WO2023026182A1

WO2023026182A1 - Sars-cov-2 vaccines

Info

Publication number: WO2023026182A1
Application number: PCT/IB2022/057882
Authority: WO
Inventors: Jerald C. Sadoff
Original assignee: Janssen Pharmaceuticals, Inc.
Priority date: 2021-08-24
Filing date: 2022-08-23
Publication date: 2023-03-02

Abstract

The present invention relates to the use of an adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for boosting the immune response against SARS CoV-2..

Description

SARS-COV-2 VACCINES INTRODUCTION The invention relates to the fields of virology and medicine. In particular, the invention relates to vaccines for the prevention of disease induced by SARS-CoV-2. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with Government support under Agreement HHSO100201700018C, awarded by HHS. The Government has certain rights in the invention. BACKGROUND Corona viruses (CoVs) are enveloped viruses responsible for respiratory tract infections and atypical pneumonia in humans. CoVs are a large family of enveloped, single- stranded positive-sense RNA viruses belonging to the order Nidovirales, which can infect a broad range of mammalian and avian species, causing respiratory or enteric diseases. Corona viruses possess large, trimeric spike glycoproteins (S) that mediate binding to host cell receptors as well as fusion of viral and host cell membranes. The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses of the genus Betacoronavirus that cause respiratory disease in humans include SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKU1, and the currently circulating SARS-CoV-2. SARS-CoV-2 is a corona virus that emerged in humans from an animal reservoir in 2019 and has rapidly spread globally. SARS-CoV-2, like MERS-CoV and SARS-CoV, is thought to originate from bats. The name of the disease caused by the virus is corona virus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases. In the case of SARS- CoV-2 the S protein is the major surface protein. The S protein forms homotrimers and is composed of an N-terminal S1 subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent cryo-EM reconstructions of the CoV trimeric S structures of alpha-, beta-, and deltacoronaviruses revealed that the S1 subunit comprises two distinct domains: an N-terminal domain (S1 NTD) and a receptor-binding domain (S1 RBD). SARS-CoV-2 makes use of its S1 RBD to bind to human angiotensin- converting enzyme 2 (ACE2) (Hoffmann et. al. (2020); Wrapp et. al. (2020)). Since the novel SARS-CoV-2 virus was first observed in humans in late 2019, over 200 million people have been infected and over 4 million have died as a result of COVID-19, in particular because SARS-CoV-2, and corona viruses more generally, lack effective treatment. Several vaccines against SARS CoV-2 have been developed and are currently being used for vaccination. For example, Ad26.COV2.S (as previously described in WO 2021/155323), has been demonstrated to be safe, immunogenic and to confer high protective efficacy against severe-critical COVID-19 disease, and to COVID-19 related hospitalization and death (Sadoff, Le Gars et al. N Engl J Med 2021; 384:1824-1835; Sadoff, Gray et al., N Engl J Med 2021; 384:2187-2201). However, as already observed for one of the other COVID-19 vaccines, vaccine mediated protection against COVID-19 may decline with time (Nanduri et al., MMWR Morb Mortal Wkly Rep 2021; 70; Rosenberg et al., MMWR Morb Mortal Wkly Rep 2021; 70; Tenforde et al., MMWR Morb Mortal Wkly Rep 2021; 70). This decline may imply that antibody levels are waning or are less effective against variants of concern, that immune priming has elicited insufficient immune memory to support anamnestic responses upon exposure to SARS-CoV-2, or a combination of these. High antibody titers are even more important in the context of emerging variants of concern that are relatively resistant to antibody mediated neutralization ( Barouch et al, N Engl J Med 2021; Jongeneelen et al., bioRxiv 2021: 2021.07.01.450707; Wu et al., bioRxiv 2021: 2021.01.25.427948.) and for which the antibody level may need to be higher to confer protection against acquisition and mild-to-moderate disease, especially in populations at high risk for COVID-19, such as the elderly population. Since COVID-19 continues to present a major threat to public health and economic systems, there is an urgent need for novel vaccination strategies that can be used to prevent coronavirus induced respiratory disease. SUMMARY OF THE INVENTION In the research that led to the present invention, it was surprisingly shown that booster vaccination, in particular a homologous booster vaccination, with Ad26.COV2.S, even at a dose as low as 1.25 x 10¹⁰ vp at 6 months after primary vaccination, gave a rapid increase in spike binding antibody levels. In 18–55-year-old adults, booster doses of either 5 x 10¹⁰vp or 1.25 x 10¹⁰vp gave rapid and strong increases in SARS CoV-2 S protein binding antibody levels that were higher than antibody levels at Day 29 post primary vaccination, the period in which the phase 3 efficacy study protection from severe COVID-19, hospitalization and death by the vaccine was demonstrated (Sadoff, Gray NEJM, supra). In adults >65 years of age, at this point only data with a booster dose of 1.25 x 10¹⁰vp were available and responses were similar to those observed with this dose level in younger adults. While the kinetics of antibody responses post boost were slightly slower in older as compared to younger adults, at Day 28 post boost, antibody levels were in the same range for both age groups. In addition, it was found that older adults in whom spike specific antibody titers had declined to undetectable levels at month 6 post primary vaccination showed similar strong responses to this low dose booster vaccination. The present invention thus relates to the use of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for boosting the immune response against SARS CoV-2 in a subject which has received a primary vaccination against SARS CoV-2, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp and is administered at least 6 months after receiving said primary vaccination. The invention further provides methods for boosting the immune response against SARS CoV-2 in a subject having received a primary vaccination against SARS CoV-2, said method comprising administering a booster dose of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp and is administered at least 6 months after receiving said primary vaccination. Thus provided is the use of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for boosting the immune response against SARS CoV-2 in a subject which has received a primary vaccination against SARS CoV-2, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp dose and is administered at least 6 months after receiving said primary vaccination. Also provided are methods for boosting the immune response against SARS CoV-2 in a subject having received a primary vaccination against SARS CoV-2, said method comprising administering a booster dose of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp and is administered at least 6 months after receiving said primary vaccination. Also provided are methods of inducing an immune response to SARS CoV-2 in a subject, the method comprising (a) administering to the subject a first composition comprising an immunologically effective amount of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for priming the immune response against SARS CoV-2 in the subject; and (b) administering to the subject a second composition comprising an immunologically effective amount of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 2 for boosting the immune response against SARS CoV-2 in the subject. In certain embodiments, the primary vaccination comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp of said recombinant adenovirus. The primary vaccination can, for example, comprise 5.0 x 10¹⁰ vp of said recombinant adenovirus. In certain embodiments, the primary vaccination consists of administering two doses of said recombinant adenovirus. The two doses can, for example, be administered about 2 to about 3 months apart. In certain embodiments, the booster dose comprises 1.25 x 10¹⁰ vp of said recombinant adenovirus. In certain embodiments, the booster dose comprises 5 x 10¹⁰ vp of said recombinant adenovirus. In certain embodiments, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2. The nucleic acid encoding the coronavirus S protein can, for example, be operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. The CMV promoter comprising at least one TetO motif can comprise a nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the recombinant adenovirus comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4. The recombinant adenovirus can, for example, have a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 region of the adenoviral genome. The recombinant adenovirus can, for example, be a recombinant human adenovirus of serotype 26. In certain embodiments, the recombinant adenovirus is administered intramuscularly. In certain embodiments, the immune response comprises the induction of SARS CoV- 2 S protein binding antibodies and/or SARS CoV-2 neutralizing antibodies. The level of SARS CoV-2 S protein binding antibodies can, for example, be increased at least 3-fold at 7 days after the booster dose as compared to pre-boost levels. The level of SARS CoV-S protein binding antibodies can, for example, be increased 6-fold at 28 days after the booster dose as compared to pre-boost levels. The level of SARS CoV-S protein binding antibodies can, for example, be increased at least 9-fold at 7 days after the booster dose as compared to the level of SARS CoV-S protein binding antibodies 28 days after the primary vaccination. In certain embodiments, the subject is 18 years or older. In certain embodiments, the subject is 65 years or older. In certain embodiments, the booster dose is administered at least 12 months after the primary vaccination. In certain embodiments, the booster dose is administered at least 18 months after the primary vaccination. In certain embodiments, the booster dose is administered at least 24 months after the primary vaccination. BRIEF DESCRIPTION OF THE FIGURES The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG. 1: Durability of neutralizing antibody responses up to 8 and 9 months following a single dose of Ad26.COV2.S (5x1010 vp) in participants 18–55 and ≥65 years old, respectively, from Phase 1/2a and 2 clinical trials. A) Phase 1/2a participants, 18–55 and >65 years of age, were administered a single dose of Ad26.COV2.S (5x10¹⁰vp) at Day 1, as primary vaccination. Serum neutralizing antibody responses against SARS-CoV-2 were evaluated by wtVNA up to 6 months post primary vaccination, in a subset of 18–55 (N=25) and >65 (N=15)-year old participants. B) Phase 2 participants, 18–55 and >65 years of age, were administered a single dose of Ad26.COV2.S (5x10¹⁰ vp) at Day 1, as primary vaccination. Serum neutralizing antibody responses against SARS-CoV-2 were evaluated by wtVNA, up to 8 and 9 months post primary vaccination in a subset of 18–55 (N=25) and >65 (N=22) year old participants, respectively. Participants 18–55 and >65 years of age are represented with blue and black lines, respectively. GMTs are depicted above each time point and response rates are illustrated at the bottom of each figure. FIG.2: Durability of spike binding antibody responses up to 6 months following a single dose of Ad26.COV2.S (5x10¹⁰ vp) and impact of a booster dose at 6 months post primary vaccination, in 18–55 year-old participants from a Phase 1 clinical trial. Phase 1 participants, age 18–55 (N=27) years of age, were administered a single dose of Ad26.COV2.S (5x10¹⁰ vp) at Day 1 and 20 participants received a booster dose of Ad26.COV2.S (5x10¹⁰ vp) at approximately 6 months (Day 183) post primary vaccination and 17 participants had data available at Day 190. Serum spike binding antibodies against SARS-CoV-2 were evaluated in an S-ELISA up to 7 days post boost (Day 190). A) Participants 18–55 years of age are represented with a blue line. GMCs are depicted above each time point and response rates are illustrated at the bottom of each figure. B) Dot plots representing the distribution of the participants per timepoint. N, GMC (ELISA Unit/mL) and percent responders are represented at the bottom of each plot. Grey open circles