FIELD OF THE INVENTION

This invention relates to biotechnology. More particularly, to methods of inducing an immune response against at least a first and a second antigen in a subject in need thereof comprising administering to the subject a first adenovirus serotype 26 (Ad26 vector) comprising a nucleic acid sequence encoding a first antigen and administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, wherein the first and second antigens are different antigens, and wherein the second Ad26 vector is administered to the subject at least two weeks after the first Ad26 vector. The methods can further comprise subsequent administration of one or more nucleic acid molecules or vectors encoding the first and/or second antigen or one or more polypeptides comprising the first and/or second antigen to the subject.

BACKGROUND OF THE INVENTION

Adenovirus 26 (Ad26) vectors have been investigated in the development of vaccines for multiple infectious diseases. Two vaccines based on Ad26, which belongs to species D, are currently widely authorized; ZABDENO (in combination with an MVA component MVBEA) for prevention of Ebola virus disease and Ad26.COV2.S which is used widely for the prevention of COVID-19 disease (Logunov et al., Lancet 397:671-81 (2021); Voysey et al., Lancet 397:99-111 (2021); Sadoff et al., N. Engl. J. Med. 19: 1824-35 (2021)). Other adenovirus vectors like ChAdOxl and Ad5 have been developed and authorized in multiple countries for prevention of COVID-19 (Folegatti et al., Lancet 396:467-78 (2020); Madhi et al., N. Engl. J. Med. 384: 1885-98 (2021)) as well. Multiple further Ad types are explored in the development of vaccine regimens targeting human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), and malaria, amongst others (Gilbert et al., Expert Rev. Vaccines 14: 1347-57 (2015); Baden et al., Lancet HIV 7:e688-e698 (2020); Salisch et al., NPJ Vaccines 4:54 (2019); Widjojoatmodjo et al., Vaccine 33:5406-14 (2015); Tiono et al., PLoS One 13:e0208328 (2018)).

Pre-existing vector-targeting immunity, especially against the same Ad type, has been hypothesized to interfere with vector-mediated delivery of the vaccine-encoded antigens, or to lead to immune-mediated clearance of immunogen-expressing cells. These mechanisms can potentially diminish expression of the antigen and reduce vaccine potency (Fausther- Bovendo et al., Hum. Vaccin. Immunother. 10:2875-84 (2014)). Pre-existing anti-vector immunity can stem from 2 sources: past exposure to the natural (or a cross-reacting) adenovirus, or from a previous vaccination with the same Ad-vector-b ackbone. In either case, pre-existing immunity to the vector may negatively influence vector-based-vaccine-induced immune responses.

Decreased immune responses in the presence of pre-existing vector-targeting immune responses have been documented for some Ad-vectored vaccines, notably type 5 (Ad5). Ad5 belongs to species C and is highly prevalent with high titers in humans, making Ad5 vectorbased vaccines less suitable for widespread use (Barouch et al., Vaccine 29:5203-9 (2011); Priddy et al., Clin. Infect. Dis. 46: 1769-81 (2008); Zhu et al., Lancet 396:479-88 (2020)). Viruses of Ad35 (type B), Ad26 and Ad48 (both type D) type have a comparatively lower natural seroprevalence and low titers in the seropositive, which varies among regions, and may thus make Ad26 interesting for vectorization (Barouch et al., Vaccine 29:5203-9 (2011)).

Published observations from clinical studies using Ad26-based EBOLA, HIV, RSV, and CO VID-19 vaccines have so far not indicated a clear negative impact of wild-type (wt) Ad26 virus-elicited anti-Ad26 neutralizing antibody (Nabs) activity in serum on Ad26- vaccine-induced immune responses after individual vaccine doses (Sadoff et al., N. Engl. J. Med. 19: 1824-35 (2021); Baden et al., Lancet HIV 7:e688-e698 (2020); Baden et al., J. Infect. Dis. 207:240-7 (2013); Baden et al., Ann. Intern. Med. 164:313-22 (2016); Baden et al., J. Infect. Dis. 218:633-44 (2018); Colby et al., Nat. Med. 26:498-501 (2020); Williams et al., J. Infect. Dis. 222:979-88 (2020); Barry et al., PLoS Med. 18:el003813 (2021)). In the presence of vaccine-elicited anti-Ad26 vector Nabs, insert-specific immune response can be boosted upon multidose Ad26 vaccine regimens with the same transgene (Sadoff et al., N. Engl. J. Med. 19: 1824-35 (2021); Ishola et al., Lancet Infect. Dis. 22:97-109 (2022)). For instance, participants in the first-in-human Phase 1 study of Janssen’s COVID-19 vaccine received Ad26.COV2.S at a dose level of 5xl0 ¹⁰ or IxlO ¹¹ vp in a 1- or 2-dose schedule with a 56-day interval between doses. Ad26 titers Nabs elicited post-dose 1 did not correlate with SARS-CoV-2 Nabs 15- and 29-days post dose 2 (Sadoff et al., N. Engl. J. Med. 19: 1824-35 (2021)).

As the number of vaccines based on viral-vector platforms and recipients of viral- vectored vaccines grow, vaccination-elicited seroprevalence will likely increase. In the future, individuals could conceivably receive multiple viral-vectored vaccines with the same vector backbone encoding different antigens over their lifetime. Therefore, it is important to understand the impact of vector-elicited vector-targeting immunity on the insert-specific immunogenicity and ultimately efficacy of subsequently administered vaccines using the same vector.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods for inducing an immune response against at least a first and a second antigen in a subject in need thereof. The methods comprise (a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier; and (b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier; wherein the first and second antigens are different antigens, and wherein the second Ad26 vector is administered to the subject at least two weeks after the first Ad26 vector. In certain embodiments, the second Ad26 vector is administered to the subject at least two (2) weeks after the first Ad26 vector. The second Ad26 vector can, for example, be administered to the subject at about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the first Ad26 vector.

In certain embodiments, the methods further comprise administering to the subject one or more nucleic acid molecules or vectors encoding the first and/or the second antigen or one or more polypeptides comprising the first and/or second antigen after administration of the first and/or second Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen can, for example, be administered to the subject: (a)(i) after administration of the first Ad26 vector and before the administration of the second Ad26 vector; or (a)(ii) at about the same time as the administration of the second Ad26 vector; or (a)(iii) after administration of the second Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen can, for example, be administered to the subject (b) after administration of the second Ad26 vector.

In certain embodiments, the subsequently administered nucleic acid molecule or vector is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi 1, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MVA, mRNA, IVT repRNA, and saRNA. The subsequently administered vector can, for example, be an Ad26 vector. In preferred embodiments, the subsequently administered vector is the same as the first Ad26 vector or the second Ad26 vector. In certain embodiments, the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject at least two (2) weeks after the first Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen can, for example, be administered to the subject at about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the first Ad26 vector.

In certain embodiments, the subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen is administered to the subject at least (2) weeks after the second Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen can, for example, be administered to the subject at about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the second Ad26 vector.

In certain embodiments, the first antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the second antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the first and second Ad26 vectors can be administered in an amount of about 5 x IO ¹⁰ adenoviral vectors, about 6 x IO ¹⁰ adenoviral vectors, about 7 x IO ¹⁰ adenoviral vectors, about 8 x IO ¹⁰ adenoviral vectors, about 9 x IO ¹⁰ adenoviral vectors, or about 1 x 10 ¹¹ adenoviral vectors. The first and second Ad26 vectors can, for example, be administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, by catheter, by lavage, or by gavage. In certain embodiments, the first and second Ad26 vector are administered in an intramuscular injection.

In certain embodiments, the subsequently administered nucleic acid molecule, vector, or polypeptide is administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, by catheter, by lavage, or by gavage. The subsequently administered nucleic acid molecule, vector, or polypeptide can, for example, be administered in an intramuscular injection.

Also provided are kits comprising: (a) a first Ad26 vector encoding a first antigen; and (b) a second Ad26 vector encoding a second antigen; wherein the first and second antigen are different antigens. In certain embodiments, the kit further comprises: (a)one or more nucleic acid molecules or vectors encoding the first antigen or one or more polypeptides comprising the first antigen; and (b) one or more nucleic acid molecules or vectors encoding the second antigen or one or more polypeptides comprising the second antigen.

In certain embodiments, the first antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the second antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the kit comprises about 5 x IO ¹⁰ adenoviral vectors, about 6 x IO ¹⁰ adenoviral vectors, about 7 x IO ¹⁰ adenoviral vectors, about 8 x IO ¹⁰ adenoviral vectors, about 9 x IO ¹⁰ adenoviral vectors, or about 1 x 10 ¹¹ adenoviral vectors of the first and second Ad26 vectors.

The one or more nucleic acid molecules or vectors is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi l, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MV A, mRNA, IVT repRNA, and saRNA. In certain embodiments, the nucleic acid molecule or vector is an Ad26 vector. In certain embodiments, the kit further comprises at least one syringe for injection of the first Ad26 vector, the second Ad26 vector, the one or more nucleic acid molecules or vectors encoding the first antigen or the one or more polypeptides comprising the first antigen, and/or the one or more nucleic acid molecules or vectors encoding the second antigen or the one or more polypeptides comprising the second antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG. 1: Summary of Study Designs and Immunization Regimens. Depicted is a generic study design of the 3 studies conducted in non-human primates (NHPs). Ad26 vaccine vectors administered during A series either as a homologous or heterologous regimen with Ad35 or MVA was followed 26 to 57 weeks later by B series of the same vectorbackbone regimen. In all studies the vaccine vectors given during A series and B series administration encoded different antigens.

In study 1, animals (n=5 per group) pre-exposed to Ad26 or Ad35 vaccine vectors encoding a RSV.FA2 antigen were dosed 55 weeks after the last vaccination in the A series with the same sequence of Ad26 or Ad35 vaccine vectors encoding ZEBOV.GP in B series (dose 1, week 0 and dose 2, week 8). Control animals received the same ZEBOV.GP - expressing homologous (n=4) or heterologous (n=5) regimens at week 0 and week 8 in B series. Details of the study design are provided in FIG. 2A.

In study 2, animals (n=6 per group) pre-exposed to Ad26 and MVA vaccine vectors encoding RSV.FA2 antigen were dosed 26 weeks after the last MVA vaccination (A series) with the same sequence of Ad26 or MVA vectors, with Ad26 encoding SUDV.GP (dose 1, week 0) and MVA-BN-Filo (dose 2, week 8), respectively during the B series. Control animals received the same SUDV.GP-expressing vaccines at week 0 and week 8 in B series. Details of the study design are provided in FIG. 2B.

In study 3, animals (n=6 per group) pre-exposed to MVA-BN-Filo and Ad26 vaccine vectors encoding ZEBOV.GP were dosed 57 weeks after the last vaccination in the A series with the same sequence of Ad26 or MVA vectors encoding a mosaic of HIV Env, Gag, and Pol antigens (dose 1, week 0 and dose 2, week 12). Control animals received the same vaccine (Ad26 then MV A) at week 0 and week 12. Details of the study design are provided in FIG. 2C.

