants were aged 18–49 years, and the mean age of Male 123/293 (42%) 65/144 (45%) 188/437 (43%) both groups was 43·98 years (SD 8·85). Time elapsed Female 170/293 (58%) 79/144 (55%) 249/437 (57%) since ChAdOx1-S administration was between 8 weeks 50–59 years 157/450 (35%) 82/226 (36%) 239/676 (35%) and 9 weeks for 411 (61%) participants and between Male 70/157 (45%) 36/82 (44%) 106/239 (44%) 10 weeks and 12 weeks for 263 (39%) participants. Female 87/157 (55%) 46/82 (56%) 133/239 (56%) In the intervention group, geometric mean titres (GMTs) of antibodies specific to the SARS-CoV-2 RBD at Time since ChAdOx1-S vaccination*, weeks day 14 were significantly (p<0·0001) higher in the 8–9 273/449 (61%) 138/225 (61%) 411/674 (61%) interventional group (7756·68 BAU/mL, 95% CI 10–12 176/449 (39%) 87/225 (39%) 263/674 (39%) 7371·53–8161·96) compared with the control group Data are n (%), mean (SD), or n/N (%). *Two participants were excluded: one because 7 weeks had elapsed since (99·84 BAU/mL, 76·93–129·59; interventional:control ChAdOx1-S vaccine, and another due to dropout on day 0. ratio 77·69, 95% CI 59·57–101·32). Immunogenic Table: Baseline characteristics response in the inter­vention group was observed at day 7 www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 5 Articles Control group Intervention group 4 Control group Intervention group A B * * * * 4 3 4 Log10 IFN-γ (pg/mL) 3 Log10 antibody titre (BAU/mL) Log10 IgG titre (BAU/mL) 3 2 2 1 2 1 0 1 0 0 14 0 0 7 14 0 7 14 Time since vaccination (days) Time since vaccination (days) Time since vaccination (days) Figure 4: IFN-γ concentrations measured in both intervention and control Figure 2: Antibody titres groups on days 0 and 14 Receptor-binding domain (anti-spike protein) antibody titres (A), and trimeric spike protein antibody titres (B), IFN=interferon. *p<0·0001. measured in both intervention and control groups on days 0, 7, and 14. *p<0·0001. appendix 3 p 3). In the intervention group, at baseline, A B GMTs of RBD antibodies and IgG against trimeric spike * protein were 71·46 BAU/mL (95% CI 59·84–85·33) and R2=0·824 98·40 BAU/mL (85·69–112·99), respectively. Results 4 4 were similar when analysed by interval between first and second dose (appendix 3 p 4). RCDCs for RBD and trimeric spike protein antibodies are shown in Log10 RBD (14 days) 3 3 appendix 3 (pp 5–6). Titres of antibodies measured by Log10 NT50 both techniques showed strong positive correlation 2 (R² 0·85; p<0·0001; appendix 3 p 7). Subgroup analysis 2 showed that immunological response at day 14 was significantly lower in men in both RBD (p=0·0162) and 1 trimeric spike protein antibodies (p<0·0001), and no 1 Control group Intervention group differences was observed in age subgroups (appendix 3 0 pp 8–9). 0 14 1 2 3 4 The functional capability of the antibodies induced in Time since vaccination (days) Log10 NT50 (14 days) the intervention group were analysed in 198 ran­ Figure 3: Neutralisation responses domly selected participants (n=129 intervention group, (A) Neutralising antibodies measured in both intervention and control groups on days 0 and 14. (B) Correlation n=69 control group). In the intervention group, between NT50 and RBD (anti-spike protein) antibody titres. NT50=titres that achieved 50% neutralisation. 96 (74%) participants showed no or very low neutralising RBD=receptor-binding domain. *p<0·0001. activity at day 0, independently of interval since prime dose (appendix 3 p 10). In comparison, 129 (100%) par­ (intervention 4353·51 BAU/mL, 95% CI 3851·58–4920·85 ticipants exhibited neutralising antibodies at day 14, vs control 90·05 BAU/mL, 69·16–117·27; p<0·0001; showing high (NT50>1:300 and <1:1000) or very high figure 2; appendix 3 p 3). When antibodies against (NT50>1:1000) activity in 126 (98%) of 129 participants SARS-CoV-2 spike protein were measured by a (appendix 3 p 11). At day 14, the GMT of neutralising chemiluminescence immunoassay technique covering antibodies increased 45-times, from 41·84 (95% CI the trimeric