represent baseline binding antibody levels, green dots indicate that participants are responders and yellow open circles indicate that participants are non-responders. FIG.3: Durability and boostability of spike binding antibody responses up to 8–9 months following a single dose of Ad26.COV2.S (5x10¹⁰vp) in 18–55 and >65-year-old participants from a Phase 2 clinical trial. Phase 2 participants 18–55 (N=50) and >65 (N=25) years of age, were administered a single dose of Ad26.COV2.S (5x10¹⁰ vp) as primary vaccination at Day 1 and 73 received a booster dose of Ad26.COV2.S (1.25x10¹⁰ vp) at 6 months (Day 169) post primary vaccination. Serum spike binding antibody responses against SARS-CoV-2 were evaluated by S-ELISA up to 28 days post boost (Day 197). Participants 18–55 and >65 years of age are represented with blue and black lines, respectively. GMCs are depicted above each time point and response rates are illustrated at the bottom of each figure. FIG. 4: Durability of Spike Binding Ab Response up to 6 Months Post Single Ad26.COV2.S Dose and Booster Dose Impact, 18-55 Year-old Participants. FIG. 5: Pseudovirion Neutralization (IC50) of Victoria/1/2020 Strain, Beta Variant, Gamma Variant, Delta Variant and Lambda Variant, Cohort 2a (N=17); PPI Set, Active Vaccine Booster Group (VAC31518COV1001). FIG. 6: SARS CoV-2 S variant psVNA GMT: Booster dose 5 x 10¹⁰ vp 6 months after first dose (COV1001 Cohort 2a). FIG. 7: Participant disposition. Abbreviations: FAS, full analysis set; NI, non- inferiority; PPI, per protocol immunogenicity; vp, viral particles. DETAILED DESCRIPTION OF THE INVENTION The term ‘recombinant’ for a nucleic acid, protein and/or adenovirus, as used herein implicates that it has been modified by the hand of man, e.g., in case of an adenovector it has altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e., it is not a naturally occurring wild type adenovirus. Nucleotide sequences herein are provided from 5’ to 3’ direction, as custom in the art. The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV-1, SARS-CoV-2 and MERS, OC43 and HKU1. As described above, SARS-CoV-2 can cause severe respiratory disease in humans. A safe and effective SARS-CoV-2 vaccine which induces a durable immune response is required to end the COVID-19 pandemic. The SARS CoV-2 viral spike (S) protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV-2. ACE2 is a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension. It is expressed in, inter alia, vascular endothelial cells, the renal tubular epithelium, and in Leydig cells in the testes. PCR analysis revealed that ACE-2 is also expressed in the lung, kidney, and gastrointestinal tract, tissues shown to harbor SARS-CoV- 2. The spike (S) protein of coronaviruses is a major surface protein and target for neutralizing antibodies in infected patients (Lester et al., Access Microbiology 2019;1) and is therefore considered a potential protective antigen for vaccine design. The adenovirus vaccine Ad26.COV2.S (Bos et al., NPJ Vaccines; article 91 (2020); WO2021/155323) has been demonstrated to be safe, immunogenic and to confer high protective efficacy against severe-critical COVID-19 disease, and to COVID-19 related hospitalization and death (Sadoff, Le Gars et al. supra; Sadoff, Gray et al., supra). In a first aspect, the present invention provides the use of an adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for boosting the immune response against SARS CoV-2 in a subject which has received a primary vaccination, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp and is administered at least 6 months after receiving said primary vaccination. According to the invention, “boosting,” “booster,” or a “booster dose” refers to (providing) another dose of a vaccine that is given to someone who built an initial protective immune response after primary vaccination. Such initial immune response may have decreased over time (referred to as waning immunity). After initial immunization, a booster dose is a re-exposure to the immunizing antigen. It is intended to increase immunity against that antigen back to protective levels, after the immune response against that antigen has declined over time or to further increase immunity as compared to after the initial immunization. Boosting can be “homologous,” i.e., the same vaccine is used in the primary vaccination regimen and as booster dose, or “heterologous,” i.e., the vaccine used in the primary vaccination is a different vaccine than the vaccine used as booster. According to the invention a “primary vaccination,” or “primary vaccination regimen,” may refer to any vaccination regimen that is currently used. In certain embodiments, the primary vaccination consists of a single dose of said adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1. The primary vaccination may, however, also comprise a vaccination regimen with two doses of an mRNA vaccine, such as, but not limited to two doses of the mRNA vaccine BNT162b2 given 21 days apart, or two doses of mRNA-1273 given 28 days apart, or two doses of the vector-based vaccine ChAdOx1-S/nCoV-19 given 4 to 12 weeks apart. According to the present invention, it has surprisingly been found that a homologous booster dose of 5 x 10¹⁰vp or 1.25 x 10¹⁰ vp of said adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1, at 6 months after primary vaccination, elicited rapid and robust increases in antibody levels that were many folds higher than pre-boost and Day 29 post dose 1 in all participants. The strong anamnestic responses after booster immunization imply robust immune memory elicited by the single dose primary vaccination. Based on recent data, administration of a booster dose resulted in increased protection against symptomatic COVID-19, increased strength and breadth of immune responses against variants and increase protection against severe/critical COVID-19. According to the invention, the coronavirus S protein encoded by said adenovirus may or may not comprise the signal peptide (or leader sequence). The signal peptide typically comprises the amino acids 1-13 of SEQ ID NO: 1. In certain embodiments, the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 1. In certain embodiments, the coronavirus S protein encoded by said adenovirus consists of an amino acid sequence of SEQ ID NO: 1 without the signal peptide. In certain embodiments, the coronavirus S protein encoded by said adenovirus consists of amino acid residues 14-1273 of SEQ ID NO: 1. In certain embodiments, the primary vaccination comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp of said recombinant adenovirus. In certain embodiments, the primary vaccination regimen consists of administering one dose of 5 x 10¹⁰ vp of said adenovirus. In certain embodiments, the primary vaccination consists of administering two doses of said recombinant adenovirus. The two doses can, for example, be administered about 2 to about 3 months apart. In certain embodiments, the booster dose comprises 1.25 x 10¹⁰ vp of said adenovirus. In certain embodiments, the booster dose comprises 5 x 10¹⁰ vp of said adenovirus. According to the invention the booster dose is administered at least 6 months after primary vaccination. In certain embodiments, the booster dose is administered at least about 6 months, 7 months, 8, months, 9, months, 10 months, 11, months, 12 months, 15 months, 18 months, 24 months, 30 months, 36 months, or any time in between after primary vaccination or later. In certain embodiments, the booster dose is administered between 6 and 12 months after the primary vaccination and subsequently an additional booster dose is administered annually. In certain embodiments, the booster dose is administered between 6 and 18 months after the primary vaccination and subsequently an additional booster dose is administered annually. In certain embodiments, the booster dose is administered between 6 and 24 months after the primary vaccination and subsequently an additional booster dose is administered annually. In certain embodiments, the booster dose is administered between 12 and 18 months after the primary vaccination and subsequently an additional booster dose is administered annually. In certain embodiments, the booster dose is administered between 12 and 24 months after the primary vaccination and subsequently an additional booster dose is administered annually. In certain embodiments, the booster dose is administered between 6 and 12 months, between 6 and 18 months, between 6 and 24 months, between 12 and 24 months, between 12 and 18 months, or between 18 and 24 months after the primary vaccination. It is understood by a skilled person that numerous different nucleic acids can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acids, to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In certain embodiments, the nucleic acid is codon optimized for expression in human cells. In certain preferred embodiments the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2. In certain embodiments of the invention, the vector is an adenovirus vector. An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to, a bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), a canine adenovirus (e.g., CAdV2), a porcine adenovirus (e.g., PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd). In the invention, a human adenovirus is meant if referred to as Ad without indication of species, e.g., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26. Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, a recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials. Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g., US6083716; WO 2005/071093; WO 2010/086189; WO 2010/085984). Hence, in other embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g., a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P. In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see, e.g., WO 2012/172277), or ChAdOx 2 (see, e.g., WO 2018/215766). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see, e.g., WO 2019/086456), or BZ1 (see, e.g., WO 2019/086466). In a preferred embodiment of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g., including Ad26. In the typical embodiment, the vector is an rAd26 virus. An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a “capsid protein” for a particular adenovirus, such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are of Ad26. One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See, for example, WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also, e.g., WO 2019/086461 for chimeric adenoviruses Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13, that include an Ad26 virus backbone having partial capsid proteins of Ptr1, Ptr12, and Ptr13, respectively) In certain preferred embodiments, the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the E1 region of the genome. For adenoviruses being derived from non-group C adenovirus, such as Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C, such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the E1 genes of Ad5, such as, for example, 293 cells, PER.C6 cells, and the like (see, e.g., Havenga, et al., 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, such adenoviruses will not be capable of replicating in non-complementing cells that do not express the E1 genes of Ad5. The preparation of recombinant adenoviral vectors is well known in the art. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention, for instance, include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety. Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded. The adenovirus vectors useful in the invention are typically replication deficient. In these embodiments, the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the E1 region. The regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding a synthetic SARS CoV2 S protein (usually linked to a promoter), or a gene encoding an SARS CoV2 S antigenic polypeptide (usually linked to a promoter) within the region. In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3, or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication. A packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with a deletion in the E1 region include, for example, PER.C6, 911, 293, and E1 A549. In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the E1 region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p.4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the synthetic SARS CoV-2 S antigens is cloned into the E1 and/or the E3 region of the adenoviral genome. In certain embodiments, the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. This allows for the cost-effective, large-scale manufacturing of adenoviral particles comprising the SARS CoV-2 S protein insert. Without intending to be limited by theory, it is believed that the SARS CoV-2 S protein leads to lower levels of adenoviral particle production. The addition of the TetO motif to the CMV promoter allows for higher levels of adenoviral particle production. As used herein, a “promoter” is a nucleic acid sequence enabling the initiation of the transcription of a gene sequence in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter. As defined above, in certain embodiments, the promoter is a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif. The TetO motif can be referred to a “regulatory sequence” or “regulatory element,” which as used herein refers to a segment of nucleic acid, typically, but not limited to DNA, that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and, thus, acts as a transcriptional modulator. A regulatory sequence often comprises nucleic acid sequences that are transcription binding domains that are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, enhancers, or repressors, etc. For example, it is possible to operably couple a repressor sequence to the promoter, which repressor sequence can be bound by a repressor protein that can decrease or prevent the expression of the transgene in a production cell line that expresses the repressor protein. This can improve genetic stability and/or expression levels of the nucleic acid molecule upon passaging and/or when this is produced at high quantities in the production cell line. Such systems have been described in the art. A regulatory sequence can include one or more tetracycline operator (TetO) motifs/sequences, such that expression is inhibited in the presence of the tetracycline repressor protein (TetR). In the absence of tetracycline, the TetR protein is able to bind to the TetO sites and to repress transcription of a transgene (e.g., SARS CoV-2 S antigen) operably linked to the TetO motifs/sequences. In certain embodiments, the nucleic acid encoding the SARS-CoV-2 S protein, when present in the adenoviral vector, is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif, such that the expression of the SARS CoV-2 S protein is inhibited in recombinant adenoviruses that are produced in the producer cell line in which the TetR protein is expressed. Expression will not be inhibited when the recombinant adenoviral vector is introduced into a subject or into cells that do not express the TetR protein. The invention, however, is not limited to use of a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif. As used herein, the term “repressor” refers to molecules (e.g., proteins) having the capability to inhibit, interfere, retard, and/or repress the production of a heterologous protein product of a recombinant expression vector (e.g., an adenoviral vector). The repressor can inhibit expression by interfering with a binding site at an appropriate location along the expression vector, such as in an expression cassette (e.g., a TetR can bind the TetO motif in the CMV promoter). Repression of vector transgene expression during vector propagation can prevent transgene instability and can increase yields of vectors having the transgene during production. A nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence. For example, one segment of DNA can be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding sequence so as to facilitate transcription of the coding sequence; a ribosome binding site that is positioned relative to a coding sequence so as to facilitate translation; or a pre-sequence or secretory leader that is positioned relative to a coding sequence so as to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of the encoded polypeptide). In other examples, the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers. In certain embodiments, the nucleic acid encoding the coronavirus S protein can, for example, be operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. In certain embodiments, the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 5, preferably the CMV promotor consists of SEQ ID NO: 5. In certain embodiments, the adenovirus comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4. The present invention thus relates to boosting the immune response against SARS CoV-2. In certain embodiments, the immune response comprises the induction of SARS CoV-2 spike (S) protein binding antibodies and/or SARS CoV-2 neutralizing antibodies. In certain embodiments, the level of SARS CoV-2 S protein binding antibodies is increased at least 3-fold, preferably at least 3.5-fold at 7 days after the booster dose as compared to pre- boost levels. In certain embodiments, the level of SARS CoV-S protein binding antibodies is increased at least 6-fold, preferably at least 6.5-fold at 28 days after the booster dose as compared to pre-boost levels. In certain embodiments, the levels of SARS CoV-2 S binding antibodies at 28 days post boost are at least 6-fold higher than 29 days after the primary vaccination. In certain embodiments, the levels of SARS CoV-2 S binding antibodies at 7 days post boost are at least 9-fold higher than the levels of SARS CoV-2 S binding antibodies 28 days after the primary vaccination. Preferably, the subject is 18 years or older, preferably between 18 and 55 years old, or 65 years or older. Administration of the adenovirus can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as by injection, e.g., intramuscular, intradermal, etc., or subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. However, it is particularly preferred according to the present invention to administer the vaccine intramuscularly. The advantage of intramuscular administration is that it is simple and well-established and does not carry the safety concerns for intranasal application in infants younger than 6 months. In one embodiment a composition is administered by intramuscular injection, e.g. into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh. The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention. EXAMPLES Example 1: Strong durability of antibody responses elicited by a single dose of Ad26.COV2.S and substantial increase following late boosting Methods Study participants and immunogenicity assessment Participants from an ongoing Phase 1/2a (study (COV1001, NCT04436276; Cohort 1a, 18-55 years old [N=25]; Cohort 2a, 18-55 years old [N=17]; Cohort 3, >65 years old [N=22]; and an ongoing Phase 2 study (COV2001; NCT04535453; 18–55 and >65 years old, (total N=73) received a single dose of Ad26.COV2.S (5x10¹⁰ viral particles [vp]) as a primary vaccination regimen. In addition, Phase 1/2a Cohort 2a and Phase 2 participants received homologous booster doses, of Ad26.COV2.S of 5 x 10¹⁰ vp or 1.25 x 10¹⁰ vp, respectively, at 6 months post primary vaccination. Spike-binding and neutralizing antibody levels were assessed by ELISA during a 6- to 9-month follow up after a single dose Ad26.COV2.S primary vaccination regimen, and after a booster dose at 6 months post primary vaccination. Neutralizing antibody titers were evaluated in a subset of participants from Phase 1/2a Cohorts 1a and 3 and Phase 2. Binding antibody geometric mean concentrations (GMCs) and neutralizing geometric mean titers (GMTs) were determined at Days 1, 15, 29, 57, 71, 85 and 169 (6 months) (Phase 1/2a Cohort 2a and Phase 2, 8 months (Phase 1/2a Cohort 1a) and 9 months (Phase 1/2a Cohort 3) post primary vaccination; binding antibody GMCs were also evaluated at days 7- and 28 post booster doses. A participant was considered a responder after primary vaccination regimen if: baseline antibody titers were below the lower limit of quantification (LLOQ) prior to vaccination and were above the LLOQ after vaccination; or if baseline antibody titers were above LLOQ prior to vaccination and were 4-fold higher than baseline titers after vaccination. All participants were monitored for solicited and unsolicited local and systemic adverse events by 7-day diary cards and 28-day follow-up for unsolicited AEs. Serious adverse events (SAEs) were collected for 6 months following the last vaccination in each study and all SAEs of special interest were collected throughout the study. All participants provided written informed consent. The trial adhered to the principles of the Declaration of Helsinki and to the Good Clinical Practice guidelines of the International Council for Harmonisation. SARS-CoV-2 wild-type virus neutralization assay Neutralizing antibodies capable of inhibiting wild type virus infections were quantified using the wild type virus microneutralization assay (MNA) that was developed and qualified by Public Health England (PHE). The virus stocks used were derived from the Victoria/1/2020 strain and the LLOQ is 58 IC₅₀. In brief, 6 two-fold serial dilutions of the inactivated human serum samples were prepared in 96-well transfer plate(s). The SARS-CoV-2 wild-type virus was subsequently added to the serum dilutions at a target working concentration (approximately 100 plaque- forming units [PFU]/well) and incubated at 37°C for 60 to 90 minutes. The serum-virus mixture was then transferred onto assay plates, previously seeded overnight with Vero E6 African green monkey kidney cells and incubated at 37°C and 5% CO₂ for 60 to 90 minutes before the addition of carboxymethyl cellulose (CMC) overlay medium and further incubation for 24 hours. Cells were then fixed and stained using an antibody pair specific for the SARS-CoV-2 RBD spike (S) protein and immunoplaques were visualized using TrueBlueTM substrate (KPL, Milford, MA, USA). Immunoplaques were counted using the Immunospot Analyzer from CTL (Cellular Technology Limited, Cleveland, OH, USA). The immunoplaque counts were exported to SoftMax Pro (Molecular Devices, San Jose, CA, USA) and the neutralizing titer of a serum sample was calculated as the reciprocal serum dilution corresponding to the 50% neutralization antibody titer (IC50) for that sample. Spike protein enzyme-linked immunosorbent assay (ELISA) SARS-CoV-2 pre-spike-specific binding antibody concentrations were determined using the human SARS-CoV-2 pre-spike IgG ELISA, an indirect ELISA that is based on antibody/antigen interactions. The SARS-CoV-2 antigen as a stabilized pre-fusion spike protein ((2P), Δfurin, T4 foldon, His-tag), derived from the first clinical isolate of the Wuhan strain (Wuhan, 2019, whole genome sequence NC_045512), was produced in ES-293 cells. The ELISA was developed and qualified for human serum at Nexelis, Laval, PQ, Canada. The lower limit of quantification of the assay is 53 ELISA Units (EU/mL). In brief, purified pre-spike Antigen was adsorbed to the wells of a microplate and diluted serum samples (test samples, standard, and quality controls) were added. Unbound sample was washed away, and enzyme-conjugated anti-human IgG added. After washing excess conjugate away, 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric substrate was added. After the established time period, the reaction was stopped. A reference standard on each test plate was used to quantify the amount of antibodies against SARS-CoV-2 pre-spike in the sample according to the unit assigned by the standard (EU/mL) Results Durability of humoral immunity after single dose Ad26.COV2.S (5x10¹⁰vp) Short-term immunogenicity after single-dose primary vaccination with Ad26.COV2.S in participants of a Phase 1/2a study was previously reported (Sadoff, Le Gars et al., N Engl J Med 2021; 384(19): 1824-35). In this Example, the levels of neutralizing antibodies after longer follow up of the participants of Cohort 1a (18–55 years old; 8 months follow-up) and Cohort 3 (>65 years old; 9 months follow-up) are reported. In participants aged 18–55 years old (Cohort 1a), neutralizing antibody responses were detectable up to at least Day 239 (8 months) after a single dose of Ad26.COV2.S, with GMTs of 226 and 21/22 (95%) participants with detectable titers, which is similar to what was observed by Day 29 post dose 1 (GMT of 224 and 96% of participants with positive titers; Figure 1A). In participants ≥65 years of age (Cohort 3), neutralizing antibodies were still detectable in 13/19 (68%) of participants by Day 268 (9 months) after one dose of Ad26.COV2.S, with