Peripheral blood mononuclear cells and serum were collected over the course of the study for immunological assays.

The dose of Ad26 and Ad35 vaccine vectors was 5xlO ¹⁰ VP and the dose of MVA was 10 ⁸ IFU. All vaccines were administered by intramuscular injection.

FIGs. 2A-2C: Immunization regimens, timelines and sampling time points per study. Filled circles represent a sampling time point. (FIG. 2A) Study 1: During the A series animals received homologous Ad26/Ad26 or heterologous Ad26/Ad35 viral vectors encoding RSV.FA2 (groups referred to as “Ad26/Ad26 repeat [rep]” and “26/35 rep,” respectively) or were untreated (groups referred to as “26/26” and “26/35,” respectively. Fifty -five weeks later, animals received homologous Ad26/Ad26 or heterologous Ad26/Ad35 viral vectors encoding ZEBOV GP antigen in the B series. (FIG. 2B) Study 2: During the A series animals received heterologous Ad26/MVA viral vectors encoding RSV.FA2 (group referred to as “26/MV Arep”) or were untreated (group referred to as “26/MV A”). Twenty-six weeks later, animals received heterologous Ad26 viral vector encoding SUDV (Sudan Gulu) GP antigen and MVA encoding SUDV, ZEBOV GP, MARV GP (MVA-BN-Filo) (B series) (FIG. 2C) Study 3: During the A series animals received heterologous MVA-BN-Filo vector and Ad26 vector encoding ZEBOV GP (group referred to as “26/MV Arep”) or were untreated (group referred to as 26/MV A”). Fifty-seven weeks later, animals received heterologous Ad26.Mos4.HIV and MVA.mBN414A viral vectors encoding HIV Env, Gag, and Pol antigens (B series).

FIGs. 3A-3D: Anti-Ad26 vector responses in animals dosed with Ad26 vectors. (FIGs. 3 A-3B) Ad26 neutralizing antibody titers were determined using an Ad26-based virus neutralization assay with sera obtained from animals in the indicated groups from study 1. Each symbol represents one animal (filled circle, animals dosed in series A with Ad26; open circle, animals not dosed in series A). Dotted lines depict upper and lower limit of detection respectively (ULoD: loglO highest serum dilution in the assay, 1/65536 for Ad26; LLoD; loglO lowest serum dilution in the assay, 1/64 for Ad26). N = 4-5 animals per group. (FIGs. 3C-3D) Ad26 Hexon-specific T-cell responses as measured by IFNy ELISpot using PBMCs stimulated with peptide pools covering Ad26 hexon sequences. The dotted line corresponds to a threshold of 50 spot-forming units (SFU)/10 ⁶ PBMCs. The lines represent the mean group response. Animals that had received an Ad26 and/or Ad35 vaccine during the A series are annotated as Ad26/Ad26rep or Ad26/Ad35rep. FIGs. 4A-4D: Ad26 neutralizing antibodies and Hexon-specific T-cell responses in Cynomolgus macaques receiving multiple doses of Ad26 viral vectors. (FIGs. 4A-4B) Ad26 neutralizing antibody responses from animals in studies 2 and 3. Each symbol represents one animal. (FIG. 4 A) Study 2: The two dotted lines depict the lower limit of detection. The upper line corresponds to a start dilution of 125 (2.11ogl0) for serum samples at week -22 and week 0, and the lower line to a start dilution of 64 (1.8 Ilog 10) for the remaining serum samples. (FIG. 4B) Study 3 : Results depicted are from baseline (week -2) prior to dosing with Ad26.Mos4.HIV, and post dosing with Ad26 and MVA encoding multiple HIV antigens at week 12 and 14. The dotted line depicts the lower limit of detection and corresponds to a serum start dilution of 32 (1.5 lloglO). The lines represent the group mean. (FIGs. 4C-4D) Hexon-specific T-cell responses as measured by IFNy. ELISpot using PBMCs stimulated with peptide pools covering Ad26 and Ad35 hexon sequences (study 1). The dotted line corresponds to a threshold of 50 spot-forming units (SFU)/10 ⁶ PBMCs. The lines represent the mean group response. Animals that had received an Ad26 and/or Ad35 and/or MVA, vaccine during the A series are annotated as Ad26/Ad26rep, Ad26/Ad35rep, or Ad26/MVArep.

FIGs. 5A-5E: Ad26 vaccine-elicited vector-immunity has minor impact on cellular immune responses induced by a first dose of the B series Ad26 vaccines in Cynomolgus macaques. IFNy ELISPOT responses in PBMC (FIGs. 5 A-5D) were determined after stimulation with pools of 15-mer peptides overlapping by 11 amino acids, covering the protein sequence of ZEBOV GP (ZEBOV) (FIG. 5 A, 5B), SUDAN GP (SUDV) (FIG. 5C), Env, Gag and Pol (FIG. 5D). Shown are background subtracted values per animal, of animals receiving Ad26 (FIGs. 5A-5B) regimen encoding ZEBOV GP, (FIG. 5C) regimen encoding SUDV GP, (FIG. 5D), regimen encoding Mos4.HIV. Animals that had received an Ad26 vaccine during the A series are annotated as Ad26/Ad26rep (FIG. 5 A), Ad26/Ad35rep (FIG. 5B), Ad26/MVArep (FIGs. 5C-5D). In FIGs. 5A to 5D the dotted line represents the assay threshold of 50 spot-forming units (SFU)/10 ⁶ PBMC. Horizontal bars depict geometric group means. In (FIG. 5E) the fold change in IFNy SFU/10 ⁶ PBMCs is depicted and corresponds to the change in response comparing pre-dose 1 to peak response post-dose 1 per animal. Ad26rep refers to animals vaccinated in the A series, and Ad26 to animals that were not vaccinated during the A series. The horizonal line is the geometric mean. For all three studies the baseline (pre-dose 1) was defined as the timepoint just prior to dosing (study 1-3: week - 2). Pairwise comparison of the difference between pre-exposed animals and unexposed animals per timepoint was performed for data in (FIGs. 5A-5D), summarized in Table 1. An ANOVA was performed over the fold changes per study and across the 3 studies over the data shown in (FIG. 5E), summarized in Table 2.

FIGs. 6A-6E: Ad26 vaccine-elicited immunity has a minor impact on humoral immune responses induced by a first dose of the B series in Cynomolgus macaques. Binding serum antibody titers were measured by ELISA for each regimen (FIGs. 6A-6E) specific for ZEBOV glycoprotein (GP) (FIGs. 6A-6B), SUDV GP (FIG. 6C), HIV Env Clade C (FIG. 6D) and HIV Env Mosl (FIG. 6E). Shown are geometric group means of the loglO EU/ml values determined relative to an assay standard. Dotted lines represent the lower limit of detection/lower limit of quantitation (LLoD/LLoQ). For FIGs. 6A and 6B the LLoQ of the human assay of 1.621ogl0 was used. For FIG. 6C the LLoD was defined as 1.481ogl0 (EU/ml) measured for the pre-dosing samples in the SUDV GP-specific assay. For FIGs. 6D and 6E, the LLOQ of the human assay of 2.191ogl0 was used, whereas the Upper limit of qualification of the human assay was 3.691ogl0. The fold change in ELISA titers is depicted in (FIG. 6F) and corresponds to the change in response comparing pre-dose 1 to peak response post-dose 1 per animal, Ad26rep refers to animals vaccinated in the A series, and Ad26 to animals that were not vaccinated during the A series. Pairwise comparison of the difference between pre-exposed animals and unexposed animals per timepoint was performed for data in (FIGs. 6A-6E), summarized in Table 3. An ANOVA was performed over the fold changes per study and across the 3 studies over the data shown in (FIG. 6F), summarized in Table 2.

FIGs. 7A-7D: Ebola Zaire/Guinea 2014 strain virus neutralization titers post-Ad26- Ad26 or Ad26-Ad35 dosing in animals with pre-existing Ad26 or Ad35 vaccine-elicited immunity. Filovirus neutralizing antibody responses against Zaire/2014 strain were measured using a pseudovirion neutralization assay as described in the material and methods. Shown are the antibody titers over time induced by (FIG. 7A) Ad26-Ad26, (FIG. 7B) Ad26- Ad35 encoding ZEBOV GP in animals previously dosed with Ad26-Ad26 (FIG. 7A) or Ad26-Ad35 (FIG. 7B) encoding RSV.FA2 (Ad26/Ad26rep or Ad26/Ad35rep) or in unexposed animals (Ad26/Ad26 or Ad26/Ad35). The fold change in neutralizing antibody titers in animals dosed with Ad26/Ad26 (FIG. 7B) or Ad26/Ad35 (FIG. 7C) was calculated. Shown are the changes in response comparing pre-dose 1 to peak response post-dose 1 per animal and pre-dose 2 to peak post-dose 2. The horizonal line is the geometric mean. Pairwise comparison of the difference between pre-exposed animals and unexposed animals per timepoint was performed for data in (FIGs. 7A-7B), summarized in Table 3. An ANOVA was performed over the fold changes over the data shown in (FIGs. 7C-7D), summarized in Table 2.

FIGs. 8A-8E: Heterologous vectors boost Ad26 vaccine-induced cellular immune responses in the presence of pre-existing Ad26 immunity in Cynomolgus macaques. IFNy ELISpot responses in PBMC (FIGs. 8A to 8D) were determined as described in Figure 5 legend with peptide pools for ZEBOV GP (FIGs. 8A-8B), SUDAN GP (FIG. 8C), HIV Env, Gag and Pol (FIG. 8D). Shown are background subtracted values per animal, of animals receiving an Ad26-Ad26 (FIG. 8 A) or Ad26-Ad35 (FIG. 8B) regimen encoding ZEBOV GP, Ad26 encoding SUDV GP, MVA encoding SUDV GP, ZEBOV GP, MARV GP (FIG. 8C), Ad26-MVA encoding Env, Gag, and Pol of HIV (FIG. 8D). Animals that had received an Ad26 vaccine during the A series are annotated as Ad26/Ad26rep (FIG. 8 A), Ad26/Ad35rep (FIG. 8B), Ad26/MVArep (FIGs. 8C-8D). In FIG. 8A-8D the dotted line represents the assay threshold of 50 spot-forming units (SFU)/10 ⁶ PBMC. In FIG. 8E, the fold change is depicted and corresponds to the change in responses comparing pre-dose 2 to peak response post-dose 2 per animal. Ad26-Xrep refers to animals vaccinated in the A series, and Ad26-X to animals that were not vaccinated during the A series. The horizonal line is the geometric mean. Pairwise comparison of the difference between pre-exposed animals and unexposed animals per timepoint was performed for data in (FIGs. 8A-8D), summarized in Table 1. An ANOVA was performed over the fold changes per study and across the 3 studies over the data shown in (FIG. 8E), summarized in Table 2.