spike protein, 14-day immunogenic response 31·28–55·96) to 1905·69 (1625·65–2233·98) in the inter­ in the interven­ tion group was statistically signifi­ cant vention group, compared with 41·81 (27·18–64·32) at (intervention 3684·87 BAU/mL, 3429·87–3958·83 vs day 14 in the control group (p<0·0001). Moreover, this control 101·2 BAU/mL, 82·45–124·22; interven­ increase was observed in all participants independently of tional:control ratio 36·41, 95% CI 29·31–45·23; baseline NT50 levels (appendix 3 p 12). GMT of neutralising p<0·0001), which was a 37-fold increase from baseline. antibodies in the control group was not significantly Likewise, titres of anti­bodies at day 7 were significantly different from baseline (GMT 50·84, 95% CI 33·56–76·99; higher in the inter­ ven­tion group (2246·25 BAU/mL, figure 3; appendix 3 p 13). RCDCs for neutralising 95% CI 2010·4–2509·78) than the control group antibodies are shown in appendix 3 (p 14) for both study (102·25 BAU/mL, 83·52–125·18; p<0·0001; figure 2; groups, and by baseline NT50 (p 15). Neutralising antibody 6 www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 Articles responses had a strong positive correlation with RBD Injection site, local antibody titres (R² 0·82; p<0·0001; figure 3). A B C Discomfort Erythema Pruritus Dynamic changes of functional spike-specific T-cell 100 response were analysed in 151 (22%) of 676 partici­pants 80 Frequency (%) (n=99 intervention group, n=52 control group), showing 60 significant levels of IFN-γ production at day 0 40 (GMT 129·63 pg/mL, 95% CI 103·51–162·35 intervention group vs 151·63 pg/mL, 114·09–201·53 control group), 20 consistent with a previous immunisation with a single 0 dose of ChAdOx1-S. On day 14, the production of IFN-γ had significantly increased in the intervention D E F Urticaria Hardness Pustule 100 group (GMT 521·22 pg/mL, 422·44–643·09; p<0·0001) compared with the control group (122·67 pg/mL, 80 Frequency (%) 88·55–169·95; p<0·0001), in which IFN-γ produc­ tion 60 remained unchanged (figure 4). RCDCs for immuno­ 40 logical response are shown in appendix 3 (p 16). 20 Reactogenicity analysis was based on solicited adverse events in 448 individuals from the intervention group, 0 with headache (n=199 [44%]), myalgia (n=194 [43%]), Systemic and malaise (n=187 [42%]), the most commonly reported G H I systemic reactions. Other systemic adverse reactions, Malaise Pyrexia Headache 100 including fever (n=11 [2%]), were less common (appendix 3 80 p 17). Injection site pain (n=395 [88%]), induration Frequency (%) 60 (n=159 [35%]), and erythema (n=139 [31%]) were the most commonly reported local reactions. Other local adverse 40 reactions were less common (appendix 3 p 17). Local and 20 systemic reactions were most frequently reported by 0 female participants (appendix 3 p 18). No differences in adverse event frequency were observed by age groups J K L Myalgia Arthralgia Chills (appendix 3 p 18). Of 1771 solicited adverse events reported 100 in the 7 days after vaccination in the intervention group, 80 most were mild (n=1210 [68%]) or moderate (n=530 [30%]), Frequency (%) 60 and self-limited. In 31 participants, the most frequent 40 severe adverse events were malaise (n=7 [23%]), myalgia (n=6 [19%]), and headache (n=5 [16%]). All these 20 participants were contacted and subsequently evaluated 0 by investigators, who did not report any serious adverse events. The severity of solicited local and systemic M N O Cough Pruritus Rash reactions was highest on day 2 after vaccination (figure 5). 