Durability of Heterologous and Homologous COVID-19 Vaccine Boosts

Key Points

Question  What is the durability of humoral and cellular immune responses in individuals who originally received the BNT162b2 vaccine and were boosted with Ad26.COV2.S or BNT162b2?

Findings  In this cohort study of 68 adults, both Ad26.COV2.S and BNT162b2 were associated with increased humoral and cellular immune responses. Boosting with Ad26.COV2.S was associated with durable antibody and T-cell responses for at least 4 months.

Meaning  A heterologous mix-and-match vaccine strategy was associated with durable antibody and T-cell responses against the SARS-CoV-2 Omicron variant.

Importance  Antibody responses elicited by current messenger RNA (mRNA) COVID-19 vaccines decline rapidly and require repeated boosting.

Objective  To evaluate the immunogenicity and durability of heterologous and homologous prime-boost regimens involving the adenovirus vector vaccine Ad26.COV2.S and the mRNA vaccine BNT162b2.

Design, Setting, and Participants  In this cohort study at a single clinical site in Boston, Massachusetts, 68 individuals who were vaccinated at least 6 months previously with 2 immunizations of BNT162b2 were boosted with either Ad26.COV2.S or BNT162b2. Enrollment of participants occurred from August 12, 2021, to October 25, 2021, and this study involved 4 months of follow-up. Data analysis was performed from November 2021 to February 2022.

Exposures  Participants who were previously vaccinated with BNT162b2 received a boost with either Ad26.COV2.S or BNT162b2.

Main Outcomes and Measures  Humoral immune responses were assessed by neutralizing, binding, and functional antibody responses for 16 weeks following the boost. CD8⁺ and CD4⁺ T-cell responses were evaluated by intracellular cytokine staining assays.

Results  Among 68 participants who were originally vaccinated with BNT162b2 and boosted with Ad26.COV2.S (41 participants; median [range] age, 36 [23-84] years) or BNT162b2 (27 participants; median [range] age, 35 [23-76] years), 56 participants (82%) were female, 7 (10%) were Asian, 4 (6%) were Black, 4 (6%) were Hispanic or Latino, 3 (4%) were more than 1 race, and 53 (78%) were White. Both vaccines were found to be associated with increased humoral and cellular immune responses, including against SARS-CoV-2 variants of concern. BNT162b2 boosting was associated with a rapid increase of Omicron neutralizing antibodies that peaked at a median (IQR) titer of 1018 (699-1646) at week 2 and declined by 6.9-fold to a median (IQR) titer of 148 (95-266) by week 16. Ad26.COV2.S boosting was associated with increased Omicron neutralizing antibodies titers that peaked at a median (IQR) of 859 (467-1838) week 4 and declined by 2.1-fold to a median (IQR) of 403 (208-1130) by week 16.

Conclusions and Relevance  Heterologous Ad26.COV2.S boosting was associated with durable humoral and cellular immune responses in individuals who originally received the BNT162b2 vaccine. These data suggest potential benefits of heterologous prime-boost vaccine regimens for SARS-CoV-2.

Messenger RNA (mRNA) vaccines for COVID-19 have demonstrated outstanding short-term immunogenicity and protective efficacy.^1,2 However, neutralizing antibody (NAb) responses have been reported to wane by 3 to 6 months after primary immunization.^3-7 Following a third mRNA dose, Omicron-specific NAbs were induced,^8-11 but these antibody responses and clinical effectiveness also declined after 3 to 6 months.^12,13 Following a fourth mRNA dose, protection against infection with SARS-CoV-2 Omicron waned after 4 weeks, although protection against severe disease lasted for at least 6 weeks.¹⁴ In contrast with mRNA COVID-19 vaccines, an adenovirus serotype 26 vector-based COVID-19 vaccine induced lower initial NAb titers compared with mRNA vaccines,^15-17 but these antibody responses and protective efficacy were durable for at least 8 months.^7,18,19

Cellular immune responses, and in particular CD8⁺ T-cell responses, may also contribute to protection, especially long-term protection against severe disease.^20-22 The SARS-CoV-2 Omicron variant largely escapes from vaccine-elicited NAbs,^8,23-25 but T-cell responses remain highly cross-reactive against Omicron.^26-28 T-cell responses are also substantially more durable than serum NAb titers.^7,22,29,30

Optimal boosting strategies for induction of Omicron-specific NAbs and CD8⁺ T-cell responses have not yet been defined and are important for long-term pandemic control. To assess the immunogenicity and durability of heterologous and homologous vaccine boost strategies, we evaluated humoral and cellular immune responses in individuals who were vaccinated at least 6 months previously with 2 immunizations of BNT162b2 (Pfizer-BioNTech) and were then boosted with either Ad26.COV2.S (Janssen) or BNT162b2.