FIGs. 9A-9F : Heterologous vectors boost Ad26 vaccine-induced humoral immune responses in the presence of pre-existing Ad26 immunity in Cynomolgus macaques. Binding serum antibody titers were measured by ELISA for each regimen (FIGs. 9A-9E) specific for ZEBOV GP (FIGs. 9A-9B), SUDV GP (FIG. 9C), HIV Clade C Env (FIG. 9D) and HIV Mosl Env (FIG. 9E), as described in Figure 6 legend. Shown are geometric group means of the log 10 EU/ml values determined relative to an assay standard. Dotted lines represent the lower limit of detection/lower limit of quantitation (LLoD/LLoQ). For FIGs. 9A and 9B, the LLoQ of the human assay of 1.621ogl0 was used. For FIG. 9C, the LLoD was defined as 1.481ogl0 (EU/ml) measured for the pre-dosing samples in the SUDV GP-specific assay. For FIGs. 9D and 9E, the LLOQ of the human assay of 2.191ogl0 was used, whereas the Upper limit of qualification of the human assay was 3.691ogl0. The fold change in ELISA titers is depictured in (FIG. 9F) and corresponds to the change in response comparing pre-dose 2 to peak response post-dose 2 per animal. Ad26-Xrep refers to animals vaccinated in the A series, and Ad26-X to animals that were not vaccinated during the A series. The horizonal line is the geometric mean. Pairwise comparison of the difference between pre-exposed animals and unexposed animals per timepoint was performed for data in (FIGs. 9A-9E), summarized in Table 3. An ANOVA was performed over the fold changes per study and across the 3 studies over the data shown in (FIG. 9F), summarized in Table 2.

FIG. 10 shows a schematic overview of the study for Group 1.

FIG. 11 shows a schematic overview of the study for Group 2.

FIG. 12 shows a schematic overview of the study for Group 3.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention. It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition. As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

As used herein, “subject” or “patient” means any animal, preferably a mammal, most preferably a human, to whom will be or has been administered a vaccine by a method according to an embodiment of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.

As used herein, the term “immune response” means that the vaccinated subject is able to control an infection or disease (e.g., a viral, bacterial, or parasitic infection, or a disease, such as cancer) with the pathogenic agent against which the vaccination was done (e.g., a viral, bacterial, or parasitic antigen, or a cancer antigen). The pathogenic agent can, for example, be an antigenic gene product or antigenic protein, or a fragment thereof. Usually, a subject in which an “immune response” against a virus, bacteria, parasite, or disease (i.e., cancer) has been generated, will not develop disease manifestations or those disease manifestations will be milder, and ultimately the subject will not die as a result of the infection and/or disease.

By “generating an immune response” or “promoting an immune response” or “provoking an immune response” is meant eliciting a humoral response (e.g., the production of antibodies) or a cellular response (e.g., the activation of T cells, macrophages, neutrophils, and/or natural killer cells) directed against, for example, one or more infective agents (e.g., a virus, a bacteria, a parasite) or a disease (e.g., a cancer) or protein targets in a subject to which the pharmaceutical composition (e.g., an immunogenic composition or vaccine) has been administered.

By “immunogen” or “antigen” is meant any polypeptide that can induce an immune response in a subject upon administration. In some embodiments, the immunogen or antigen is encoded by a nucleic acid molecule that may be incorporated into, for example, a polynucleotide or vector of the invention, for subsequent expression of the immunogen or antigen (e.g., a gene product of interest, or fragment thereof (e.g., a polypeptide)). The term “immunogenic composition” as used herein, is defined as material used to generate an immune response and may confer immunity after administration of the immunogenic composition to a subject.

By “isolated” is meant separated, recovered, or purified from a component of its natural environment. For example, a nucleic acid molecule or polypeptide of the invention may be isolated from a component of its natural environment by 1% (2%, 3%, 4%, 5%, 6%, 7%, 8% 9% 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or 90%) or more.

“Nucleic acid molecule” or “polynucleotide,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after synthesis, such as by conjugation with a label.

The nucleic acid molecules of the invention may be further optimized, such as by codon optimization, for expression in a targeted mammalian subject (e.g., human).

By “heterologous nucleic acid molecule” is meant a nucleotide sequence that can encode proteins derived or obtained from pathogenic organisms, such as viruses, which can be incorporated into a polynucleotide or vector of the invention. Heterologous nucleic acids can also encode synthetic or artificial proteins, such as immunogenic epitopes, constructed to induce immunity. An example of a heterologous nucleic acid molecule is one that encodes one or more immunogenic peptides or polypeptides derived from a Zika virus (ZIKV). The heterologous nucleic acid molecule is one that is not normally associated with the other nucleic acid molecules found in the polynucleotide or vector into which the heterologous nucleic acid molecule is incorporated.

A “nucleic acid vaccine” or “DNA vaccine” refers to a vaccine that includes a heterologous nucleic acid molecule under the control of a promoter for expression in a subject. The heterologous nucleic acid molecule can be incorporated into an expression vector, such as a plasmid or an adenoviral vector.

The term “vaccine” as used herein, is defined as material used to provoke an immune response and that confers immunity for a period of time after administration of the vaccine to a subject. 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.

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 can be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.

Nucleic Acid Molecules, Polypeptides, and Vectors of the Invention

Antigens

In embodiments of the invention, the first and second antigens are different antigens, and each antigen can, for example, be selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

A filovirus antigen can, for example, be an antigen from an Ebola virus or the genetically related Marburg virus. Filovirus antigens can, for example, include an envelope glycoprotein, a nucleoprotein (NP), matrix proteins VP24 and VP40, presumed nonstructural proteins VP30 and VP35, and the viral polymerase. In certain embodiments, the nucleic acid molecules or vectors encode the transmembrane form of the viral glycoprotein (GP), the secreted form of the viral glycoprotein (ssGP), or the viral nucleoprotein (NP).

The present invention also includes combinations of antigenic proteins as either the first or second antigen. For example, and without limitation, nucleic acid molecules encoding GP, ssGP and NP of the Zaire, Sudan, Marburg and Ivory Coast/Tai forest Ebola strains may be combined in any combination, in one vaccine composition. Filovirus antigens are known in the art, see, e.g., WO2016/036955, WO2016/036971, WO2016/187613, WO2018/011768, WO2018/185732. Each reference is herein incorporated by reference in its entirety.

Zika virus antigens for use in the invention can include, but are not limited to, a M- Env, prM-Env, prM-Env.dTM, prM-Env.dStem, Env, Env.dTM, and/or Env.d Stem or a portion thereof. Zika virus antigens are described at least in WO2017/214596, W02019/099970, and US2021/0317477. Each reference is herein incorporated by reference in its entirety.

Human Immunodeficiency Virus (HIV) antigens for use in the invention can include, but are not limited to, envelope antigens, polymerase antigens, gag polyprotein antigen, and fusion proteins thereof. HIV antigens are described at least in WO2010/059732, WO2014/107744, W02010042942, WO2016/049287, WO2018/045267, WO2018/229711, WO20 19/018724, WO2019/055888, and US2019/0231866. Each reference is herein incorporated by reference in its entirety.

Coronavirus antigens for use in the invention can include, but are not limited to, the spike protein (S), nucleocapsid protein (N), viral polyprotein (Plpro), membrane protein (M), envelope protein (E), non-structural protein 5 (nsp5), non-structural protein 3 (nsp3), and hemagglutinin esterases. Coronavirus antigens are described at least in WO2021/155323, which is herein incorporated by reference in its entirety, and V’kovski et al., Nat. Rev. Microbiol., pgs 1-16 (2020).

Influenza virus antigens for use in the invention can include, but are not limited to, nucleoprotein (NP), polymerase PA, polymerase PB1, polymerase PB2, matrix protein Ml, matrix protein M2, non-structural protein 1 (NS1), non-structural protein 2 (NS2), and non- structural protein PB1-F2, hemagglutinin, and neuraminidase. Influenza virus antigens are described at least in US2019/0015500, WO2016/005480, US2017/0189519, WO20 16/005482, US Pat. No. 10,738,083, US Pat. No. 10,517,941, and US Pat. No. 10,703,803. Each reference is herein incorporated by reference in its entirety.

Respiratory syncytial virus (RSV) antigens for use in the invention can include, but are not limited to, RSV glycoprotein (G), RSV fusion protein (F), matrix protein (M), phosphoprotein (P), nucleoprotein (N), polymerase (L), non-structural protein (NS1), non- structural protein (NS2), and non-structural protein (SH). RSV antigens are described at least in WO2017/174564, US2019/0125854, WO2017/174568, and US2019/0119330. Each reference is herein incorporated by reference in its entirety. Flavivirus antigens for use in the invention can include, but are not limited to, capsid protein (C), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and polymerase protein (NS5). Flaviviruses can include, but are not limited to, dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV) and tick-borne encephalitis virus (TBEV). Flavivirus antigens are described at least in Diosa-Toro et al., Virol. J. 17:60 (2020).

Bacterial antigens for use in the invention can include, but are not limited to, bacterial surface proteins (i.e., proteins expressed on surface of bacteria), bacterial lipopolysaccharides (LPS), and bacterial lipopolysaccharide proteins (LPSP). These bacterial antigens can be expressed or are on the surface of the bacteria. Bacterial antigens are known in the art and are described at least in Cunningham, Microbiol. Spectr. 7(4) (2019); Shepherd and McLaren, Int. J. Mol. Sci. 21(17):6144 (2020); Gnauck et al., Int. Rev. Immunol. 35(3): 189-218 (2016); Morrison, Rev. Infect. Dis. 5 Supp. 4:S733-47 (1983).

Parasitic antigens for use in the invention can include, but are not limited to, malarial antigens, protozoa antigens, helminth antigens, tapeworm antigens, fluke antigens, hookworm antigens, pinworm antigens, and trichinosis worm antigens. Parasitic antigens are known in the art.

Cancer antigens for use in the invention can include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA4), CD47, PD-1, PD-L1, 0X40, CD 137, CD80, CD40, B and T lymphocyte attenuator (BTLA), KIR, T cell receptor (TCR), LAG3, CD27, T cell membrane 3 (TIM3), DLL3, VEGF, EGFR, and c-Met. Cancer antigens are known in the art and are described at least in Pardoll, Nat. Rev. Cancer 12(4):252-64 (2012); Roudko et al., Front. Immunol. 11 :27 (2020).

According to embodiments of the invention, the antigen or immunogen can be isolated from, or derived from, a pathogen, such as a virus (e.g., filovirus, adenovirus, arbovirus, astrovirus, coronavirus, coxsackie virus, cytomegalovirus, Dengue virus, Epstein- Barr virus, hepatitis virus, herpesvirus, human immunodeficiency virus, human papilloma virus, human T-lymphotropic virus, influenza virus, JC virus, lymphocytic choriomeningitis virus, measles virus, molluscum contagiosum virus, mumps virus, norovirus, parovirus, poliovirus, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, rotavirus, rubella virus, smallpox virus, varicella zoster virus, West Nile virus, Zika virus, etc.), a bacteria (e.g., Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc.), a fungus (e.g., Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, Aspergillus species, etc.), a protozoan (e.g., Plasmodium, Leishmania, Trypanosome, cryptosporidiums, isospora, Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinii, etc.), or a cancer (e.g., bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, etc.).