GMTs of 114. This represents a 2.3-fold decrease in GMTs compared to Day 29 post dose 1 (GMT of 258) in participants ≥65 years of age (Figure 1A). From Cohort 2a of our Phase 1/2a study, which is described in the supplementary material of Sadoff et al., (supra) 17 participants 18–55 years of age had undetectable binding antibody levels at Day 8 post prime vaccination but could be measured at the next timepoint (Day 29; GMC, 418 [95% CI, 322–554], with 100% responders; Figure 2A and 2B). This is in the same range as the observation in adults 18–55 years old in our Phase 2 study (Figure 3). By month 6, GMCs in participants in Cohort 2a had increased to 900 (441–1643) with 100% of participants still having detectable binding antibodies (Figure 2A). In a Phase 2 study, (COV2001), a single dose of Ad26.COV2.S elicited neutralizing antibody responses by Day 15 in 21 out of 22 participants aged 18-55 years (96% responders; GMTs of 244158-277]) and in 10 of 15 participants ≥65 years of age (67% responders; GMT: 119 [66- 217]; Figure 1B). These responses further increased by Day 29 in both age groups (18-55 (100% responders and GMT:277 [211- 365]; ≥ 65 year old (100% responders; GMT:240 [179-322] Figure 1B). Up to Day 85, neutralizing antibody responses remained stable in participants aged 18-55 years (95% responders and GMT: 243) while they decreased modestly to a GMT of 186 (99-349) in participants ≥ 65 years old, with 77% still above the LLOQ of the assay. Neutralizing antibody levels in participants 18-55 years old 6 months after vaccination, were in a similar range as Day 29 levels (GMT: 200 [106-378]; 84% responders). In adults ≥65-years old, the GMT of neutralizing antibody at 6 months after primary vaccination was 134 (68-266) (with 69% responders). Binding antibody levels also gradually increased from baseline to Day 15 to Day 29 and remained stable up to Day 85 in both age groups ((18–55-years:GMC: 572 [420-780], ≥ 65-years : GMC: 313[201-86], with 96% above LLOQ of the assay in both groups; Figure 3). GMCs in participants ≥ 65 years old were slightly lower at all timepoints, compared to those aged 18-55 years. At 6 months after primary vaccination, GMCs of binding antibodies had declined to 416 (294- 588) and 234 (136- 403) with 93% and 86% of participants still having titers above the LLOQ of the assay in those aged 18-55 and ≥ 65 years, respectively. Binding antibodies at 6 months primary vaccination were undetectable in 2 out 44 participants aged 18–55 years and in 4 of 29 participants ≥ 65 years old. Humoral immune responses after homologous boosting with Ad26.COV2.S at a dose of 5 x 10¹⁰ vp. Clinical trial participants in the phase 1/2a study (18–55 years of age; N=17) who had received a primary vaccination with a single dose of Ad26.COV2.S at a dose level of 5x10¹⁰ vp were given a homologous booster vaccination 6 months later with a dose of 5x10¹⁰ vp. By day 7 post boost, all participants demonstrated a robust increase in binding antibody levels of 4.7-fold (GMC: 3779 [2741-4243]) compared to immediate pre-boost binding antibody levels and a 9-fold increase in GMCs compared to Day 29 binding antibody levels after the initial immunization (Figure 2). Humoral immune responses after low-dose homologous boosting with Ad26.COV2.S In a Phase 2 trial, a lower homologous booster dose of 1.25x10¹⁰vp Ad26.COV2.S was given at 6 months in 44 participants of 18-55 years of age and 29 participants ≥ 65 years of age (Figure 3). This lower booster dose surprisingly also elicited a rapid and high increase of 3.6-fold increase in binding antibody levels at 7 days post boost (GMCs 1,719 [1321- 2236]) as compared to immediate pre-boost binding antibody levels. Levels of binding antibodies had further increased by Day 28 post boost (GMC: 2,444 [1855-3219]), representing 6.4-fold and 6.9-fold increases compared to Day 29 levels after the primary vaccination and immediate pre-boost antibody levels, respectively. While the kinetics of the post low-dose booster were slower in the ≥ 65 years old adults, the magnitude of the response by Day 28 was similar in both younger and older adults studied (Figure 3). Safety of a booster dose of Ad26.COV2.S In Cohort 2 of the Phase 1/2a study (COV1001) in 17 participants, both post-primary regimen and post-booster reactogenicity appeared similar as compared to the previously reported Cohort 1 reactogenicity (Sadoff, le Gars et al, NJEM, supra). Reactogenicity following the boost may have been more comparable to the reactogenicity previously reported from Cohort 1 following a second dose at 57 days after the initial dose, which was lower than following the initial priming dose, but the limited number of participants precludes precision in these measurements. In the Phase 2 study (COV2001), when booster doses of 1.25x10¹⁰ vp Ad26.COV2.S were given at 6 months after the primary single dose immunization of 5x10¹⁰ vp in 81 participants, solicited AEs after the primary dose versus after the booster were 67.9% versus 54%, respectively, and for grade 3 solicited AEs, 1.2% versus 0%, for solicited local AEs, 51.9% versus 57%, for solicited local AEs of grade 3 or more 0% versus 0%, for solicited systemic AEs, 66.7% versus 28%, and for solicited systemic AEs of grade 3 or more, 1.2% vs 0%. Example 2. COV1001 - Ad26.CoV2.S Booster at 6 months In Cohort 2a (Group 2) of study COV1001, immunogenicity of a booster dose after the primary vaccination regimen was evaluated in healthy adults aged ≥18 to ≤55 years. Participants received Ad26.COV2.S at the selected dose level of 5×10¹⁰ vp as the first dose and received Ad26.COV2.S at a dose level of 5×10¹⁰ vp as the booster, 6 months (Day 183) after primary vaccination. Boosting with Ad26.COV2.S (5×1010 vp) 6 months after primary vaccination induced a substantial and rapid increase of humoral immune responses (see Figure 4). 7 days after the booster (Day 190), binding antibody concentrations demonstrated a substantial and rapid increase to 3,779 (N=17), representing a GMI from pre-booster levels of 4.2-fold. Similarly, neutralizing antibodies (psVNA) increased 3.5-fold compared to pre-boosting (GMT of 156 versus 33 on Day 190 and Day 183, respectively). 28 days after the booster, (Day 211), a further increase in binding antibody concentrations to 5,108 (N=15) was observed, representing a GMI from pre-booster levels of 5.4-fold. Similarly, neutralizing antibodies (psVNA) increased 5.0-fold compared to pre- boosting (GMT of 241 versus 33 on Day 211 and Day 183, respectively). In participants who did not receive a booster dose 6 months after primary vaccination, GMC and responder rates remained stable between Day 29 and Day 183 and decreased slightly at Day 190 and Day 211 compared to the Day 29 responses, confirming the previously described immunogenicity results on the durability of the response induced by a single dose of Ad26.COV2.S (Figure 1). A 5 x 10¹⁰ vp booster dose at 6 months elicited a rapid increase in binding Abs 7 days post boost, compared to immediate pre-boost levels and compared to Day 29 post dose 1 levels in 18–55 yo participants, exceeding levels of human convalescent sera reported earlier by about 4-fold (Sadoff 2021, supra). Moreover, a post-hoc NI analysis was performed on the 17 participants from COV1001 Cohort 2a/group 2. This post hoc analysis calculated the fold increase in the ELISA assay (Nexelis) and the psVNA assay (Janssen Vaccines Discovery) from 28 days post dose 1 to 7 days post booster dose (Day 190) and from 28 days post dose 1 to 28 days post booster dose (Day 211). While the study did not include a pre-specified non-inferiority objective, the results of this post-hoc analysis can be interpreted in relation to standard non- inferiority criteria such as a lower limit of the 95% confidence interval being above 0.67. The calculated fold-increases, were: - For the ELISA assay: 9.04 (N=17; 95% CI: 5.86; 13.96) and 11.99 (N=15; 95% CI: 7.92;18.16) 7 and 28 days post booster dose, respectively. - For the psVNA assay: 7.20 (N=17; 95% CI: 4.80; 10.81) and 11.01 (N=15; 95% CI: 7.96; 15.21) 7 and 28 days post booster, respectively. Note that values below the LOD at 28 days post-dose 1 were imputed with the LOD. The lower limit of each of these CIs was above 1, thereby also meeting standard non- inferiority criteria such as a lower limit of the 95% CI above 0.67, for both assays at both timepoints. Example 3. Immunogenicity of a 2-dose Vaccination Against Variants of Concern Samples from a subset of Cohort 2a participants from study COV1001 who had received a 6 month booster vaccination (n=17) were measured for neutralizing antibodies against the SARS-CoV-2 Beta, Gamma (P.1 lineage), Delta, and Lambda (C.37 lineage) variants by a psVNA conducted by JBDA. Graphical representation of neutralizing responses (geometric means) to the original strain and to the 4 variants, as measured by the developed Janssen Bioassay Development and Automation (JBDA) psVNA, prior to booster and at Day 183 and Day 190 are shown in Figure 5. Post boost, neutralizing antibodies to the original strain were numerically higher than those seen against the variants, although some overlap of the 95% CIs was seen with both the Delta and Lambda variants at Day 191. This exploratory analysis showed that neutralizing antibodies against the Beta variant were numerically lower than those against the other variants, with some overlap of the 95% CIs at the post boost timepoints. Neutralizing antibodies against the Delta and Lambda variant were comparable, and numerically higher than those against the Gamma and Beta variants, although overlap of the 95% CIs was apparent. Samples were also measured for binding antibodies against the SARS-CoV-2 original strain, Beta variant and Delta variant. This analysis utilized developed ELISAs conducted by JBDA. Binding rapidly increased after a booster dose of 5×1010vp of Ad26.COV2.S. By Day 190 (7 days post booster) for the Beta variant, binding antibodies remained at 100% and the mean EC50 was now 3.04, representing a mean increase from pre-booster levels of 0.73. For the Delta variant, binding antibodies also remained at 100% and the mean EC50 was now 3.02, representing a mean increase from pre-booster levels of 0.67. For both variants, these responses remained stable to Day 211, the last timepoint tested. A high correlation between the Nexelis ELISA and the JBDA ELISA was seen at all the timepoints (Spearman values >0.90). This suggests that despite differences in the S protein from the Beta or Delta variant and reference strain (Victoria/1/2020), the Nexelis S-ELISA could potentially be used as a surrogate for the Beta and Delta variant S ELISAs. Example 4: Efficacy of the Ad26.COV2.S Booster Dose An Ad26.COV2.S booster dose administered 2 months after the primary Ad26.COV2.S dose substantially increases protection, especially against symptomatic COVID-19 (see Table 1 and 2), including when caused by SARS-CoV-2 variants of concern (see Table 3). The primary analysis results of Janssen’s 2-dose efficacy study COV3009 includes data from 7484 participants who received 2 doses of Ad26.COV2.S and 7008 participants who received 2 doses of placebo in the PP set. Median follow-up time after the second dose in the double blind phase was 36 days (0-172 days), with 29.3% of participants in the per protocol set with at least 2 months of follow-up after the 2nd dose. Sequencing data were available from 68.0% of cases in the double-blind phase of the study. The reference sequence was only present in 6.0% of the sequenced strains overall and 22.9% in the US. In the US, where the dominant strain during the double-blind phase of COV3001 (single dose regimen) was the reference strain, VE was stable when comparing the COV3001 primary and final analysis. After the Ad26.COV2.S booster dose (COV3009), VE against symptomatic COVID-19 was 94% in the US. The number of cases in COV3009 that were infected with the Alpha or Mu variant were sufficient to allow a variant specific analysis of vaccine efficacy, which demonstrated that a homologous booster of Ad26.COV2.S increases protection against symptomatic infection for different variants. (Table 3). At the analysis cut-off date in June 2021, the Delta variant was not yet prominent in our study population, so vaccine efficacy for this variant are not available. Table 11: VE After Ad26.COV2.S Booster Dose 2 Months after First Dose (COV3009) and After Single Ad26.COV2.S Dose (COV3001) *