100 80 Discussion Frequency (%) 60 This is, to our knowledge, the first report to show that a 40 COVID-19 heterologous vaccination schedule induces an immune response in humans and is associated with an 20 acceptable and manageable reactogenicity profile. The 0 1 2 3 4 5 6 7 early response obtained 7 days after the second dose, and confirmed at day 14, showed a boost effect linked to the P Q Time since vaccination (days) Nausea Vomiting 100 heterologous scheme. In particular, there was a robust coherence between the immune response evaluated by 80 Frequency (%) titres of specific antibodies against SARS-CoV-2 spike 60 protein and the proportional increase of the functional 40 Severity of adverse event capacity of neutralisation in the corresponding test. Severe 20 There was strong positive correlation observed between Moderate 0 Mild the two immunoassays and the pseudovirus neutralisa­ 1 2 3 4 5 6 7 1 2 3 4 5 6 7 tion assay. Immune cellular response 14 days after the Time since vaccination (days) Time since vaccination (days) booster vaccine also provides support for the effectiveness of the heterologous approach. The immune response Figure 5: Solicited local and systemic adverse reactions in first 7 days after vaccination www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 7 Articles with the heterologous vaccination schedule was within response has been described with homologous mRNA the range of those previously reported using homologous vaccine schedules.19–21 schedules. Data from the Oxford COVID Vaccine Trial The solicited adverse events profile in CombiVacS is Group showed that after a second dose of ChAdOx1-S, similar to that after homologous vaccination with humoral response was associated with a ten-times ChAdOx1-S13 or BNT162b2,22 and in a cohort of health- increase of anti-SARS-CoV-2 spike protein IgG stan­ care workers in Germany.23 However, our findings differ dardised ELISA titres, as compared with one dose.13 from those reported by Shaw and colleagues.3 Shaw Additionally, in phase 1/2 BNT162b2 trials,14 RBD anti­ and colleagues describe an increase in systemic bodies also increased from 1536 U/mL to 16 166 U/mL reactogenicity after the booster dose in heterologous after the second dose of BNT162b2, and neutralising vaccine schedules, compared with homologous vaccine antibody titres increased from 29 GMT to 437 GMT. In schedules—particularly in a self-reported feeling of phase 1/2 CX-024414 trials,15 RBD antibodies increased feverishness.3 Although participants in our study were from 93 231 GMT to 558 905 GMT, 2 weeks after the younger than in Shaw and colleagues’ study, we reported second vaccine.15 a lower frequency of reactogenicity events, which might In the present study, the time between the doses was be explained—at least in part—by the difference in also likely to have had a role in immunogenicity and administration intervals (ie, 28 days3 vs 8–12 weeks). reactivity; the window to receive the second dose ranged Notwithstanding, comparisons between studies should from 8 weeks to 12 weeks, which was longer than that be cautious due to the differences between the studies— used in earlier homologous approaches. Two studies12,13 ie, time from the prime dose, evaluation of some and a pooled analysis of four randomised trials from the adverse events (fever vs feverishness) or age differences. Oxford COVID Vaccine Trial Group16 showed that the The absence of an active control group did not allow for longer the interval between the first and second doses of a direct comparison with reactogenicity elicited by ChAdOx1-S, the higher SARS-CoV-2 IgG spike protein- homologous ChAdOx1-S vaccination. specific response. This effect was more evident in Regarding the higher frequency of adverse events individuals younger than 55 years,16 but was also described reported by women in our study, women have been in people older than 80 years who were vaccinated under reported to have a stronger immune response to vaccines an extended interval between two doses of BNT162b2.17 than men; conversely, they also present more frequent and We also found that neutralising activity (determined severe adverse events.24,25 Unfortunately, sex-disaggregated using a pseudovirus assay) increased after BNT162b2 data about immunogenicity and reactogenicity have not immunisation in all participants, independently of been reported in COVID-19 vaccines trials to date. NT50 at baseline. 