The invention also features recombinant vectors including any one or more of the polynucleotides described above. The vectors of the invention can be used to deliver a nucleic acid expressing an antigen of the invention, and include mammalian, viral, and bacterial expression vectors. The mammalian, viral, and bacterial vectors of the invention can be genetically modified to contain one or more nucleic acid molecules encoding a first and/or second antigen of the invention.

Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that can be used to deliver a nucleic acid expressing an antigen of the invention include adenovirus (e.g., Ad2, Ad5, Adi 1, Adl2, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (also known as AdC68)), and modified vaccinia ankara virus (MV A). These adenovirus vectors can be derived from, for example, human, chimpanzee (e.g., ChAdl, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd27.1, ChAd28.1, ChAd29, ChAd30, ChAd31.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2, ChAd39, ChAd40.1, ChAd41.1, ChAd42.1, ChAd43, ChAd44, ChAd45, ChAd46, ChAd48, ChAd49, ChAd49, ChAd50, ChAd67, or SA7P), or rhesus adenoviruses. The viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into the antigens to elicit the immune response.

Adenoviral vectors disclosed in International Patent Application Publications W02006/040330 and W02007/ 104792, each incorporated by reference herein, are particularly useful as vectors of the invention. These adenoviral vectors can encode and/or deliver one or more of the antigens disclosed herein to elicit an immune response. In some embodiments, one or more recombinant adenovirus vectors can be administered to the subject in order to express more than one type of antigen disclosed herein, e.g., a first and a second antigen.

Adenoviruses

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; in the invention a human adenovirus is meant if referred to Ad without indication of species, e.g., the brief notation “Ad5” means the same as HAdV5, which is human adenovirus serotype 5), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV).

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, the 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 or 50. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of one of the serotypes 26 or 35.

An advantage of these serotypes is a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9):4654-63, both of which are incorporated by reference herein in their entirety. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Preparation of rAd35 vectors is described, for example, in US Patent No. 7,270,811, in WO 00/70071, and in Vogels et al., (2003) J Virol 77(15): 8263-71, all of which are incorporated by reference herein in their entirety. Exemplary genome sequences of Ad35 are found in GenBank Accession AC 000019 and in Fig. 6 of WO 00/70071.

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 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mital, 2006, Vaccine 24: 849- 62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other preferred 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.

Adenoviral Vectors Ad26 and Ad35

In a preferred embodiment according to the invention the adenoviral vectors comprise capsid proteins from two rare serotypes: Ad26 or Ad35. In the typical embodiment, the vector is an Ad26 virus.

Thus, the vectors that can be used in the invention comprise an Ad26 or Ad35 capsid protein (e.g., a fiber, penton or hexon protein). One of skill will recognize that it is not necessary that an entire Ad26 or Ad35 capsid protein be used in the vectors of the invention. Thus, chimeric capsid proteins that include at least a part of an Ad26 or Ad35 capsid protein can be used in the vectors of the invention. The vectors of the invention can also comprise capsid proteins in which the fiber, penton, and hexon proteins are each derived from a different serotype, so long as at least one capsid protein is derived from Ad26 or Ad35. In preferred embodiments, the fiber, penton and hexon proteins are each derived from Ad26 or Ad35.

One of skill 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 can combine the absence of pre-existing immunity of the Ad26 and Ad35 serotypes 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.

In certain embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad35 or from Ad26 (i.e., the vector is Ad35 or Ad26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome. For the adenoviruses of the invention, being derived from Ad26, 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 El 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). In certain embodiments, the adenovirus is a human adenovirus of serotype 35, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. In certain embodiments, the adenovirus is a human adenovirus of serotype 26, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. For the Ad35 adenovirus, it is typical to retain the 3’ end of the E1B 55K open reading frame in the adenovirus, for instance the 166 bp directly upstream of the pIX open reading frame or a fragment comprising this such as a 243 bp fragment directly upstream of the pIX start codon, marked at the 5’ end by a Bsu36I restriction site, since this increases the stability of the adenovirus because the promoter of the pIX gene is partly residing in this area (see, e.g. Havenga et al, 2006, supra; WO 2004/001032). The preparation of recombinant adenoviral vectors is well known in the art.

Preparation of Ad26 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. Preparation of rAd35 vectors is described, for example, in US Patent No. 7,270,811 and in Vogels et al., (2003) J Virol 77(15): 8263-71. An exemplary genome sequence of Ad35 is found in GenBank Accession AC 000019.

In an embodiment of the invention, the vectors useful for the invention 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 defective. In these embodiments, the virus is rendered replication-defective by deletion or inactivation of regions critical to replication of the virus, such as the El region. The regions can be substantially deleted or inactivated by, for example, inserting the gene of interest (usually linked to a promoter). 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. 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 amount of adenovirus vectors of the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6, 911, 293, and El A549. MVA vectors

MVA vectors useful for the present invention utilize attenuated virus derived from Modified Vaccinia Ankara virus which is characterized by the loss of their capabilities to reproductively replicate in human cell lines. The MVA vectors can express a wide variety of antigens disclosed herein.

MVA has been generated by more than 570 serial passages on chicken embryo fibroblasts of the dermal vaccinia strain Ankara [Chorioallantois vaccinia virus Ankara virus, CVA; for review see Mayr et al. (1975), Infection 3, 6-14] that was maintained in the Vaccination Institute, Ankara, Turkey for many years and used as the basis for vaccination of humans. However, due to the often severe post-vaccination complications associated with vaccinia viruses, there were several attempts to generate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded in attenuating CVA by over 570 continuous passages in CEF cells [Mayr et al. (1975)]. It was shown in a variety of animal models that the resulting MVA was avirulent [Mayr, A. & Danner, K. (1978), Dev. Biol. Stand. 41: 225-234], As part of the early development of MVA as a pre-smallpox vaccine, there were clinical trials using MVA-517 in combination with Lister Elstree [Stickl (1974), Prev. Med. 3: 97-101; Stickl and Hochstein-Mintzel (1971), Munch. Med. Wochenschr. 113: 1149-1153] in subjects at risk for adverse reactions from vaccinia. In 1976, MVA derived from MVA-571 seed stock (corresponding to the 571st passage) was registered in Germany as the primer vaccine in a two-stage parenteral smallpox vaccination program. Subsequently, MVA-572 was used in approximately 120,000 Caucasian individuals, the majority children between 1 and 3 years of age, with no reported severe side effects, even though many of the subjects were among the population with high risk of complications associated with vaccinia (Mayr et al. (1978), Zentralbl. Bacteriol. (B) 167:375-390). MVA- 572 was deposited at the European Collection of Animal Cell Cultures as ECACC V94012707. As a result of the passaging used to attenuate MV A, there are a number of different strains or isolates, depending on the number of passages conducted in CEF cells. For example, MVA-572 was used in a small dose as a pre-vaccine in Germany during the smallpox eradication program, and MVA-575 was extensively used as a veterinary vaccine. MVA as well as MVA- BN lacks approximately 15% (31 kb from six regions) of the genome compared with ancestral CVA virus. The deletions affect a number of virulence and host range genes, as well as the gene for Type A inclusion bodies. MVA-575 was deposited on December 7, 2000, at the European Collection of Animal Cell Cultures (ECACC) under Accession No. V00120707. The attenuated CVA-virus MVA (Modified Vaccinia Virus Ankara) was obtained by serial propagation (more than 570 passages) of the CVA on primary chicken embryo fibroblasts.

Even though Mayr et al. demonstrated during the 1970s that MVA is highly attenuated and avirulent in humans and mammals, certain investigators have reported that MVA is not fully attenuated in mammalian and human cell lines since residual replication might occur in these cells [Blanchard et al. (1998), J. Gen. Virol. 79: 1159-1167; Carroll & Moss (1997), Virology 238:198-211; U.S. Patent No. 5,185,146; Ambrosini et al. (1999), J. Neurosci. Res. 55: 569], It is assumed that the results reported in these publications have been obtained with various known strains of MVA, since the viruses used essentially differ in their properties, particularly in their growth behavior in various cell lines. Such residual replication is undesirable for various reasons, including safety concerns in connection with use in humans.

Strains of MVA having enhanced safety profiles for the development of safer products, such as vaccines or pharmaceuticals, have been developed by Bavarian Nordic. MVA was further passaged by Bavarian Nordic and is designated MVA-BN, a representative sample of which was deposited on August 30, 2000 at the European Collection of Cell Cultures (ECACC) under Accession No. V00083008. MVA-BN is further described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699), both of which are incorporated by reference herein in their entirety.

MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. MVA-BN is strongly adapted to primary chicken embryo fibroblast (CEF) cells and does not replicate in human cells. In human cells, viral genes are expressed, and no infectious virus is produced. MVA-BN is classified as Biosafety Level 1 organism according to the Centers for Disease Control and Prevention in the United States. Preparations of MVA-BN and derivatives have been administered to many types of animals, and to more than 2000 human subjects, including immune-deficient individuals. All vaccinations have proven to be generally safe and well tolerated. Despite its high attenuation and reduced virulence, in preclinical studies MVA-BN has been shown to elicit both humoral and cellular immune responses to vaccinia and to heterologous gene products encoded by genes cloned into the MVA genome [E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300; A. Cosma et al. (2003), Vaccine 22(l):21-9; M. Di Nicola et al. (2003), Hum. Gene Ther. 14(14): 1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res., 10(16):5381-5390],

“Derivatives” or “variants” of MVA refer to viruses exhibiting essentially the same replication characteristics as MVA as described herein but exhibiting differences in one or more parts of their genomes. MVA-BN as well as a derivative or variant of MVA-BN fails to reproductively replicate in vivo in humans and mice, even in severely immune suppressed mice. More specifically, MVA-BN or a derivative or variant of MVA-BN has preferably also the capability of reproductive replication in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in the human keratinocyte cell line HaCat [Boukamp et al (1988), J. Cell Biol. 106: 761-771], the human bone osteosarcoma cell line 143B (ECACC Deposit No. 91112502), the human embryo kidney cell line 293 (ECACC Deposit No. 85120602), and the human cervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL- 2). Additionally, a derivative or variant of MVA-BN has a virus amplification ratio at least two-fold less, more preferably three-fold less than MVA-575 in Hela cells and HaCaT cell lines. Tests and assay for these properties of MVA variants are described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699).

The term “not capable of reproductive replication” or “no capability of reproductive replication” is, for example, described in WO 02/42480, which also teaches how to obtain MVA having the desired properties as mentioned above. The term applies to a virus that has a virus amplification ratio at 4 days after infection of less than 1 using the assays described in WO 02/42480 or in U.S. Patent No. 6,761,893, both of which are incorporated by reference herein in their entirety.