This cohort study was approved by the Beth Israel Deaconess Medical Center (BIDMC) institutional review board. Enrollment of participants occurred from August 12 to October 25, 2021, and this study involved 4 months of follow-up. A specimen biorepository at BIDMC obtained samples from 68 individuals who received the BNT162b2 vaccine at least 6 months prior to boost. Participants either continued follow-up in the biorepository and were boosted with 30 ug BNT162b2 or were enrolled in the COV2008 clinical trial (NCT04999111) and were boosted with 5.0 × 10¹⁰, 2.5 × 10¹⁰, or 1.0 × 10¹⁰ viral particles Ad26.COV2.S. An additional 15 participants were also enrolled in the COV2008 study. The Advarra institutional review board approved the COV2008 study. All participants provided informed consent (verbal consent for the specimen biorepository and written consent for the COV2008 clinical trial). Race and ethnicity data were obtained by self-report. Race and ethnicity are not biological constructs; however, they were assessed for this study given their clinical and public health relevance in the context of disparities in COVID-19 disease outcomes and vaccination and to allow readers to determine generalizability.

Individuals were excluded if they had a history of SARS-CoV-2 infection, received other COVID-19 vaccines, or received immunosuppressive medications. Participants were excluded from the immunologic analysis if they had a positive nucleocapsid serology by electrochemiluminescence assay (ECLA), a confirmed breakthrough COVID-19 infection, or an additional COVID-19 vaccine outside of the study protocol. This report followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.

Humoral immune responses were assessed by pseudovirus NAb assays, live virus NAb assays, enzyme-linked immunosorbent assays, ECLA, antibody-dependent phagocytosis, antibody-dependent neutrophil phagocytosis, and antibody-dependent complement deposition (eMethods in the Supplement). Cellular immune responses were assessed by intracellular cytokine staining assays and receptor-binding domain (RBD)–specific B cell staining (eMethods in the Supplement).

Descriptive statistics were calculated using Prism statistical software version 8.4.3 (GraphPad Software). Data are presented as individual values with medians, or as box-and-whisker plots with medians and ranges. Immune responses were compared with 2-tailed Mann-Whitney tests. P < .05 was considered significant. Data analysis was performed from November 2021 to February 2022.

In this cohort study, 68 participants who were originally vaccinated at least 6 months previously with 2 immunizations of BNT162b2 were enrolled and were boosted with either Ad26.COV2.S (41 participants; median [range] age, 36 [23-84] years) or BNT162b2 (27 participants; median [range] age, 35 [23-76] years); 56 participants (82%) were female, 7 (10%) were Asian, 4 (6%) were Black, 4 (6%) were Hispanic or Latino, 3 (4%) were more than 1 race, and 53 (78%) were White. (Table). Participants were excluded if they had a positive nucleocapsid serology by ECLA, a confirmed breakthrough COVID-19 infection, or any additional COVID-19 vaccine. No serious adverse effects were observed following heterologous or homologous boosting in this study.