moderate to severe/critical COVID-19 per the case definitions in Error! Reference source not found.. Table 2. VE Against Symptomatic COVID-19: Single Dose (COV3001) vs Booster Dose 2 Months After First Dose (COV3009).

Table 3. VE Against Symptomatic COVID-19 by Variant: Single Dose (COV3001) vs Booster Dose 2 Months After First Dose (COV3009).

Immunogenicity of the Ad26.COV2.S Booster Dose The immunogenicity of an Ad26.COV2.S booster dose was measured over different time intervals throughout the different AD26.COV2.S clinical studies. Overall, the data show that an Ad26.COV2.S booster dose administered 2 to 6 or more months after the primary regimen increases immunogenicity versus the 1-dose regimen and induces a strong and broad immune response that is expected to confer extended durable protection against COVID-19, including variants of concern. A post-hoc NI analysis was performed on 17 participants from COV1001 who received a booster dose 6 months after the first dose. While the study did not include a pre- specified non inferiority objective, the fold increase in the ELISA assay and the psVNA assay from 28 days post dose 1 to 7 days post booster dose (Day 190) and from 28 days post dose 1 to 28 days post booster dose (Day 211) have met the standard NI criteria. Using an internally developed ‘fit for purpose’ pseudovirus neutralization assay specific for the variants of concern, a substantial increase in variant-specific neutralizing antibodies was observed, including the Delta variant (see Figure 5). By 28 days post boost, all 17 participants evaluated had detectable neutralizing antibodies against the Delta variant. A larger interval between the primary vaccination with Ad26.COV2.S and a homologous booster dose resulted in a larger increase in humoral immune responses (ELISA titers) versus the 1-dose regimen (see Table 4), for both participants 1855 years of age and ≥65 years of age, going from a 4-6 fold increase (both age groups) with a 2-month boost to a 12-fold increase with a 6-month boost (younger age group only). Table 4. Spike Binding Antibody Levels: Booster Dose 2-3 Months (COV2001) vs 6 Months (COV1001) After First Dose

The final analysis of COV3001 demonstrate that the single dose Ad26.COV2.S primary regimen provides substantial protection against severe COVID-19 disease, hospitalization and death and maintains a favorable benefit-risk profile. Durable protection against observed COVID-19 and COVID-19 related hospitalizations was confirmed by large real-world- effectiveness studies in the US and South Africa, including in calender time that the delta variant was highlight prevalent. Therefore, the single dose Ad26.COV2.S regimen continues to be an important tool in the fight against COVID 19. An Ad26.COV2.S booster dose administered 2 months after the primary Ad26.COV2.S dose substantially increases protection, especially against symptomatic COVID-19, including when caused by SARS-CoV-2 variants of concern. Studies COV1001 and COV2001 indicate that a larger interval between the primary vaccination with Ad26.COV2.S and a homologous booster dose resulted in a larger increase in humoral immune responses (ELISA titers) versus the 1-dose regimen, for both participants 1855 years of age and ≥65 years of age, going from a 4-6 fold increase (both age groups) with a 2-month boost to a 12-fold increase with a 6-month boost (younger age group only). A total of 9,379 participants ≥18 years of age, including 2,383 participants ≥60 years of age, have received 2 doses of Ad26.COV2.S 5×1010 vp in clinical studies, with the booster administered after an interval of 2 months to ≥6 months. Overall, Ad26.COV2.S has an acceptable reactogenicity profile after both the first dose and booster, with the reactogenicity post-booster being similar or milder than post-dose 1. No new safety concerns have been identified after an Ad26.COV2.S booster. Example 5. Safety, Reactogenicity, and Immunogenicity of Ad26.COV2.S as Homologous or Heterologous COVID-19 Booster Vaccination In this randomized, double-blind, multicenter phase 2 trial, participants ≥18 years received an Ad26.COV2.S booster dose (5×10¹⁰ viral particles [vp], 2.5×10¹⁰ vp, or 1×10¹⁰ vp) ≥6 months after primary vaccination with either single-dose Ad26.COV2.S (homologous boost; n=774) or two-dose BNT162b2 (heterologous boost; n=758). Primary endpoints were non-inferiority of neutralizing antibody (nAb) responses to the reference SARS-CoV-2 strain (D614G) and leading VOCs at Day 15 after boosting vs Day 29 after primary vaccination. Reactogenicity and safety were also assessed. Methods Trial Design This phase 2 randomized, double-blind, multicenter trial (ClinicalTrials.gov Identifier: NCT04999111) was conducted at 21 sites in the United States. Two cohorts were enrolled with participants who received single-dose primary vaccination with Ad26.COV2.S (cohort 1) or a 2-dose primary vaccination with BNT162b2 (cohort 2), and within each cohort, participants were randomly assigned to receive a single booster injection of 5×10¹⁰ vp, 2.5×10¹⁰ vp, or 1×10¹⁰ vp Ad26.COV2.S. The 5×10¹⁰ vp dose level of Ad26.COV2.S was selected based on previous phase 1 data demonstrating robust immunogenicity and acceptable safety and reactogenicity. Because a lower booster dose level has potential advantages, including a lower incidence of resultant AEs, booster dose levels of 2.5×10¹⁰ vp and 1×10¹⁰ vp were also evaluated. The purpose of this study was to demonstrate that the neutralizing antibody responses to SARS-CoV-2 following homologous or heterologous boosting with Ad26.COV2.S were noninferior to responses elicited by primary vaccination with Ad26.COV2.S or BNT162b2. NI of immune responses to the D614G strain (“reference strain,” highly related to the original Wuhan strain) and to the Delta (B.1.617.2) variant, which was the leading VOC at the time of primary analysis, were evaluated in this study. Prespecified NI tests were performed for responder rate (cohort 1) or seropositivity rate (cohort 2). NI in cohort 1 was assessed with respect to the GMR of 14-day post-boost GMT to 28-day post-prime GMT, and NI in cohort 2 was assessed using the ratio of 14-day post-booster GMT to 14- to 60-day post-prime GMT. Non-powered descriptive NI analyses were prespecified for other VOCs that might arise and were conducted for Beta (B.1.351) and Omicron (B.1.529 or BA.1). Neutralizing antibody titers against the reference strain and Delta, Beta, and Omicron variants were assessed 14 days (Day 15) and 28 days (Day 29) following boosting. Trial Participants Participants were adults aged ≥18 years who had received either a single dose of 5×1010 vp of Ad26.COV2.S on day 1 of enrolling in the phase 3 ENSEMBLE trial (NCT04505722) or the 2-dose primary regimen of BNT162b2 (30 µg dose) in study NCT04368728 or post-authorization at least 6 months prior to enrollment in this study. Participants could be enrolled in cohort 1 if they were currently enrolled in ENSEMBLE with no major protocol deviations in that trial, day 1 and day 29 serum samples were available, and blood samples 28 days post–primary vaccination were collected within the permitted visit window. All enrolled cohort 2 participants had received 2 primary regimen doses of BNT162b221 to 42 days apart post-authorization. At one study site, participants could be enrolled in cohort 2 if serum samples collected 15 to 60 days post–primary vaccination were available; such samples were not available at the remaining 20 sites. Randomization and Procedures Participants in each cohort were initially assigned 1:1:1 into 3 dose level groups with the use of randomly permuted blocks in an interactive web response system. When the 1×10¹⁰ vp group in each cohort was fully enrolled, randomization continued in a 1:1 ratio for the 5×10¹⁰ vp and 2.5×10¹⁰ vp groups. In cohort 2, Ad26.COV2.S was administered to 6 sentinel participants to monitor for unexpected severe adverse reactions. No clinically significant safety findings were reported, and randomization and vaccination continued as planned. Signs and symptoms of COVID-19 were actively surveilled using an electronic clinical outcome assessment measure. All participants with COVID-19-like signs or symptoms meeting the prespecified criteria for suspected COVID-19, and all participants with at least 1 positive RT-PCR test for SARS-CoV-2 within 5 days of symptom onset underwent prespecified COVID-19 procedures. The first 330 randomized participants in each cohort were assigned to 1 of 4 blood collection subsets for immunogenicity assessments. Once the subsets were enrolled, subsequently enrolled participants were not assigned to a subset. Blood collections for immunogenicity samples from all participants were scheduled on days 1, 15, 121, 181, and 361. Immunogenicity samples were collected from subsets (n=27) on days 2, 8, 29, and 71. Immunogenicity Assessments Venous blood samples were collected for assessment of humoral immunogenicity via a psVNA validated and performed by Monogram Biosciences. In brief, HIV-1 pseudovirions expressing SARS-CoV-2 spike protein of the reference strain or VOCs (Beta, Delta, and Omicron variants) were prepared by co-transfecting HEK293 producer cells with an HIV-1 genomic vector and a SARS-CoV-2 envelope expression vector. Inhibition of luciferase activity in angiotensin-converting enzyme 2 receptor–expressing target cells, mediated by functional anti-SARS-CoV-2 antibodies with neutralizing activity, was assessed. Antibody titers were reported as the reciprocal of the serum dilution/concentration conferring 50% inhibition (IC50) of pseudovirus infection. A sample was considered positive if neutralizing antibody titers were >LLOQ for each psVNA measurement. A participant was considered a responder if at least one of the following pre-booster conditions was satisfied: pre–primary vaccination titer was <LLOQ and post–primary vaccination titer was ≥4×LLOQ; pre–primary vaccination titer was >LLOQ and post–primary vaccination titer was ≥4×pre-primary vaccination titer. After the booster, a participant was considered a responder if at least one of the following post-booster conditions was satisfied: pre-booster vaccination titer was <LLOQ and post-booster vaccination titer was ≥4×LLOQ; pre-booster vaccination titer was >LLOQ and post-booster vaccination titer was ≥4×pre-booster vaccination titer. Safety Assessments Solicited local and systemic AEs were collected for 7 days after vaccination through an electronic diary, and unsolicited AEs were recorded for 28 days after vaccination. SAEs, AEs leading to study or vaccine discontinuation, and AEs of special interest (ie, TTS (11)) were recorded throughout the study. Analysis Sets and Statistical Analysis The full analysis set (FAS) for safety/reactogenicity analyses included all participants with documented administration of Ad26.COV2.S in this study. The PPI population included all vaccinated participants for whom post-baseline immunogenicity data were available but excluded participants with major protocol deviations expected to impact the immunogenicity outcomes. Samples obtained after natural SARS-CoV-2 infection were excluded. The NI analysis set for hypothesis testing included all PPI participants who were SARS-CoV-2 seronegative at pre-boost (based on N-serology). Two participants who had received BNT162b2 primary vaccination were incorrectly enrolled into the 2.5×10¹⁰ vp dose level group of cohort 1. One participant who had received mRNA-1273 (Spikevax, Moderna) primary vaccination was incorrectly enrolled in the 5×10¹⁰ vp dose level group of cohort 2. These participants were excluded from the NI and PPI set but were included in the FAS. The non-powered descriptive NI analyses on other VOCs were also performed on the NI analysis set. The primary immunogenicity endpoints were designed to sequentially evaluate NI of responses after boosting with 3 different doses of Ad26.COV2.S in participants primed with either Ad26.COV2.S (5×10¹⁰ vp) or BNT162b2 versus the neutralizing antibody responses to the primary single-dose regimen of Ad26.COV2.S. Sample size was chosen to provide sufficient power for the NI comparison of 6 hierarchical hypotheses controlling for family- wise error rate at α=0.025. If the primary hypothesis 1a in cohort 1 was met, then the remaining hypotheses were to be tested independently with a Bonferroni correction to correct for type 1 error. If a primary endpoint met an NI criterion, superiority criteria were assessed (exploratory endpoint). No formal statistical testing of safety data was planned. Safety data were analyzed descriptively by vaccine group. Study Approval The protocol and amendments were approved by institutional review boards (Advarra Inc., of Columbia, Maryland, USA, for all sites except the University of Kentucky [University of Kentucky Medical Institutional Review Board] and Massachusetts General Hospital [Mass General Brigham IRB]). All participants provided written informed consent prior to participation. The trial adhered to the principles of the Declaration of Helsinki and to the Good Clinical Practice guidelines of the International Council for Harmonisation. Results Participant Demographics Study enrollment began August 6, 2021, and the primary analysis data cutoff was December 15, 2021. Overall, 1532 participants were randomized and boosted with one of 3 dose levels by group (Figure 7). Among those enrolled in cohort 1, 774 received a homologous boost of Ad26.COV2.S (full analysis set [FAS]; 5×1010 vp, n=330; 2.5×1010 vp, n=328; 1×1010 vp, n=116). Fifteen (1.9%) participants in cohort 1 prematurely discontinued study participation (participant withdrawal, n=8; lost to follow up, n=4; other, n=3). In cohort 2, 758 received a heterologous boost of Ad26.COV2.S (FAS, 5×1010 vp, n=326; 2.5×1010 vp, n=326; 1×1010 vp, n=106). Eight (1.1%) participants in cohort 2 discontinued study participation (lost to follow-up, n=3; physician decision, n=1; participant withdrawal, n=4). The majority of participants in both cohorts were white (>80%); more participants in cohort 1 were over the age of 60 compared with those in cohort 2 (Table 1).