The term “fails to reproductively replicate” refers to a virus that has a virus amplification ratio at 4 days after infection of less than 1. Assays described in WO 02/42480 or in U.S. Patent No. 6,761,893 are applicable for the determination of the virus amplification ratio.

The amplification or replication of a virus is normally expressed as the ratio of virus produced from an infected cell (output) to the amount originally used to infect the cell in the first place (input) referred to as the “amplification ratio”. An amplification ratio of “1” defines an amplification status where the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells, meaning that the infected cells are permissive for virus infection and reproduction. In contrast, an amplification ratio of less than 1, i.e., a decrease in output compared to the input level, indicates a lack of reproductive replication and therefore attenuation of the virus.

The advantages of MVA-based vaccine include their safety profile as well as availability for large scale vaccine production. Preclinical tests have revealed that MVA-BN demonstrates superior attenuation and efficacy compared to other MVA strains (WO 02/42480). An additional property of MVA-BN strains is the ability to induce substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA- prime/vaccinia virus boost regimes.

The recombinant MVA-BN viruses, the most preferred embodiment herein, are considered to be safe because of their distinct replication deficiency in mammalian cells and their well-established avirulence. Furthermore, in addition to its efficacy, the feasibility of industrial scale manufacturing can be beneficial. Additionally, MVA-based vaccines can deliver multiple heterologous antigens and allow for simultaneous induction of humoral and cellular immunity.

MVA vectors useful for the present invention can be prepared using methods known in the art, such as those described in WO/2002/042480 and WO/2002/24224, the relevant disclosures of which are incorporated herein by references.

In another aspect, replication deficient MVA viral strains may also be suitable such as strain MVA-572, MVA-575 or any similarly attenuated MVA strain. Also suitable may be a mutant MVA, such as the deleted chorioallantois vaccinia virus Ankara (dCVA). A dCVA comprises del I, del II, del III, del IV, del V, and del VI deletion sites of the MVA genome. The sites are particularly useful for the insertion of multiple heterologous sequences. The dCVA can reproductively replicate (with an amplification ratio of greater than 10) in a human cell line (such as human 293, 143B, and MRC-5 cell lines), which then enable the optimization by further mutation useful for a virus-based vaccination strategy (see WO 2011/092029).

In vitro transcribed (IVT) repRNAs

RepRNAs useful in the invention are derived from alphaviruses, which are singlestrand positive-sense RNA viruses. In one embodiment, a RepRNA that can be used in the invention contains a 7- methylguanosine cap, a 5’ UTR, an RNA-dependent RNA polymerase (RdRp) polyprotein P1234 (i.e., nonstructural proteins, nsPs), a subgenomic promoter element, a variable region of interest from which an antigenic protein is expressed, a 3’ UTR, and a poly (A) tail.

RepRNAs useful in the invention can be derived from any self-replicating positive strand RNA virus, for example RepRNAs can be derived from the virus families of Togaviridae or Arteriviridae, such as Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Buggy Creek virus, Chikungunya virus, Eastern Equine Encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Middleburg virus, Mucambo virus, Ndumu virus, O’nyong-nyong virus, Pixuna virus, Ross River virus, S.A. AR86, Sagiyama virus, Semliki Forest virus, Sindbis virus, Una virus, Venezuelan Equine Encephalitis virus, Western Equine Encephalitis virus, Whataroa virus, African pouched rat arterivirus, DeBrazza’s monkey arterivirus, Equine arteritis virus, Kibale red colobus virus, Kibale red-tailed guenon virus, Lactate dehydrogenase-elevating virus, Mikumi yellow baboon virus 1, Pebjah virus, Porcine reproductive and respiratory syndrome virus. In preferred embodiments, repRNAs of the invention are derived from Venezuelan Equine Encephalitis (VEE) virus. Examples of preferred repRNA backbones of the invention include those described by Frolov et al., (J. Virol. 73(5):3854-65 (1999).

The preparation of in vitro transcribed (IVT) RNA is well known in the art, and standard IVT and purification procedures can be used to prepare IVT repRNAs useful in the invention in view of the present disclosure.

Preparation of IVT repRNA is described, for example, in US2011/0300205 and US2013/0195968, the relevant content of which is incorporated herein by reference. For example, repRNA molecules can be prepared by IVT of a DNA that encodes the selfreplicating RNA molecule using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, etc. IVT can use a cDNA template created and propagated in plasmid from bacteria, or created synthetically, such as by gene synthesis and/or PCR-based methods. Appropriate capping addition reactions can be used as required, and the poly-A can be encoded within the DNA template or added by a poly-A reaction. Suitable synthetic methods can be used alone, or in combination with one or more other methods (e.g., recombinant DNA or RNA technology), to produce an IVT repRNA molecule of the invention. Suitable methods for de novo synthesis are well-known in the art and can be adapted for particular applications.

Typically, an IVT repRNA useful in the invention is produced using a DNA molecule from which the repRNA can be transcribed. Thus, the invention also provides isolated nucleic acid molecules that encode repRNAs 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.

IVT repRNAs useful in the invention can be formulated into suitable delivery systems for administration. In preferred embodiments, IVT repRNAs useful in the invention are formulated into non-virion particles for administration. Suitable non-virion particles are described, for example, in US2011/0300205 and US2013/0195968, the relevant content of which is incorporated herein by reference. For example, useful delivery systems include liposomes, polymer particles, non-toxic and biodegradable microparticles, electroporation, injection of naked RNA, and cationic submicron oil-in-water emulsions. In preferred embodiments, IVT repRNAs useful in the invention are formulated in lipid nanoparticle (LNP) compositions (see, e.g., Semple et al., 2010, Nat Biotechnol. 28(2): 172-176, the relevant content of which is incorporated herein by reference).

Pharmaceutical Compositions

In another general aspect, the invention relates to pharmaceutical compositions comprising nucleic acid molecules or vectors (e.g., adenoviral vectors) encoding an antigen of interest (e.g., a first and/or second antigen of the invention) or polypeptides comprising the antigen of interest (e.g., a first and/or second antigen of the invention) and a pharmaceutically acceptable carrier. Nucleic acid molecules, vectors, and polypeptides of the invention and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.

By “pharmaceutical composition” is meant any composition that contains a therapeutically or biologically active agent, such as an immunogenic composition or vaccine of the invention (e.g., a nucleic acid molecule, vector, and/or polypeptide of the invention), preferably including a nucleotide sequence encoding an antigenic gene product of interest, or fragment thereof, that is suitable for administration to a subject and that treats or prevents a disease (e.g., a viral, bacterial, or parasitic infection, or a cancer) or reduces or ameliorates one or more symptoms of the disease (e.g., viral titer, viral spread, infection, bacterial load, parasitic load, or a symptom of a cancer (e.g., pain, weight loss, fatigue, fever, sores, unusual bleeding, skin changes, etc.)). For the purposes of this invention, pharmaceutical compositions include vaccines, and pharmaceutical compositions suitable for delivering a therapeutic or biologically active agent can include, for example, tablets, gelcaps, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Any of these formulations can be prepared by well-known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21 ^st ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.

As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical composition can be used in the invention.

Pharmaceutically acceptable acidic/anionic salts for use in the invention include, and are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate and triethiodide. Organic or inorganic acids also include, and are not limited to, hydriodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharinic or trifluoroacetic acid.

Pharmaceutically acceptable basic/cationic salts include, and are not limited to aluminum, 2-amino-2-hydroxymethyl-propane-l,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”), ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

In some embodiments of the invention, the first and second Ad26 vectors are administered in an amount of about 1 x IO ¹⁰ , about 2 x IO ¹⁰ , about 3 x IO ¹⁰ , about 4 x IO ¹⁰ , about 5 x IO ¹⁰ , about 6 x IO ¹⁰ , about 7 x IO ¹⁰ , about 8 x IO ¹⁰ , about 9 x IO ¹⁰ , about 1 x 10 ¹¹ , about 2 x 10 ¹¹ , about 3 x 10 ¹¹ , about 4 x 10 ¹¹ , or about 5 x 10 ¹¹ viral vectors (or particles) per dose. In certain embodiments of the invention, the first and second Ad26 vectors are administered in an amount of about 1 x IO ¹⁰ adenoviral vectors (or particles) to about 5 x 10 ¹¹ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the first and second Ad26 vectors are administered in an amount of about 5 x IO ¹⁰ adenoviral vectors (or particles) to about 1 x 10 ¹¹ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the first and second Ad26 vectors are administered in an amount of about 5 x IO ¹⁰ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the first and second Ad26 vectors are administered in an amount of about 1 x 10 ¹¹ adenoviral vectors (or particles) per dose.

In some embodiments of the invention, pharmaceutical compositions are provided comprising the adenoviral vectors of the invention in an amount of about 1 x IO ¹⁰ , about 2 x IO ¹⁰ , about 3 x IO ¹⁰ , about 4 x IO ¹⁰ , about 5 x IO ¹⁰ , about 6 x IO ¹⁰ , about 7 x IO ¹⁰ , about 8 x IO ¹⁰ , about 9 x IO ¹⁰ , about 1 x 10 ¹¹ , about 2 x 10 ¹¹ , about 3 x 10 ¹¹ , about 4 x 10 ¹¹ , or about 5 x 10 ¹¹ viral vectors (or particles) per dose. In certain embodiments of the invention, the pharmaceutical composition comprises about 1 x IO ¹⁰ adenoviral vectors (or particles) to about 5 x 10 ¹¹ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the pharmaceutical composition comprises about 5 x IO ¹⁰ adenoviral vectors (or particles) to about 1 x 10 ¹¹ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the pharmaceutical composition comprises about 5 x IO ¹⁰ adenoviral vectors (or particles) per dose. In certain embodiments of the invention, the pharmaceutical composition comprises about 1 x 10 ¹¹ adenoviral vectors (or particles) per dose.

In another embodiment of the invention, the MVA vector is administered (e.g., intramuscularly) in a volume ranging between about 100 pl to about 10 ml of saline solution containing a dose of about 1X1O ⁷ TCIDSO to IxlO ⁹ TCID50 (50% Tissue Culture Infective Dose) or Inf.U. (Infectious Unit). Preferably, the MVA vector is administered in a volume ranging between 0.25 and 1.0 ml. More preferably the MVA vector is administered in a volume of 0.5 ml.

Typically, the MVA vector is administered in a dose of about IxlO ⁷ TCID50 to IxlO ⁹ TCID50 (or Inf.U.) to a human subject during one administration. In a preferred embodiment, the MVA vector is administered in an amount of about 5xl0 ⁷ TCID50 to 5xl0 ⁸ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about 5xl0 ⁷ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about 1x10 ⁸ TCID50 (or Inf.U.). In another preferred embodiment, the MVA vector is administered in an amount of about 1.9xl0 ⁸ TCIDso for Inf.U). In yet another preferred embodiment, the MVA vector is administered in an amount of about 4.4xl0 ⁸ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about 5x10 ⁸ TCID50 (or Inf.U.).