NAb responses were assessed following the boost immunization by pseudovirus neutralization assays^25,31 and authentic live virus neutralization assays.³² Six months following initial BNT162b2 vaccination, pseudovirus NAb titers were detectable but low against the WA1/2020, Delta, and Beta variants but largely undetectable against Omicron (Figure 1A and 1B). Ad26.COV2.S boosted median (IQR) Omicron BA.1 NAb titers from 21 (20-32) at week 0 to 591 (272-881) at week 2, a peak of 859 (467-1838) at week 4, and a decline of 2.1-fold to 403 (208-1130) at week 16 following the boost (Figure 1A). BNT162b2 boosted median (IQR) Omicron BA.1 NAb titers from 20 (20-27) at week 0 to 1018 (699-1646) at week 2, 345 (230-972) at week 4, and declined by 6.9-fold to 148 (95-266) at week 16 (Figure 1B). At week 16, the median (IQR) BA.1 (403 [208-1130]) and BA.2 (308 [179-1273]) pseudovirus NAb titers were higher following the Ad26.COV2.S boost compared with the BNT162b2 boost (median [IQR], BA.1, 148 [95-266]; BA.2, 146 [96-265]) (Figure 1C and 1D and eFigure1 in the Supplement). Authentic live virus NAb assays showed similar profiles to the pseudovirus NAb assays (Figure 2A and 2B). At week 16, Omicron BA.1 live virus NAb titers were higher following the Ad26.COV2.S boost compared with the BNT162b2 boost (median [IQR], 887 [554-1709] vs 511 [289-838]) (Figure 2C and 2D).

Both Ad26.COV2.S and BNT162b2 also boosted RBD-specific binding antibody responses by ECLA³³ (eFigure 2 in the Supplement) and enzyme-linked immunosorbent assays (eFigure 3 in the Supplement). Fc functional antibody responses³⁴ were also evaluated given their potential role in protection and were comparable following the Ad26.COV2.S and BNT162b2 boosts, with relatively preserved antibody-dependent cellular phagocytosis but reduced antibody-dependent neutrophil phagocytosis and antibody-dependent complement deposition responses against Omicron BA.1 compared with WA1.2020 (eFigure 4 in the Supplement).

SARS-CoV-2–specific CD8⁺ and CD4⁺ T-cell responses were assessed by pooled peptide Spike-specific intracellular cytokine staining assays.²⁷ Ad26.COV2.S boosted median Omicron BA.1 Spike-specific interferon (IFN)–γ CD8⁺ T-cell responses from medians (IQRs) of 0.017% (0.009%-0.030%) at week 0 to 0.093% (0.045%-0.181%) at week 2 and 0.081% (0.042%-0.200%) at week 16 (Figure 3A) and IFN-γ CD4⁺ T cell responses from medians (IQRs) of 0.030% (0.011%-0.047%) at week 0 to 0.092% (0.046%-0.123%) at week 2 and 0.065% (0.027%-0.110%) at week 16 (Figure 3B). BNT162b2 boosted median Omicron BA.1 Spike-specific IFN-γ CD8⁺ T-cell responses from medians (IQRs) of 0.023% (0.010%-0.050%) at week 0 to 0.033% (0.018%-0.094%) at week 2 and 0.024% (0.007%-0.063%) at week 16 (Figure 3A) and IFN-γ CD4⁺ T-cell responses from medians (IQRs) of 0.027% (0.015%-0.070%) at week 0 to 0.039% (0.028%-0.090%) at week 2 and 0.027% (0.015%-0.063%) at week 16 (Figure 3B). At week 16, median (IQR) Omicron T cell responses following the Ad26.COV2.S boost (CD8, 0.081% [0.042%-0.200%]; CD4, 0.065% [0.027%-0.110%]) were higher compared with the BNT162b2 boost (CD8, 0.024% [0.007%-0.063%]); CD4, 0.027% [0.015%-0.063%]) (Figure 3C). CD8⁺ and CD4⁺ T-cell responses were comparable for Omicron BA.1 and WA1/2020, consistent with prior studies.^26,27

Omicron BA.1 RBD-specific memory B-cell responses were also assessed by flow cytometry.³⁵ At week 16, BA.1 RBD-specific memory B cells were 0.053% after the Ad26.COV2.S boost and 0.047% after the BNT162b2 boost (eFigure 5 in the Supplement). Omicron BA.1 memory B cells were lower than WA1/2020 memory B cells (eFigure 5 in the Supplement). Memory B cells showed primarily an activated memory phenotype at week 2 following the boost but transitioned back to a resting memory phenotype by week 16 (eFigure 6 in the Supplement).