Table 1. Summary of Participant Demographics and Characteristics (Full Analysis Seta)

^aThe full analysis set included all participants with a documented study vaccine administration (Ad26.COV2.S). bAt the 2.5×10¹⁰ vp dose level, 2 participants were incorrectly enrolled in cohort 1, as they had received 2 doses of Pfizer BNT162b2 vaccine as their primary vaccination. cAt 5×10¹⁰ vp dose level, 1 participant was incorrectly enrolled in cohort 2, as the participant had received 2 doses of Moderna mRNA-1273 as their primary vaccination. dRace and ethnicity were self-reported by participants. eBody-mass index (BMI) is the weight in kilograms divided by the square of the height in meters. Total number of participants with available BMI measurements differed in the 5×10¹⁰ vp group (n=328) and the 2.5×10¹⁰ vp group (n=327) of cohort 1. fProphylactic use of antipyretics was not permitted, but investigators/study staff were permitted to advise participants to take antipyretics at the first sign of solicited symptoms post-vaccination and document medication usage. Abbreviations: vp, viral particles. Following booster administration, 12.5% of participants in cohort 1 used analgesics or antipyretics compared with 30.2% in cohort 2, with ibuprofen and paracetamol being those most commonly used. The median time between primary vaccination and booster vaccination ranged between 206 and 283 days Table 2). Table 2. Interval between Primary Vaccination and Booster Vaccination (Per Protocol Immunogenicity Set)

Homologous Boost – Primary Objectives Primary objectives 1a, 1c, and 1d were to demonstrate NI of Day 15 neutralizing antibody responses to the reference strain after a booster dose of 5×1010 vp, 2.5×1010 vp, and 1×1010 vp Ad26.COV2.S, respectively, compared with Day 29 responses after primary vaccination (5×1010 vp Ad26.COV2.S). Primary objective 1b was to demonstrate NI of the Day 15 neutralizing antibody response to the leading VOC (Delta) elicited by a booster dose of 5×1010 vp versus the Day 29 responses following Ad26.COV2.S primary vaccination. Responder rates met statistical NI if the two-sided 100*(1-2*α)% CI of the difference between post-booster and post-prime responder rate was above -10%. The geometric mean ratio (GMR) of post-booster geometric mean titer (GMT) to post-prime GMT met statistical NI if the two-sided 100*(1-2*α)% CI of the estimated GMR was above 0.67 and GMR >0.8.NI testing of the primary objectives for homologous boosting was conducted in the NI set of cohort 1, which included all baseline-seronegative participants in the per protocol immunogenicity (PPI) set. NI criteria, in terms of responder rates and GMRs, were met for all 4 primary objectives (Table 3). Corresponding GMTs (95% CI), responder rates, and seropositivity rates are presented for each endpoint, ie, Day 29 post-prime compared with Day 15 post-boost.

Table 3. Non-inferiority Assessment of Neutralizing Antibody Titers (psVNA IC₅₀) by 14 Days Post–homologous (Objectives 1a-1d) or Heterologous (Objectives 2a-2d) Ad26.COV2.S Boost (Day 15; Non-inferiority Analysis Seta)