In another embodiment, the IVT repRNA is administered (e.g., intramuscularly) in a volume ranging between about lOOpl to about 10ml containing a dose of <200pg, <100pg, <50pg, or <10pg IVT repRNA, but expression can be seen at much lower levels, e.g., <lpg, <100ng, <10ng, or <lng IVT repRNA per dose. Preferably, the IVT repRNA is administered in a volume ranging between 0.25ml and 1.0ml. More preferably the IVT repRNA is administered in a volume of 0.5ml.

Typically, the IVT repRNA is administered in an amount of about 10-100pg per dose. In a preferred embodiment, the IVT repRNA is administered in an amount of about lOpg per dose. In another preferred embodiment, the IVT repRNA is administered in an amount of about 25 pg per dose. In another preferred embodiment, the IVT repRNA is administered in an amount of about 50pg per dose. In another preferred embodiment, the IVT repRNA is administered in an amount of about 75pg per dose. In another preferred embodiment, the IVT repRNA is administered in an amount of about lOOpg per dose.

In certain embodiments, the subject is administered a single dose of the pharmaceutical composition comprising the first antigen and a single dose of the pharmaceutical composition comprising the second antigen. In certain embodiments, the pharmaceutical composition comprising the second antigen can be administered to the subject about two (2) weeks, about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the pharmaceutical composition comprising the first antigen.

In certain embodiments, the subject is administered a double dose of the pharmaceutical composition comprising the first antigen and a double dose of the pharmaceutical composition comprising the second antigen. When administering a double dose, the first and second dose of the pharmaceutical composition comprising the first antigen can be administered to the subject about two (2) weeks, about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after apart. When administering a double dose, the first and second dose of the pharmaceutical composition comprising the second antigen can be administered to the subject about two (2) weeks, about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after apart.

The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation comprising water. The liquid formulation can comprise a solution, a suspension, an emulsion, a microemulsion, a gel, and the like. An aqueous formulation typically comprises at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w/w of water.

In one embodiment, the pharmaceutical composition can be formulated as an injectable which can be injected, for example, via a syringe or an infusion pump. The injection can be delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously, for example.

In another embodiment, the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use. Solid dosage forms can include tablets, such as compressed tablets, and/or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The pharmaceutical composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.

The dosage forms can be immediate release, in which case they can comprise a water- soluble or dispersible carrier, or they may be delayed release, sustained release, or modified release, in which case they may comprise water-insoluble polymers that regulate the rate of dissolution of the dosage form in the gastrointestinal tract.

In other embodiments, the pharmaceutical composition can be delivered intranasally, intrabuccally, or sublingually.

The pH in an aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0. In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer may be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4- hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2- phenyl ethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservative may be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of isotonic agents include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), an alditol (such as glycerol, 1,2-propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol), polyethyleneglycol (e.g. PEG400), and mixtures thereof. Another example of an isotonic agent includes a sugar. Non-limiting examples of sugars may be mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta- HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one — OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. Pharmaceutical compositions comprising each isotonic agent listed in this paragraph constitute alternative embodiments of the invention. The isotonic agent can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific isotonic agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy-/hydroxycellulose and derivates thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulphur-containing substances such as monothioglycerol), or thioglycolic acid. The stabilizer can be present individually or in the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term “surfactant” refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactant can, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants. The surfactant can be present individually or in the aggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid), and/or benzamidine hydrochloric acid (HC1). The protease inhibitor can be present individually or in the aggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.

The pharmaceutical composition of the invention can comprise an amount of an amino acid base sufficient to decrease aggregate formation of the polypeptide during storage of the composition. The term “amino acid base” refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), or analogues thereof. Any amino acid can be present either in its free base form or in its salt form. Any stereoisomer (i.e., L, D, or a mixture thereof) of the amino acid base may be present. The amino acid base can be present individually or in the combination with other amino acid bases, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of these specific amino acid bases constitute alternative embodiments of the invention.

It is also apparent to one skilled in the art that the therapeutically effective dose of a nucleic acid molecule or vector (e.g., adenoviral vectors) encoding an antigen described herein, or a polypeptide comprising an antigen disclosed herein or a pharmaceutical composition thereof will vary according to the desired effect. Therefore, optimal dosages to be administered can be readily determined by one skilled in the art and will vary with the particular nucleic acid molecule, vector, or polypeptide used, the mode of administration, the strength of the preparation, and the advancement of the disease condition (e.g., viral infection, bacterial infection, parasitic infection, or cancer). In addition, factors associated with the particular subject being treated, including subject age, weight, diet and time of administration, will result in the need to adjust the dose to an appropriate therapeutic level.

The pharmaceutically-acceptable salts of the adenoviral particles of the invention include the conventional non-toxic salts or the quaternary ammonium salts which are formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate, dodecylsulfate, hydrochloride, hydrobromide, lactate, maleate, methanesulfonate, nitrate, oxalate, pivalate, propionate, succinate, sulfate and tartrate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamino salts and salts with amino acids such as arginine. Also, the basic nitrogen-containing groups may be quaternized with, for example, alkyl halides. The pharmaceutical compositions of the invention can be administered by any means that accomplish their intended purpose. As used herein, by “administering” is meant a method of giving a dosage of a pharmaceutical composition to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered, and the severity of the condition being treated).

Methods of use

The present invention provides methods for generating an immune response against a first and second antigen in a human subject in need thereof. The methods comprise administering to the subject a pharmaceutical composition comprising nucleic acid molecules or vectors encoding the first and/or second antigen or polypeptides comprising the first and/or second antigen and a pharmaceutically acceptable carrier. The methods are for preventing, treating, delaying the onset of, or ameliorating an infection or any one or more symptoms of said infection (e.g., a viral, bacterial, or parasitic infection) or a disease or any one or more symptoms of said disease (e.g., cancer), the method comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition of the invention.

According to particular embodiments, an immunogenic or effective or protective amount refers to the amount of an antigen which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the infection (e.g., viral, bacterial, or parasitic infection) or disease (e.g., cancer) to be treated or a symptom associated therewith; (ii) reduce the duration of the infection or disease to be treated, or a symptom associated therewith; (iii) prevent the progression of the infection or disease to be treated, or a symptom associated therewith; (iv) cause regression of the infection or disease to be treated, or a symptom associated therewith; (v) prevent the development or onset of the infection or disease to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the infection or disease to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the infection or disease to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the infection or disease to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the infection or disease to be treated, or a symptom associated therewith; (xi) inhibit or reduce the infection or disease to be treated, or a symptom associated therewith in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

Examples of symptoms of diseases caused by a viral infection, that can be prevented using the compositions of the invention include, for example, fever, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia. These symptoms, and their resolution during treatment, can be measured by, for example, a physician during a physical examination or by other tests and methods known in the art.

The immunogenic or effective amount or dosage can vary according to various factors, such as the infection or disease to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to the infection or disease, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the infection or disease. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the infection or disease. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the infection or disease. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the virus infection or disease. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the infection or disease in the subject.

Single or multiple administrations of the compositions of the present invention can be given (pre- or post-exposure and/or pre- or post-diagnosis) to a subject (e.g., one administration or administration two or more times). For example, subjects who are particularly susceptible to, for example, an infection can require multiple administrations of the compositions of the present invention to establish and/or maintain protection against the infection. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, for example, measuring amounts of neutralizing secretory and serum antibodies. The dosages can then be adjusted or repeated as necessary to trigger the desired level of immune response. For example, the immune response triggered by a single administration (prime) of a composition of the invention may not be sufficiently potent and/or persistent to provide effective protection. Accordingly, in some embodiments, repeated administration (boost), such that a prime boost regimen is established, can significantly enhance humoral and cellular responses to the antigen of the composition.

Alternatively, the efficacy of treatment can be determined by monitoring the level of the antigenic or therapeutic gene product, or fragment thereof, expressed in a subject (e.g., a human) following administration of the pharmaceutical compositions of the invention. For example, the blood or lymph of a subject can be tested for antigenic or therapeutic gene product, or fragment thereof, using, for example, standard assays known in the art.

Immunogenicity of the pharmaceutical compositions of the invention can be improved if it is co-administered with an immunostimulatory agent and/or adjuvant. Suitable adjuvants well-known to those skilled in the art include, for example, aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.

The term “immunostimulatory agent” refers to substances (e.g., drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. An immunostimulatory agent can, for example, include a cytokine (e.g., the granulocyte macrophage colony-stimulating factor) and interferon (e.g., IFN-a and/or IFN-y).

The term “adjuvant” is defined as a pharmacological or immunological agent that modifies the effect of other agents (e.g., the antigens disclosed herein) while having few if any direct effects when administered alone. An adjuvant can be one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the adenoviral particles of the invention.

Prime-Boost Regimens

Provided herein are methods for inducing an immune response against at least a first and a second antigen in a subject in need thereof. The methods comprise (a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier; and (b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier; wherein the first and second antigens are different antigens, and wherein the second Ad26 vector is administered to the subject at least two weeks after the first Ad26 vector.

The methods can, for example, comprise (a) administering to the subject a first composition comprising an immunologically effective amount of a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid encoding a first antigen, together with a pharmaceutically acceptable carrier; and (b) administering to the subject a second composition comprising an immunologically effective amount of a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier; wherein the first and second antigens are different antigens, and wherein the second Ad26 vector is administered to the subject at least two weeks after the first Ad26 vector.

In certain embodiments, the methods further comprise administering to the subject one or more nucleic acid molecules or vectors encoding the first and/or the second antigen or one or more polypeptides comprising the first and/or second antigen after administration of the first and/or second Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen can, for example, be administered to the subject: (a)(i) after administration of the first Ad26 vector and before the administration of the second Ad26 vector; or (a)(ii) at about the same time as the administration of the second Ad26 vector; or (a)(iii) after administration of the second Ad26 vector. The subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen can, for example, be administered to the subject (b) after administration of the second Ad26 vector.