14 days after immunisation, NT50 was Although the data are preliminary, thromboembolitic above 1:1000 in 75% and above 1:300 in 98% of par­ events associated with ChAdOx1-S,26 and anaphylaxis ticipants in the intervention group. A study has reported associated with mRNA vaccines,27 are more frequent in that neutralisation level is highly predictive of immune females. Our data show a numerically higher incidence of protection, and perhaps also vaccine efficacy.4 Baseline unsolicited adverse events, and also a higher immune NT50 was low in our study (42–51). Levels reported in response in women than men. The trial is ongoing, and a earlier studies range from an NT50 of 88 to 140 at day 28,13,14 full analysis of the effect of sex and age on reactogenicity showing a sharp decrease (NT50 40–70) at day 56, after the and immuno­genicity will be reported on completion of first vaccine dose.14 Our baseline findings appear to be the trial. consistent with these results. Analysing by interval since Finally, in figures 2 and 3, the presence of individuals first dose, we found that baseline NT50 was numerically, with elevated antibody titres at the time of randomisation but not significantly, lower in participants included at is evident. If we can eliminate individual variability as weeks 10–12 (NT50 36) compared with participants in the cause of these titres, we hypothesise we enrolled weeks 8–9 (NT50 51). Notwithstanding, our data are individuals who had been inadvertently infected some limited and interpretations from indirect comparisons time before the start of the trial. In that case, the titres must be made cautiously. obtained in these individuals would depend directly on a Additionally, our results indicate that the use of heterologous combination of antigens, because they have BNT162b2 as a second dose in a heterologous scheme been exposed to wild-type SARS-CoV-2 and ChAdOx1-S, increases the cellular immunity responses obtained which would confirm our findings. However, this after the initial dose of ChAdOx1-S. To date, second hypothesis will be assessed in our study population. doses of ChAdOx1-S in homologous schedules have A limitation of the study is the absence of a control failed to show an improvement in the cellular response group completing the homologous ChAdOx1-S scheme. obtained after an initial dose,12,13,18 suggesting that At the time of the clinical trial design, the use of cellular response is maintained irrespective of vaccina­ ChAdOx1-S had been suspended in Spain. Considering tion interval, age, and sex after a two-dose homologous that supplies of other vaccine modalities were not yet vaccination strategy with ChAdOx1-S. The enhancer able to cope with the existing demand, we used BNT162b2 effect of the second dose on the cellular immune because the supplies we had were adequate enough for a 8 www.thelancet.com Published online June 25, 2021 https://doi.org/10.1016/S0140-6736(21)01420-3 Articles potential application in real life of the eventual results Instituto de Salud Carlos III, cofinanced with FEDER funds. emanating from the study. Consequently, we evaluated CombiVacS was designed under the umbrella of the VACCELERATE project. VACCELERATE and INsTRuCT received funding from the EU’s the immune response of one heterologous scheme Horizon 2020 Research and Innovation Programme (grant agreement compared with the suboptimal vaccination situation, by numbers 101037867 and 860003). The Health Institute Carlos III is the which those persons affected by the suspension could be Spanish partner in the VACCELERATE project. The authors thank all left if they did not receive a second dose. We do not know trial participants, the international data safety monitoring board, and the trial steering committee (appendix 3 pp 26–27). The authors thank whether the immunogenic response observed in our Esther Prieto for editorial assistance and writing support (employed by study will result in better efficacy and effectiveness—a Hospital Universitario La Paz; funded by the Instituto de Salud fact that should be