Evaluations of COVID-19 vaccine boosters have to date largely focused on early NAb responses shortly after boosting. Multiple studies have shown that a third mRNA vaccine effectively induces Omicron-specific NAb titers.^8-11 However, serum antibody responses and clinical effectiveness have been reported to wane by 4 months following an mRNA vaccine boost.^12,13 Following a fourth mRNA vaccine, protective efficacy against infection with SARS-CoV-2 Omicron has been shown to wane even more quickly.¹⁴ Such rapid waning of immunity has led to recommendations for frequent mRNA vaccine boosting, which may be challenging to sustain as a long-term policy in the developed world and difficult to implement in the developing world. The development of boosting strategies with improved durability would, therefore, be desirable.

In the present study, both heterologous Ad26.COV2.S and homologous BNT162b2 boosting were associated with increased Omicron-specific humoral and cellular immune responses in individuals who were vaccinated at least 6 months previously with BNT162b2. BNT162b2 boosting was associated with a rapid increase of Omicron NAbs that peaked at week 2 and declined 6.9-fold by week 16. In contrast, Ad26.COV2.S boosting was associated with increased Omicron NAb titers that peaked later at week 4 but only declined 2.1-fold by week 16, suggesting improved durability of the heterologous boosting approach (Figure 1B). We speculate that the differences in the kinetics of the immune responses may be related to differences in the kinetics of immunogen expression in vivo. These observations are consistent with recent reports of the durability of immune responses and protection following initial Ad26.COV2.S vaccination.^7,18 These data also extend the results of a prior mix-and-match study, which evaluated a shorter boosting interval of 3 to 4 months, although this prior study only reported responses at 2 to 4 weeks following the boost.³⁶

An Ad26.COV2.S booster dose was also associated with increased Omicron-specific CD8⁺ T-cell responses following the boost immunization. Preclinical studies have suggested that CD8⁺ T-cell responses contribute to protection against SARS-CoV-2, particularly when antibody responses are subprotective.²⁰ Moreover, cellular immune responses have shown greater durability and more cross-reactivity against SARS-CoV-2 variants than serum NAb responses,^7,15,29,30 suggesting their importance for protection against virus variants such as Omicron that largely escape NAb responses. Consistent with these data are recent studies that have reported that BNT162b2 and Ad26.COV2.S provided 70% and 85% efficacy, respectively, against hospitalization with the SARS-CoV-2 Omicron variant in South Africa^37,38 in the absence of high levels of Omicron-specific NAbs, suggesting the importance of other immune responses in protection against severe disease.

This study has several limitations. First, the small size of the study, lack of randomization, and female predominance of the study population suggest that larger studies should be performed. Second, participants were enrolled at a single site in Boston, and, thus, generalizability will require further study. Third, immunogenicity data are shown for 4 months following immunization, and, thus, additional follow-up time will be required to assess long-term durability.

Our findings show that both heterologous and homologous COVID-19 vaccines boost Omicron-specific antibody and T-cell responses in BNT162b2-vaccinated individuals. NAb and CD8⁺ T-cell responses were higher following the Ad26.COV2.S boost compared with the BNT162b2 boost at week 16. These data suggest potential immunologic benefits of mix-and-match heterologous COVID-19 vaccine regimens and emphasizes the importance of durability for COVID-19 vaccine boosting strategies. Future studies could explore reduced booster doses as well as Omicron-containing boosters.

Accepted for Publication: June 23, 2022.

Published: August 10, 2022. doi:10.1001/jamanetworkopen.2022.26335

Open Access: This is an open access article distributed under the terms of the CC-BY-NC-ND License. © 2022 Tan CS et al. JAMA Network Open.

Corresponding Author: Dan H. Barouch, MD, PhD, Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, 330 Brookline Ave, E/CLS-1043, Boston, MA 02115 (dbarouch@bidmc.harvard.edu).

Author Contributions: Drs Collier and Barouch had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Tan and Collier are co–first authors.

Concept and design: Tan, Collier, Schmidt, Le Gars, Barouch.

Acquisition, analysis, or interpretation of data: Tan, Collier, Yu, Liu, Chandrashekar, McMahan, Jacob-Dolan, He, Roy, Hauser, Munt, Mallory, Mattocks, Powers, Meganck, Rowe, Hemond, Apraku Bondzie, Jaegle, Baric, Alter, Le Gars, Sadoff, Barouch.

Drafting of the manuscript: Tan, Collier, He, Roy, Alter, Barouch.