^aThe NI analysis set included all PPI participants who were SARS-CoV-2 seronegative at baseline (based on the serological test for SARS-CoV-2–specific nucleocapsid antibodies [N-serology]). bTiter geometric mean ratio was calculated by dividing the post-booster geometric mean titer by the post– primary geometric mean titer. Abbreviations: GMR, geometric mean ratio; IC50, half maximal inhibitory concentration; PPI, per protocol immunogenicity population; psVNA, pseudovirus neutralization assay; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; vp, viral particle. After primary vaccination with 5×10¹⁰ vp Ad26.COV2.S, the Day 29 GMT against the reference strain (objective 1a; 5×10¹⁰ vp booster dose group) was 102 (88–118), with a responder rate of 15.9% (12.1–20.4) and seropositivity rate of 47.7% (42.1–53.3). By Day 15 after the 5×10¹⁰ vp homologous boost, the GMT was 1130 (989–1291). Responder and seropositivity rates increased to 63.4% (57.7–68.9) and 96.6% (93.9–98.4), respectively. GMTs against the Delta variant (objective 1b; 5×10¹⁰ vp booster dose group) were below the lower limit of quantitation (LLOQ) on Day 29 after primary vaccination, with a responder rate of 9.4% (6.4–13.2) and seropositivity rate of 21.8% (17.4–26.7). By Day 15 following the booster dose, the GMT was 471 (411–539). Responder and seropositivity rates increased to 56.7% (50.9–62.4) and 91.9% (88.3–94.8), respectively. In the 2.5×10¹⁰ vp booster dose group (objective 1c), post-prime Day 29 GMT against the reference strain was 99 (86–113), with a responder rate of 16.6% (12.7–21.1) and seropositivity rate of 48.4% (42.8–54.1). By Day 15 after booster vaccination, the GMT was 915 (792–1058). Responder and seropositivity rates increased to 57.9% (52.1–63.6) and 94.9% (91.8–97.1), respectively. In the 1×10¹⁰ vp booster dose group (objective 1d), GMT against the reference strain was <LLOQ by Day 29 after primary vaccination, with a responder rate of 11.3% (6.2–18.6) and a seropositivity rate of 40.9% (31.8–50.4). The GMT at Day 15 following the booster increased to 734 (564–954), with increases also observed in responder rate (64.5% [53.9– 74.2]) and seropositivity rate (91.4% [83.8–96.2]). Homologous Boost – Secondary and Exploratory Objectives The neutralizing immune responses against VOCs at the lower booster dose levels (2.5×10¹⁰ vp and 1×10¹⁰ vp) were assessed as secondary objectives. GMTs against Delta at Day 29 (post-prime) were <LLOQ, as were immediate pre-boost GMTs. By Day 15 following boosting, GMTs against Delta in the 2.5×10¹⁰ vp Ad26.COV2.S and 1×10¹⁰ vp booster dose level groups were 391 (335–456) and 316 (244–409), respectively. Responder rates were 49.2% (43.3–55.0) and 51.6% (41.0–62.1), with seropositivity rates of 89.6% (85.5–92.8) and 86.0% (77.3–92.3). GMTs against Beta were <LLOQ at Day 29 (post-prime), with responder rates of 2.2% (0.9–4.5), 1.9% (0.7–4.0), and 0% (0–3.2) at the 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp dose levels, respectively; seropositivity rates were 5.3% (3.1–8.4), 4.1% (2.2–6.8), 1.7% (0.2–6.1). At Day 15 post-boost, all dose levels elicited a significant increase in neutralizing antibodies against Beta (GMTs: 5×10¹⁰ vp, 274 [240–314]; 2.5×10¹⁰ vp, 237 [205–275]; 1×10¹⁰ vp, 156 [125–194]), and increases in responder rates (5×10¹⁰ vp, 40.9% [35.3–46.8]; 2.5×10¹⁰ vp, 32.0% [26.7–37.6]; 1×10¹⁰ vp, 18.3% [11.0–27.6]) and in seropositivity rates (5×10¹⁰ vp, 81.5% [76.7–85.8]; 2.5×10¹⁰ vp, 77.4% [72.3–82.1]; 1×10¹⁰ vp, 72.0% [61.8– 80.9]). The GMT against Omicron at Day 29 (post-prime) was also <LLOQ, with responder and seropositivity rates of 0%. Omicron was generally resistant to neutralization after the booster, with a Day 15 GMT of 82 (<LLOQ–110), responder rate of 13.3% (5.1–26.8), and seropositivity rate of 53.3% (37.9–68.3) at the 5×10¹⁰ vp booster dose level. Data were available from too few participants (n = 5) in the 2.5×10¹⁰ vp and 1×10¹⁰ vp dose level groups to evaluate responses at the lower dose levels. Neutralizing antibody responses against the reference strain and VOCs were assessed at Day 29 post-boost in a subset of participants (n≈27) and were consistent with Day 15 results. At Day 29 after boosting, GMTs against the reference strain were 993 (688–1434), 893 (546–1459), and 887 (559–1408) at the 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp dose levels, respectively. Responder rates and seropositivity rates were 68.8% (50.0–83.9) and 96.9% (83.8–99.9) in the 5×10¹⁰ vp group, 63.6% (45.1–79.6) and 93.9% (79.8–99.3) in the 2.5×10¹⁰ vp group, and 75.0% (53.3–90.2) and 95.8% (78.9–99.9) in the 1×10¹⁰ vp group. By Day 29 post-boost, GMTs against Delta were 455 (320–647), 328 (197–545), and 333 (183–608) at the 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp dose levels, respectively. Responder and seropositivity rates against Delta at Day 29 following the homologous booster were 59.4% (40.6–76.3) and 96.9% (83.8–99.9) in the 5×10¹⁰ vp group, 36.4% (20.4–54.9) and 87.9% (71.8–96.6) in the 2.5×10¹⁰ vp group, and 62.5% (40.6–81.2) and 83.3% (62.6– 95.3) in the 1×10¹⁰ vp group. Day 29 post-boost GMTs against Beta were 269 (188–384), 201 (127–321), and 190 (116–311). Responder and seropositivity rates against Beta at Day 29 post-boost were 40.6% (23.7–59.4) and 87.5% (71.0–96.5) in the 5×10¹⁰ vp group, 18.2% (7.0–35.5) and 75.8% (57.7–88.9) in the 2.5×10¹⁰ vp group, and 29.2% (12.6–51.1) and 75.0% (53.3–90.2) in the 1×10¹⁰ vp group. GMTs against Omicron at Day 29 post-boost were 74 (<LLOQ–104) at the 5×10¹⁰ vp dose level. The responder and seropositivity rates against Omicron at Day 29 post-boost were 6.7% (0.8–22.1) and 50.0% (31.3–68.7), respectively, in the 5×10¹⁰ vp group. A small proportion of cohort 1 participants (15/755; 2%) were SARS-CoV-2 seropositive pre–homologous booster on Day 1 of this study. Seropositive participants generally had higher GMTs against the reference strain and the Delta and Beta variants both pre-boost and at Day 15 compared with seronegative participants. By Day 15 after the homologous boost, the majority of seropositive participants exceeded the upper limit of quantitation (ULOQ). Overall, a trend for lower titers and responder rates against the reference strain in participants aged ≥60 years, compared with younger adults, was observed at the 2.5×10¹⁰ vp and 1×10¹⁰ vp booster dose levels, but not for the 5×10¹⁰ vp dose level. Trends for neutralizing antibody titers, seropositivity, and responder rates suggest dose-response for the Beta variant in both younger and older adults (≥60 years) and in older adults for the Delta variant. Overall, responses against VOCs were generally lower in older adults compared to younger. This study was not powered for superiority testing, but it was prespecified that if any of the primary objectives met NI criteria they would be tested for superiority. The 95% CI for objectives 1a and 1b and the 97.5% CI for objectives 1c and 1d excluded 1 and 0, respectively, indicating that superiority criteria were met for the Day 15 post-boost response in terms of GMR and difference in responder rate compared to primary vaccination (Table 3). Heterologous Boost – Primary Objectives Primary objectives 2a, 2c, and 2d were to demonstrate the NI of Day 15 neutralizing antibody response to the reference strain after a booster dose of 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp Ad26.COV2.S, respectively, compared with Day 29 post–primary vaccination responses following two doses of BNT162b2. Primary objective 2b was to demonstrate NI of the Day 15 neutralizing antibody response to the leading VOC (Delta) elicited by a booster dose of 5×10¹⁰ vp versus Day 29 post–primary vaccination (BNT162b2) responses. Baseline neutralizing antibody titers for cohort 2 participants prior to primary vaccination with BNT162b2 were not available, and comparisons to pre-boost responses were based on seropositivity rates, rather than responder rates, in samples collected from external suppliers. NI criteria in terms of neutralizing antibody responses were met for all 4 cohort 2 primary objectives (Table 3). Within 14–60 days after BNT162b2 primary vaccination, GMT against the reference strain (objective 2a; 5×10¹⁰ vp booster dose group) in cohort 2 was 1291 (1095–1521), with a seropositivity rate of 92.0 (88.4–94.7). By Day 15 after the heterologous boost, the GMT was 4439 (4027–4893). Responder and seropositivity rates reached 97.0% (94.3–98.6) and 100.0% (98.8–100.0), respectively. GMTs against the Delta variant (objective 2b; 5×10¹⁰ vp booster dose group) were 507 (426–604) after BNT162b primary vaccination, with a seropositivity rate of 83.9% (79.4–87.8). At Day 15 post-boost, the GMT increased to 2318 (2049–2623). Responder and seropositivity rates were 93.6% (90.2–96.1) and 99.7% (98.2–100.0), respectively. After boosting with 2.5×10¹⁰ vp, the Day 15 GMT against the reference strain (objective 2c) was 3566 (3212–3958), with a responder rate of 90.5% (86.5–93.6) and seropositivity rate of 100.0% (98.8–100.0). By Day 15 after the heterologous boost with 1×10¹⁰ vp Ad26.COV2.S, the GMT against the reference strain (objective 2d) was 3218 (2582–4010), with a responder rate of 89.8% (81.5–95.2) and seropositivity rate of 98.9% (93.8–100). Overall, the neutralizing antibody response against the reference strain increased with increasing booster dose level. Heterologous Boost – Secondary and Exploratory Objectives As in cohort 1, the neutralizing antibody responses against the Delta variant elicited by lower booster dose levels were assessed as secondary objectives. By Day 15 after the heterologous boost, GMTs against Delta in the 2.5×10¹⁰ vp Ad26.COV2.S and 1×10¹⁰ vp Ad26.COV2.S dose level groups were 1872 (1646–2129) and 1761 (1372–2261), respectively. Responder rates were 91.2% (87.3–94.1) and 86.4% (77.4–92.8), with seropositivity rates of 100.0% (98.8–100.0) and 98.9% (93.8–100.0). The GMT against Beta was 142 (122–165) following primary vaccination with BNT162b2, with a seropositivity rate of 53.7% (48.0–59.3). By Day 15, heterologous boosting at all dose levels elicited a significant increase in neutralizing antibodies (5×10¹⁰ vp, 1649 [1432–1900]; 2.5×10¹⁰ vp, 1310 [1135–1511]; 1×10¹⁰ vp, 1055 [787–1412]), responder rates (5×10¹⁰ vp, 88.2% [84.0–91.7]; 2.5×10¹⁰ vp, 82.7% [77.8–86.8]; 1×10¹⁰ vp 78.4% [68.4–86.5]), and seropositivity rates (5×10¹⁰ vp, 97.7% [95.2–99.1]; 2.5×10¹⁰ vp, 98.0 [95.6– 99.3]; 1×10¹⁰ vp, 94.3% [87.2–98.1]). After primary vaccination, the GMT against Omicron was <LLOQ (evaluated ≥6 months post-prime); by Day 15 post-boost with 5×10¹⁰ vp Ad26.COV2.S, the GMT against Omicron was 526 (357–776), with responder and seropositivity rates of 68.9% (53.4–81.8) and 95.6% (84.9–99.5). In the subset of samples analyzed for neutralizing antibody responses at Day 29 following heterologous boosting, GMTs against the reference strain were 6221 (4905–7890), 4808 (3496–6612), and 4410 (3067–6341) at the 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp booster dose levels. Responder rates were 100% (90.0–100.0), 97.0% (84.2–99.9), and 95.7% (78.1–99.9); seropositivity rates were 100.0% across dose levels. By Day 29 after boosting, GMTs against Delta were 3414 (2358–4944), 2348 (1584– 3480), and 1803 (1138–2855) at the 5×10¹⁰ vp, 2.5×10¹⁰ vp, and 1×10¹⁰ vp dose levels, respectively. Responder rates were 97.1% (85.1–99.9) in the 5×10¹⁰ vp group, 97.0% (84.2– 99.9) in the 2.5×10¹⁰ vp group, and 87.0% (66.4–97.2) in the 1×10¹⁰ vp group; seropositivity rates reached 100.0% across dose levels. GMTs against Beta at Day 29 post-boost were lower than observed for Delta (5×10¹⁰ vp, 2144 [1426–3226]; 2.5×10¹⁰ vp, 1614 [1031–2529]; 1×10¹⁰ vp, 1116 [579–2152]). Responder and seropositivity rates against Beta at Day 29 post-boost were 94.3% (80.8–99.3) and 100.0% (90.0–100.0) in the 5×10¹⁰ vp group, 78.8% (61.1–91.0) and 100.0% (89.4– 100.0) in the 2.5×10¹⁰ vp group, and 82.6% (61.2–95.0) and 91.3% (72.0–98.9) in the 1×10¹⁰ vp group. GMTs against Omicron at Day 29 post-boost were 752 (496–1140) at the 5×10¹⁰ vp dose level. The responder rate against Omicron was 77.1% (59.9–89.6), with a seropositivity rate of 100.0% at the 5×10¹⁰ vp dose level. Among the small proportion of participants who were SARS-CoV-2 seropositive pre- boost, the GMTs observed post-boost (Day 15) were higher than those in seronegative participants; however, seropositives also had high GMTs pre-boost, resulting in low GMIs from pre-boost levels at Day 15. Overall, neutralizing antibody titers against the reference strain were higher in younger adults (18–59 years) than in older adults (≥60 years), after heterologous booster vaccination at all 3 dose levels. In general, responder rates and neutralizing antibody titers against variants were also higher in younger adults than in older adults following heterologous boosting at all 3 dose levels. Superiority was also assessed as an exploratory objective for cohort 2. The 97.5% CI of objectives indicated that superiority criteria were met with respect to GMR at 14 days post- boost compared to primary vaccination. For seropositivity rate, the superiority criterion was met for objectives 2a, 2b, and 2c; the 97.5% CI for objective 2d included 0, precluding any conclusions for superiority of the 1x10¹⁰ vp booster dose level (Table 3). Homologous Versus Heterologous Boost Responses to the reference strain were 3.9 and 6.3 times higher in the heterologous group versus the homologous group, by Day 15 and Day 29 post-boost, respectively (Table 4). By Day 15 post-boost, responses against Delta were 4.9 times higher in the heterologous group compared with the homologous group; by Day 29, responses to the heterologous boost were 7.5 times higher than those elicited by the homologous boost. Post-boost Day 15 and Day 29 responses against Beta were 6.0 and 8.0 times higher, respectively, for heterologous boosting. Heterologous boosting also increased responses against Omicron compared with homologous boosting; responses at Day 15 and Day 29 were 6.4 and 10.2 times greater, respectively, in the heterologous group. Overall, independent of variant tested, the heterologous boost elicited higher neutralizing antibody responses than did the homologous boost, even at the lowest booster dose level. Trends toward higher responses observed in the heterologous versus homologous boosting group were also independent of age. Table 4. Comparison of Neutralizing Antibody Geometric Mean Titers at 14 Days Post- Boost (Day 15) or 28 Days Post-Boost (Day 29) With 5×10¹⁰ vp Ad26.COV2.S Against the SARS-CoV-2 Reference Strain and Variants of Concern