taken into account when considering Carlos III, grant number PCT20/00018). All contributors thank our results in decisions regarding vaccination pro­ Raquel Yotti for her thorough, critical review of the manuscript, and her contributions to the overall conceptualisation of the clinical trial. grammes. The adverse events in our study could also be underestimated because of the small sample size and the References 1 Kardani K, Bolhassani A, Shahbazi S. Prime-boost vaccine strategy short period of observation. We estimate that the 4-week against viral infections: mechanisms and benefits. Vaccine 2016; delay in the vaccination of the control group did not pose 34: 413–23. any ethical issue, since the data reported to date suggest 2 Andreas Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S. Thrombotic thrombocytopenia after that the immune response would not be worse than in ChAdOx1 nCov-19 vaccination. N Engl J Med 2021; 384: 2092–101. the 8–12-week interval.16,17 3 Shaw RH, Stuart A, Greenland M, Liu X, Van-Tam JSN, Snape MD. In summary, our study shows a 14-day robust humoral Heterologous prime-boost COVID-19 vaccination: initial reactogenicity data. Lancet 2021; 397: 2043–46. and cellular immune response after a second dose of 4 Spencer AJ, McKay PF, Belij-Rammerstorfer S, et al. Heterologous BNT162b2 in individuals primed with ChAdOx1-S vaccination regimens with self-amplifying RNA and adenoviral 8–12 weeks earlier. The trial is ongoing; thus, the results of COVID vaccines induce robust immune responses in mice. Nat Commun 2021; 12: 2893. this and future studies comparing homologous and hetero­ 5 Logunov DY, Dolzhikova IV, Shcheblyakov DV, et al. Safety and logous vaccination schedules will allow direct comparisons efficacy of an rAd26 and rAd5 vector-based heterologous and substantiate COVID-19 vaccination decision making. prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in Russia. Lancet 2021; Contributors 397: 671–81. Trial conceptualisation was done by CB-I, AMB, AJC, JA, and JF. 6 Meyer B, Torriani G, Yerly S, et al. Validation of a commercially AJC, IF, and AAg developed the study methods. AMB, MP-O, LC, MJB, available SARS-CoV-2 serological immunoassay. JG-P, MC, AP, MG-P, EA-A, MA, FD-F, AAs, NI-A, LEB-M, CP-I, JO, Clin Microbiol Infect 2020; 26: 1386–94. and JRA were study investigators. MTGM, DL, and AGC ensured data 7 Xiong X, Qu K, Ciazynska KA, et al. A thermostable, closed accuracy. AMB, AJC, MP-O, DL, AGC, JO, JA, and JF were responsible SARS-CoV-2 spike protein trimer. Nat Struct Mol Biol 2020; for statistical analysis. CB-I, AMB, MP-O, LC, MC, MJB, AP, JO, JA, JF, 27: 934–41. and JRA supervised the study. CB-I was responsible for funding 8 Kalimuddin S, Tham CY, Qui M, et al. Early T cell and binding acquisition. CB-I, AMB, AJC, MP-O, JO, JA, and JF wrote the original antibody responses are associated with COVID-19 RNA vaccine draft of the Article. All authors reviewed and edited the manuscript, efficacy onset. Med 2021; published online April 8. https://doi.org/ and approved the manuscript for submission. All authors reviewed and 10.1016/j.medj.2021.04.003. approved the original draft. All authors had full access to the full data in 9 DiaSorin. LIAISON SARS-CoV-2 TrimericS IgG assay. https://www. the study and accept responsibility to submit for publication. diasorin.com/sites/default/files/allegati_prodotti/liaisonr_sars- cov-2_trimerics_igg_assay_m0870004408_a_lr.pdf (accessed Declaration of interests June 22, 2021). CB-I is the deputy general manager of the Instituto de Salud Carlos III. 10 Le Bert N, Clapham HE, Tan AT, et al. Highly functional virus- JRA has received fees from Janssen, outside of the submitted work. specific cellular immune response in asymptomatic SARS-CoV-2 AMB is principal investigator of clinical trials sponsored by infection. J Exp Med 2021; 218: e20202617. GlaxoSmithKline, Daiichi-Sankyo, Janssen, and Farmalider, outside of 11 Wolfe R, Carlin JB. 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