Critical revision of the manuscript for important intellectual content: Tan, Collier, Yu, Liu, Chandrashekar, McMahan, Jacob-Dolan, Hauser, Munt, Mallory, Mattocks, Powers, Meganck, Rowe, Hemond, Apraku Bondzie, Jaegle, Baric, Schmidt, Le Gars, Sadoff, Barouch.

Statistical analysis: Collier, Liu, McMahan, Munt, Mattocks, Alter, Barouch.

Obtained funding: Baric, Schmidt, Barouch.

Administrative, technical, or material support: Tan, Collier, Yu, Liu, Chandrashekar, McMahan, Jacob-Dolan, He, Hauser, Mallory, Mattocks, Rowe, Hemond, Apraku Bondzie, Jaegle, Baric, Le Gars, Barouch.

Supervision: Tan, Liu, McMahan, Jacob-Dolan, Schmidt, Alter, Barouch.

Conflict of Interest Disclosures: Dr Hauser reported receiving grants from the National Institute of Allergy and Infectious Diseases (F30 AI60908) during the conduct of the study. Dr Baric reported receiving personal fees as a member of the scientific advisory board from Adagio and as a member of the scientific advisory board from VaxArt outside the submitted work; in addition, Dr Baric had a patent for SARS-CoV 2 nLUC indicator viruses issued and used for high-throughput neutralization assays. Dr Alter reported receiving personal fees from Systems Seromyx and Leyden Labs and grants from Sanofi, BioNTech, Pfizer, and Merck outside the submitted work. Dr Sadoff reported receiving grants from the Biomedical Advanced Research and Development Authority (BARDA) (grant to Janssen) both during the conduct of the study and outside the submitted work; in addition, Dr Sadoff had a patent for invention of COVID-19 vaccine pending with all rights assigned to Janssen. Dr Barouch reported receiving personal fees from Pfizer, SQZ, Celsion, Avidea, Laronde, and Meissa; equity from Vector Sciences; and grants from Defense Advanced Research Projects Agency, BARDA, Gates Foundation, Medical Research Council, Henry Jackson Foundation, Pharm-Olam, Gilead, Legend, CureVac, Sanofi, Novavax, Intima, Alkermes, and Zentalis outside the submitted work; in addition, Dr Barouch had a patent for COVID-19 vaccine licensed to Janssen. No other disclosures were reported.

Funding/Support: The authors acknowledge Janssen Vaccines & Prevention, National Institutes of Health (NIH) grant CA260476, the Massachusetts Consortium for Pathogen Readiness, the Ragon Institute, and the Musk Foundation (Dr Barouch). The authors also acknowledge NIH grant CA260543 (Dr Baric) and the Reproductive Scientist Development Program from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and Burroughs Wellcome Fund HD000849 (Dr Collier).

Role of the Funder/Sponsor: Janssen, the sponsor of the COV2008 clinical trial, was involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation and review of the manuscript. Janssen was not involved in the decision to submit the manuscript for publication and did not have the right to veto publication or to control the decision regarding to which journal the paper was submitted.

Additional Contributions: We thank Daritza Germosen, BS, and Brandon Davis, MS (both of Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard), and Lorraine Bermudez Rivera, BS, Caroline McGrath, BS, Kathryn E. Stephenson, MD, MPH, Samuel J. Vidal, MD, PhD, Tochi Anioke, BS, Julia Barrett, BS, Benjamin Chung, BS, Makda S. Gebre, MS, Nicole Hachmann, BS, Michelle Lifton, BS, Jessica Miller, BS, Felix Nampanya, BS, Olivia Powers, BS, Michaela Sciacca, BS, Daniel Sellers, BS, Laura Shih, BS, Mazuba Siamatu, BA, Nehalee Surve, BS, Yuan Tian, BS, Lisa H. Tostanoski, PhD, Huahua Wan, MS, Haley VanWyk, BS, John D. Ventura, PhD, and Cindy Wu, BS (all from Beth Israel Deaconess Medical Center), for generous advice, assistance, and reagents. The authors also thank the study participants, the Center for Virology and Vaccine Research, the Department of Obstetrics and Gynecology, and MesoScale Discovery for providing the electrochemiluminescence assays kits.