severe acute respiratory syndrome coronavirus-2. Safety In general, after both homologous and heterologous boosting in both cohorts, the frequency of solicited local and systemic AEs was lower in participants aged ≥60 years compared to participants aged ≥18 to 59 years at all dose levels. Most unsolicited AEs were grade 1 or 2 in severity. There were no serious AEs (SAEs) related to the study vaccine reported in cohort 1, whereas 1 participant in the 2.5×10¹⁰ vp group of cohort 2 reported 5 related SAEs (asthenia, headache, nausea, fatigue, and myalgia, all grade 3; asthenia onset occurred 1 day after the booster and led to hospitalization; all SAEs reported by this participant had resolved at the time of the analysis). No participant reported an event that met the pre–established criteria for thrombosis with thrombocytopenia syndrome (TTS; defined as a thrombotic/thromboembolic event in conjunction with a platelet count below 150,000/µL) (12); however, 6 events of thrombocytopenia (without thrombosis) were reported in 5 participants in the 5×10¹⁰ vp group of cohort 1. Day 15 platelet counts of the thrombocytopenia cases ranged from 62,000/µL to 180,000/µL. No deaths or unsolicited AEs leading to study discontinuation were reported in either cohort. Summary Each of the 3 booster dose levels examined met prespecified hierarchical non- inferiority and superiority criteria. By Day 15 post–homologous boost with 1×10¹⁰, 2.5×10¹⁰, or 5×10¹⁰ vp, geometric mean titers (GMTs [95% CI]) against the reference strain increased dose-dependently to 734 (564–954), 915 (792–1058), and 1130 (989–1291), respectively. By Day 15 post–heterologous boost, GMTs against the reference strain were higher than post– homologous boost, with dose-dependent increases to 3218 (2582–4010), 3566 (3212–3958), and 4439 (4027–4893), respectively. Dose responses to a booster dose were observed for nAb titers against all tested VOCs. After boosting, nAb titers were generally higher in younger versus older adults. Boosting was well tolerated, although solicited systemic adverse events were more frequent following heterologous (64.2%, 71.2%, and 81.9%) versus homologous boosting (51.7%, 49.4%, and 57.0%). Conclusion: Ad26.COV2.S boosts nAb responses to the reference strain and VOCs in individuals who completed primary vaccination with Ad26.COV2.S or BNT162b2.

Claims

CLAIMS 1. A use of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for boosting the immune response against SARS CoV-2 in a subject which has received a primary vaccination against SARS CoV-2, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp dose and is administered at least 6 months after receiving said primary vaccination.

2. The use according to claim 1, wherein the primary vaccination consists of administering said recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1.

3. The use according to claim 1 or 2, wherein the primary vaccination comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp of said recombinant adenovirus.

4. The use according to claim 3, wherein the primary vaccination dose comprises 5.0 x 10¹⁰ vp of said recombinant adenovirus.

5. The use according to any one of claims 1 to 4, wherein the primary vaccination consists of administering two doses of said recombinant adenovirus.

6. The use according to claim 5, wherein the two doses are administered about 2 to about 3 months apart.

7. The use according to any one of claims 1 to 6, wherein the booster dose comprises 1.25 x 10¹⁰ vp of said recombinant adenovirus.

8. The use according to any one of claims 1 to 6, wherein the booster dose comprises 5 x 10¹⁰ vp of said recombinant adenovirus.

9. The use according to any one of claims 1 to 8, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2.

10. The use according to any one of claims 1 to 9, wherein the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.

11. The use according to claim 10, wherein the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 5.

12. The use according to any one of claims 1 to 11, wherein the recombinant adenovirus comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4.

13. The use according to any one of claims 1 to 12, wherein the recombinant adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 region of the adenoviral genome.

14. The use according to any one of claims 1 to 13, wherein the recombinant adenovirus is a recombinant human adenovirus of serotype 26.

15. The use according to any one of claims 1 to 14, wherein the recombinant adenovirus is administered intramuscularly.

16. The use according to any one of claims 1 to 15, wherein the immune response comprises the induction of SARS CoV-2 S protein binding antibodies and/or SARS CoV-2 neutralizing antibodies.

17. The use according to claim 16, wherein the level of SARS CoV-2 S protein binding antibodies is increased at least 3-fold at 7 days after the booster dose as compared to pre-boost levels.

18. The use according to claim 16, wherein the level of SARS CoV-S protein binding antibodies is increased 6-fold at 28 days after the booster dose as compared to pre- boost levels.

19. The use according to claim 16, wherein the level of SARS CoV-S protein binding antibodies is increased at least 9-fold at 7 days after the booster dose as compared to the level of SARS CoV-S protein binding antibodies 28 days after the primary vaccination.

20. The use according to any one of the claims 1 to 19, wherein the subject is 18 years or older.

21. The use according to any one of the claims 1 to 19, wherein the subject is 65 years or older.

22. The use according to any one of the claims 1 to 20, wherein the booster dose is administered at least 12 months after the primary vaccination.

23. The use according to any one of the claims 1 to 20, wherein the booster dose is administered at least 18 months after the primary vaccination.

24. The use according to any one of the claims 1 to 20, wherein the booster dose is administered at least 24 months after the primary vaccination.

25. A method for boosting the immune response against SARS CoV-2 in a subject having received a primary vaccination against SARS CoV-2, said method comprising administering a booster dose of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1, wherein the booster dose comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp and is administered at least 6 months after receiving said primary vaccination.

26. The method according to claim 25, wherein the primary vaccination consists of administering said recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1.

27. The method according to claim 25 or 26, wherein the primary vaccination comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp of said recombinant adenovirus.

28. The method according to claim 27, wherein the primary vaccination dose comprises 5.0 x 10¹⁰ vp of said recombinant adenovirus.

29. The method according to any one of claims 25 to 28, wherein the primary vaccination consists of administering two doses of said recombinant adenovirus.

30. The method according to claim 29, wherein the two doses are administered about 2 to about 3 months apart.

31. The method according to any one of claims 25 to 30, wherein the booster dose comprises 1.25 x 10¹⁰ vp of said recombinant adenovirus.

32. The method according to any one of claims 25 to 30, wherein the booster dose comprises 5 x 10¹⁰ vp of said recombinant adenovirus.

33. The method according to any one of claims 25 to 32, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2.

34. The method according to any one of claims 25 to 33, wherein the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.

35. The method according to claim 34, wherein the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 5.

36. The method according to any one of claims 25 to 35, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 4.

37. The method according to any one of claims 25 to 36, wherein the recombinant adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 region of the adenoviral genome.

38. The method according to any one of claims 25 to 37, wherein the recombinant adenovirus is a recombinant human adenovirus of serotype 26.

39. The method according to any one of claims 25 to 38, wherein the recombinant adenovirus is administered intramuscularly.

40. The method according to any one of claims 25 to 39, wherein the immune response comprises the induction of SARS CoV-2 S protein binding antibodies and/or SARS CoV-2 neutralizing antibodies.

41. The method according to claim 40, wherein the level of SARS CoV-2 S protein binding antibodies is increased at least 3-fold at 7 days after the booster dose as compared to pre-boost levels.

42. The method according to claim 40, wherein the level of SARS CoV-2 S protein binding antibodies is increased 6-fold at 28 days after the booster dose as compared to pre-boost levels.

43. The method according to claim 40, wherein the level of SARS CoV-S protein binding antibodies is increased at least 9-fold at 7 days after the boost dose as compared to the level of SARS CoV-S protein binding antibodies 28 days after the primary vaccination.

44. The method according to any one of the claims 25 to 43, wherein the subject is 18 years or older.

45. The method according to any one of the claims 25 to 43, wherein the subject is 65 years or older.

46. The method according to any one of the claims 25 to 45, wherein the booster dose is administered at least 12 months after the primary vaccination.

47. The method according to any one of the claims 25 to 45, wherein the booster dose is administered at least 18 months after the primary vaccination.

48. The method according to any one of the claims 25 to 45, wherein the booster dose is administered at least 24 months after the primary vaccination.

49. A method of inducing an immune response to SARS CoV-2 in a subject, the method comprising: a. administering to the subject a first composition comprising an immunologically effective amount of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 1 for priming the immune response against SARS CoV-2 in the subject; and b. administering to the subject a second composition comprising an immunologically effective amount of a recombinant adenovirus comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 2 for boosting the immune response against SARS CoV-2 in the subject.

50. The method according to claim 49, wherein the first composition comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp.

51. The method according to claim 50, wherein the first composition comprises 5 x 10¹⁰ vp.

52. The method according to any one of claims 49 to 51, wherein the second composition comprises between 1.0 x 10¹⁰ and 1 x 10¹¹ vp.

53. The method according to claim 52, wherein the second composition comprises 1.25 x 10¹⁰ vp.

54. The method according to claim 52, wherein the second composition comprises between 5.0 x 10¹⁰ vp.

55. The method according to any one of claims 49 to 54, wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 2.

56. The method according to any one of claims 49 to 55, wherein the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.

57. The method according to claim 56, wherein the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 5.

58. The method according to any one of claims 49 to 57, wherein the recombinant adenovirus comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4.

59. The method according to any one of claims 49 to 58, wherein the recombinant adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 region of the adenoviral genome.

60. The method according to any one of claims 49 to 59, wherein the recombinant adenovirus is a recombinant human adenovirus of serotype 26.

61. The method according to any one of claims 49 to 60, wherein the recombinant adenovirus is administered intramuscularly.

62. The method according to any one of claims 49 to 61, wherein the immune response comprises the induction of SARS CoV-2 S protein binding antibodies and/or SARS CoV-2 neutralizing antibodies.

63. The method according to claim 62, wherein the level of SARS CoV-2 S protein binding antibodies is increased at least 3-fold at 7 days after the administration of the second composition as compared to prior to the administration of the second composition.

64. The method according to claim 62, wherein the level of SARS CoV-S protein binding antibodies is increased 6-fold at 28 days after the administration of the second composition as compared to prior to the administration of the second composition.

65. The method according to claim 62, wherein the level of SARS CoV-S protein binding antibodies is increased at least 9-fold at 7 days after the second composition is administered as compared to the level of SARS CoV-S protein binding antibodies 28 days after the administration of the first composition.

66. The method according to any one of the claims 49 to 65, wherein the subject is 18 years or older.

67. The method according to any one of the claims 49 to 65, wherein the subject is 65 years or older.

68. The method according to any one of the claims 49 to 67, wherein the second composition is administered at least 12 months after the first composition.

69. The method according to any one of the claims 49 to 67, wherein the second composition is administered at least 18 months after the first composition.

70. The method according to any one of the claims 49 to 67, wherein the second composition is administered at least 24 months after the first composition.