In certain embodiments, the subsequently administered nucleic acid molecule or vector is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi 1, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MV A, mRNA, IVT repRNA, and saRNA. The subsequently administered vector can, for example, be an Ad26 vector. In preferred embodiments, the subsequently administered vector is the same as the first Ad26 vector or the second Ad26 vector.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of the first Ad26 vector encoding the first antigen (Ad26-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of the second Ad26 vector encoding the second antigen (Ad26- Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of a first MVA vector encoding the first antigen (MVA-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of the second Ad26 vector encoding the second antigen (Ad26- Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of the first Ad26 vector encoding the first antigen (Ad26-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of an MVA vector encoding the second antigen (MVA-Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of the first Ad26 vector encoding the first antigen (Ad26-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of a polypeptide comprising the second antigen (Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart. In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of a polypeptide comprising the first antigen (Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of the second Ad26 vector encoding the second antigen (Ad26- Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise simultaneously administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) together with a polypeptide comprising a first antigen (Agl); and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of the second Ad26 vector encoding the second antigen (Ad26-Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of the first Ad26 vector encoding the first antigen (Ad26-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) together with a polypeptide comprising a second antigen (Ag2). The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of a first IVT repRNA vector encoding the first antigen (IVT repRNA-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of the second Ad26 vector encoding the second antigen (Ad26-Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart. In certain embodiments, the methods comprise administering to the subject a first Ad26 vector encoding a first antigen (Ad26-Agl) as a priming composition followed by subsequent administration of the first Ad26 vector encoding the first antigen (Ad26-Agl) as a boosting composition at least two (2) weeks apart; and administering to the subject a second Ad26 vector encoding a second antigen (Ad26-Ag2) as a priming composition followed by subsequent administration of an IVT repRNA vector encoding the second antigen (IVT repRNA-Ag2) as a boosting composition at least two (2) weeks apart. The first and second Ad26 vectors encoding the first and second antigens, respectively, can be administered to the subject at least two (2) weeks apart.

Kits

Also provided are kits comprising: (a) a first Ad26 vector encoding a first antigen; and (b) a second Ad26 vector encoding a second antigen; wherein the first and second antigen are different antigens. In certain embodiments, the kit further comprises: (a) one or more nucleic acid molecules or vectors encoding the first antigen or one or more polypeptides comprising the first antigen; and (b) one or more nucleic acid molecules or vectors encoding the second antigen or one or more polypeptides comprising the second antigen.

In certain embodiments, the first antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the second antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

In certain embodiments, the kit comprises about 5 x IO ¹⁰ adenoviral vectors, about 6 x IO ¹⁰ adenoviral vectors, about 7 x IO ¹⁰ adenoviral vectors, about 8 x IO ¹⁰ adenoviral vectors, about 9 x IO ¹⁰ adenoviral vectors, or about 1 x 10 ¹¹ adenoviral vectors of the first and second Ad26 vectors.

The one or more nucleic acid molecules or vectors is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi l, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MV A, mRNA, and saRNA. In certain embodiments, the nucleic acid molecule or vector is an Ad26 vector.

In certain embodiments, the kit further comprises at least one syringe for injection of the first Ad26 vector, the second Ad26 vector, the one or more nucleic acid molecules or vectors encoding the first antigen or the one or more polypeptides comprising the first antigen, and/or the one or more nucleic acid molecules or vectors encoding the second antigen or the one or more polypeptides comprising the second antigen.

EMBODIMENTS

The invention provides also the following non-limiting embodiments.

Embodiment l is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier; wherein the first and second antigens are different antigens, and wherein the second Ad26 vector is administered to the subject at least two weeks after the first Ad26 vector.

Embodiment 2 is the method of embodiment 1, wherein the second Ad26 vector is administered to the subject at least four (4) weeks after the first Ad26 vector.

Embodiment 3 is the method of embodiment 2, wherein the second Ad26 vector is administered to the subject at about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the first Ad26 vector.

Embodiment 4 is the method of any one of embodiments 1-3, further comprising administering to the subject one or more nucleic acid molecules or vectors encoding the first and/or the second antigen or one or more polypeptides comprising the first and/or second antigen after administration of the first and/or second Ad26 vector.

Embodiment 5 is the method of embodiment 4, wherein:

(a)(i) the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject after administration of the first Ad26 vector and before the administration of the second Ad26 vector; or

(a)(ii) the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject at about the same time as the administration of the second Ad26 vector; or

(a)(iii) the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject after administration of the second Ad26 vector; and/or

(b) the subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen is administered to the subject after administration of the second Ad26 vector.

Embodiment 6 is the method of embodiment 4 or 5, wherein the subsequently administered nucleic acid molecule or vector is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi 1, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MV A, mRNA, IVT repRNA, and saRNA.

Embodiment 7 is the method of embodiment 6, wherein the subsequently administered vector is an Ad26 vector, preferably wherein the subsequently administered vector is the same as the first Ad26 vector or the second Ad26 vector.

Embodiment 8 is the method of any one of embodiments 4-7, wherein the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject at least two (2) weeks after the first Ad26 vector.

Embodiment 9 is the method of any one of embodiments 4-7, wherein the subsequently administered nucleic acid molecule or vector encoding the first antigen or polypeptide comprising the first antigen is administered to the subject at about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the first Ad26 vector.

Embodiment 10 is the method of any one of embodiments 4-7, wherein the subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen is administered to the subject at least (2) weeks after the second Ad26 vector.

Embodiment 11 is the method of any one of embodiments 4-7, wherein the subsequently administered nucleic acid molecule or vector encoding the second antigen or polypeptide comprising the second antigen is administered to the subject at about four (4) weeks, about eight (8) weeks, about twelve (12) weeks, about three (3) months, about six (6) months, about nine (9) months, about one (1) year, or about (2) years after the second Ad26 vector.

Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the first antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

Embodiment 13 is the method of any one of embodiments 1 to 12, wherein the second antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the first and second Ad26 vectors are administered in an amount of about 5 x IO ¹⁰ adenoviral vectors, about 6 x IO ¹⁰ adenoviral vectors, about 7 x IO ¹⁰ adenoviral vectors, about 8 x IO ¹⁰ adenoviral vectors, about 9 x IO ¹⁰ adenoviral vectors, or about 1 x 10 ¹¹ adenoviral vectors.

Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the first and second Ad26 vectors are administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, by catheter, by lavage, or by gavage.

Embodiment 16 is the method of embodiment 15, wherein the first and second Ad26 vector are administered in an intramuscular injection.

Embodiment 17 is the method of any one of embodiments 4-16, wherein the subsequently administered nucleic acid molecule, vector, or polypeptide is administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, by catheter, by lavage, or by gavage.

Embodiment 18 is the method of embodiment 17, wherein the subsequently administered nucleic acid molecule, vector, or polypeptide is administered in an intramuscular injection.

Embodiment 19 is a kit comprising: a) a first Ad26 vector encoding a first antigen; and b) a second Ad26 vector encoding a second antigen; wherein the first and second antigen are different antigens.

Embodiment 20 is the kit of embodiment 19, further comprising: c) one or more nucleic acid molecules or vectors encoding the first antigen or one or more polypeptides comprising the first antigen; and d) one or more nucleic acid molecules or vectors encoding the second antigen or one or more polypeptides comprising the second antigen.

Embodiment 21 is the kit of embodiment 19 or 20, wherein the first antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

Embodiment 22 is the kit of embodiment 19 or 20, wherein the second antigen is selected from the group consisting of a filovirus antigen, a Zika virus antigen, a human immunodeficiency virus antigen, a coronavirus antigen, an influenza virus antigen, a respiratory syncytial virus antigen, a flavivirus antigen, a bacterial antigen, a parasitic antigen, and a cancer antigen.

Embodiment 23 is the kit of any one of embodiments 19-22, wherein the kit comprises about 5 x IO ¹⁰ adenoviral vectors, about 6 x IO ¹⁰ adenoviral vectors, about 7 x IO ¹⁰ adenoviral vectors, about 8 x IO ¹⁰ adenoviral vectors, about 9 x IO ¹⁰ adenoviral vectors, or about 1 x 10 ¹¹ adenoviral vectors of the first and second Ad26 vectors.

Embodiment 24 is the kit of any one of embodiments 20-24, wherein the nucleic acid molecule or vector is selected from the group consisting of Ad26, Ad35, Ad2, Ad5, Adi 1, Adl2, Ad24, Ad34, Ad40, Ad48, Ad49, Ad50, Ad52, Pan9, MV A, mRNA, IVT repRNA and saRNA.

Embodiment 25 is the kit of embodiment 24, wherein the nucleic acid molecule or vector is an Ad26 vector. Embodiment 26 is the kit of any one of embodiments 19-25, further comprising at least one syringe for injection of the first Ad26 vector, the second Ad26 vector, the one or more nucleic acid molecules or vectors encoding the first antigen or the one or more polypeptides comprising the first antigen, and/or the one or more nucleic acid molecules or vectors encoding the second antigen or the one or more polypeptides comprising the second antigen.

Embodiment 27 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first Ad26 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second Ad26 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 28 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject an MVA vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second Ad26 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 29 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first Ad26 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject an MVA vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 30 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first Ad26 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 31 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second Ad26 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 32 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) simultaneously administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier, together with the first antigen; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second Ad26 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 33 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first Ad26 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) simultaneously administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier, together with the second antigen; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 34 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject a first IVT repRNA vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second Ad26 vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the second Ad26 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 35 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first Ad26 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject an IVT repRNA vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 36 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject a first Ad35 vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject a second Ad35 vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

Embodiment 37 is a method for inducing an immune response against at least a first and a second antigen in a subject in need thereof, the method comprising: a) administering to the subject a first adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a first antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject the first MVA vector comprising a nucleic acid sequence encoding the first antigen as a boosting composition at least two (2) weeks apart; and b) administering to the subject a second adenovirus serotype 26 (Ad26) vector comprising a nucleic acid sequence encoding a second antigen, together with a pharmaceutically acceptable carrier as a priming composition; followed by administering to the subject a second MVA vector comprising a nucleic acid sequence encoding the second antigen as a boosting composition at least two (2) weeks apart; wherein the first and second antigens are different antigens, and wherein the first and second Ad26 vectors are administered to the subject at least two weeks apart.

EXAMPLES

Example 1: Sequence use of Ad26-based vaccines demonstrated limited impact of immunogenicity of second vaccination in nonhuman primates.

Materials and Methods

Ethics statement

The 3 studies were conducted in facilities assured by the National Institute of Health Office of Animal Welfare and accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC). The first study was performed at Advanced BioSience Laboratories Inc. (Rockville, MD; purchased by BIOQU AL Inc., Rockville, MD, US, BIOQUAL: Accreditation # 000624, US Department of Agriculture [USDA] license #51-R-0036). The second study was performed at Charles River Laboratories Edinburgh Ltd (East Lothian, United Kingdom: OLAW Foreign assurance: F16-00018 [A5020-01] and Establishment License PEL PCD 60/8607). The third study was performed at Charles River Laboratories, Inc. (Reno, US, AALAC accredited since 1997 (000918) Office of Laboratory Animal Welfare (OLAW) Animal Welfare Assurance #A4112-01 USDA, Research Registration 14-R-0144).

All animal research protocols were approved by the Institutional Animal Care and Use Committees at each center and the studies were conducted in compliance with the Animal Welfare Act, Public Health Service Policy on Humane Care and Use of Laboratory Animals and other federal statutes and regulations relating to animals and experiments involving animals. Import and export permits for vectors and NHP bio-specimens were obtained in compliance with US federal regulations and in accordance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora as overseen by the US Fish and Wildlife Service. Adenoviral Vectors

Replication-incompetent, El/E3-deleted recombinant Ad26 and Ad35 vectors were engineered using the AdVac® system as described elsewhere (Widjojoatmodjo et al., Vaccine 33:5406-14 (2015); Zahn et al., PLos One 7:e44115 (2012)), and as described in detail for Ad26.ZEBOV, Ad35.ZEBOV, Ad26.SUDV, and Ad26.Mos4.HIV (Baden et al., Lancet HIV 7:e688-98 (2020); Barouch et al., Lancet 392:232-43 (2018); Callendret et al., PLoS One 13:e0192312 (2018)). Ad26.Mos4.HIV consists of 4 Ad26 vectors: Ad26.Mos. LEnv (encodes a mosaic insert of the Env protein sequence), Ad26.Mos2S.Env (encodes modified Mos2 HIV-1 Env protein sequence), Ad26.Mosl.Gag-Pol (encodes Mosl, HIV-1 Gag and Pol protein), and Ad26.Mos2.Gag-Pol (encodes Mos2 HIV-1 Gag and Pol protein).

Briefly, codon optimized genes encoding the relevant transgene were inserted into the El -position of the Ad genomes under transcriptional control of the human cytomegalovirus promoter and the SV-40 polyadenylation sequence.

Cloning, rescue and manufacturing of the replication deficient Ad vectors using the complementing cell line PER.C6®was described previously (Zahn et al., PLoS One 7:e44115 (2012)) Virus particle (vp) titers in the viral preparations were quantified by measurement of optical density at 260 nm (Maizel et al., Virol. 36:115-25 (1968)). The vp/IU ratio was between 1 : 1 and 6: 1 for the viral preparations. Ad-mediated expression of the various transgenes was confirmed by Western blot analysis of cell-culture lysates from infected A549 cells or by PCR. MVA Vectors

MVA-BN-Filo is a trivalent recombinant MVA strain Bavarian Nordic (MVA-BN) based filovirus vaccine directed against Marburgvirus and Ebolavirus infection. The full- length coding sequences for GP antigens of MARV Musoke, EBOV Mayinga and SUDV Gulu as well as the nucleoprotein antigen from Tai Forest ebolavirus were codon optimized, synthesized (GeneArt, Regensburg, Germany) and inserted into MVA-BN and were generated as previously described (Callendret et al., PLoS One 13:e0192312 (2018)). MVA- mBN414A is a monovalent vaccine compromising a single MVA-BN® vector genetically engineered to express Mosl. Env, Mos2S.Env, Mosl.Gag-Pol, and Mos2.Gag-Pol HIV-1 proteins sequences, which was produced as described previously (Baden et al., J. Infect. Dis. 218:633-44 (2018)). MVA-RSV.FA2 (MVA-mBN235A) is a monovalent vaccine comprising a single MVA-BN® vector genetically engineered to express the F fusion protein of RSV strain A2 under the synthetic early-late promoter PrS. Primary chicken embryo fibroblast cells used for recombinant live, attenuated MVA virus-vectored vaccine generation and production were prepared from embryonated eggs and maintained in serum free conditions. Animals and housing

For study 1, a total of 19, 5-6 year old, healthy female cynomolgus macaques (Macaco fascicularis) of Vietnamese origin (body weight 3-8 kg at study start) were rolled over from a previous study (Salisch et al., NPJ Vaccines 4:54 (2019)). The animals were originally purchased from Covance (Alice, TX). Five of the animals had received previous vaccination with an Ad26-Ad26 homologous regimen, and 5 had previously received an Ad26-Ad35 heterologous regimen. The insert encoded by these vectors was RSV.FA2 (Salisch et al., NPJ Vaccines 4:54 (2019)). Nine other animals were vaccine-naive upon enrollment.

For study 2, 12 4-5 year-old healthy female cynomolgus macaques of Mauritian origin (body weight 3-7 kg at study start) were rolled over from a previous study and were purchased by Charles River Edinburg. Six of the animals had previously received 2 doses of an Ad26 vector expressing a fusion protein of RSV.FA2 and Gaussian luciferase, and a dose of MVA encoding RSV.FA2, and 6 were vaccine-naive upon enrollment.

For study 3, 12 4-7 year-old healthy male and female cynomolgus macaques of Chinese origin (body weight 3-7 kg at study start) were rolled over from a previous study and were purchased by Charles River Reno NV and Alpha Genesis Inc. Six of the animals had previously received an MVA-Ad26 heterologous regimen. The inserts of those vectors were ZEBOV GP for Ad26 and MVA-BN-Filo. Six other animals were vaccine-naive upon enrollment.

All animals were kept in a BSL-2 facility under specific pathogen-free conditions after screening negative for Mycobacterium tuberculosis, simian immunodeficiency virus, simian retrovirus, and simian T-lymphotropic virus. Screening included Herpes B virus and measles serology.

Animals in study 1 and study 3 were pair-housed in groups of 2 or 3 animals in stainless steel cages placed in study-dedicated, USDA and OLAW approved rooms, while animals in study 2 were social housed in groups of 6 in two story gang pens, also OLAW- approved. Animals in all 3 studies were kept under controlled, recorded environmental conditions of humidity, temperature, and light (12-hour light cycle). For all 3 studies animals of the same sex and study group per cage were co-housed, except for brief, procedure-related periods. Animals were provided with sensory and cognitive environmental enrichment including manipulatable objects and foraging devices. Three times a day, animals were fed a standard NHP diet that consisted mainly of high-protein monkey biscuits, but included primatreats, soft dough diet, and a selection of fresh fruit, peanuts, cereals, or other treats. Tap water was provided ad libitum through an automated system. Animal well-being was monitored daily by husbandry staff and routine animal health surveillance, including evaluation of blood chemistry and hematology, was provided by veterinary staff. Pre-set humane endpoints were used to define study -unrelated sacrifice criteria by a veterinarian. All measures were taken to minimize pain, distress and suffering and all procedures were performed by trained personnel.

Study design and animal procedures

For study 1 and study 3, all animal procedures were performed under anesthesia either with ketamine (10-15 mg/kg intramuscularly) or Dormitor (0.015 mg/kg intramuscularly).

For study 2, animals were trained, therefore vaccination and blood sampling were performed without the use of anesthesia.

Animals were assigned to the study treatment groups based on vaccine A series administered, receiving a vector-backbone homologous regimen matching the initial regimen for the B series (e.g., an Ad26-Ad26 regimen twice). The initial vaccinations were given at either 12-week (study 1 and 2) or 8-week (study 3) intervals. Treatment groups in the present study were named according to the administered vector backbones with ‘rep’ indicating that the sequence was identical to that received previously. The interval between the last dose of the first study (A series) and the first dose of the subsequent vaccination (B series) was 55 weeks for study 1, 26 weeks for study 2, and 57 weeks for study 3 (FIG. 1).

Animals in study 1 were divided into 4 study groups with 4-5 animals per group (FIG. 2A). The animals received 2 doses (5xlO ^lo vp) of either Ad26.ZEBOV (2-dose homologous regimen, 26/26 rep group) or Ad26.ZEBOV followed by Ad35.ZEBOV (2-dose heterologous regimen, 26/35 rep group) with an 8-week interval between doses. Control animals who had not received any prior treatment received either a 2-dose homologous Ad26-Ad26 regimen (26/26 group), or a 2-dose heterologous Ad26-Ad35 regimen (26/35 group).

Animals in study 2 were divided into 2 study groups (pre-exposed or unexposed) with 6 animals per group (FIG. 2B). Both groups received a 2-dose heterologous vaccination regimen of 5xlO ¹⁰ vp of Ad26.SUDV at dose 1 followed 8 weeks later by 10 ⁸ vp units (ifu) of MVA-BN-Filo (26/MVA rep group and 26/MVA group).

Animals in study 3 were divided into 2 study groups (pre-exposed or unexposed) with 6 animals per group (FIG. 2C). Both groups received a 2-dose heterologous vaccination regimen of 5xlO ¹⁰ vp of Ad26.Mos4.HIV followed 12 weeks later by 10 ⁸ vp units (ifu) of MVA-mBN414A (26/MVA rep group and 26/MVA group).

All vaccines were administered in a 0.5 mL volume, intramuscularly in the quadriceps with the indicated vector particle-doses in formulation buffer. Venous blood for PBMC isolation or serum was collected from the femoral vein. Blood volumes taken did not exceed 12 ml/kg within 30 days and a maximum of 9 ml/kg at each individual bleeding time point. Processing of peripheral blood

Serum samples were prepared from clotted blood drawn into serum tubes after spinning at 1900 G for 5 minutes at room temperature (RT). Serum was stored at -80°C until time of analysis. PBMCs were isolated from whole blood drawn into anticoagulantcontaining tubes (EDTA) by Ficoll density gradient centrifugation. Blood was diluted 1 : 1 with D-PBS without Ca ²⁺ or Mg ²⁺ (Quality Biological; Gaithersburg, MD), underlayed with an equal volume of Ficoll-Paque Plus (GE Healthcare; Little Chalfont, UK), spun at 1750 G for 40 minutes at RT. Buffy layers were transferred into a fresh tube, washed 3 times with D- PBS, spun at 393 G for 5 min at RT. When needed, lysis of residual red blood cells (RBCs) in RBC Lysis Solution (Qiagen; Hilden, Germany), or ACK lysis buffer (Lonza Bio Whittaker; Basel, Switzerland) for 10-15 minutes at RT was performed. Lysis was stopped by addition of excess D-PBS and tubes spun at 1750 G for 5 minutes at RT. Viable cell numbers were subsequently determined by Trypan Blue exclusion using a Countess Automated Cell Counter (Thermo Fisher Scientific; Hampton, NH) or using ViaCount reagent with a third generation GUAVA® EASYCYTE™ cytometer. For cells processed directly for IFNy ELISpot, cells were adjusted to a concentration of 2xl0 ⁶ cells/ml in RPML10 (RPMI complemented with 10% fetal bovine serum (FBS), Fisher Scientific, Hampton, NH), lOmM Hepes buffer (Quality Biological, Gaithersburg, MD), 2mM L-glutamine (Quality Biological), lOOpg/ml penicillin/streptomycin (Quality Biological) and kept on ice until analysis. Alternatively, cells were adjusted to a concentration of 5x10 ⁶ cells/ml in CryoStor® and samples were aliquoted and transferred into liquid nitrogen until use.

Frozen cells were thawed in a 37°C water bath, washed with RPML10, spun at 400G for 5 minutes at RT, and subsequently washed twice. Cells were counted as indicated above using ViaCount reagent/Guava. Cells were adjusted to a concentration of 10 ⁷ /ml and cultured in T-25 flasks in a 37°C, water jacked 5% CO2 incubator for 18 to 24 hours. Following incubation, cells were counted using ViaCount reagent/Guava and adjusted to a concentration of 2.5xl0 ⁶ cells/ml in RPML10 and kept on ice until analysis. Adenoviral neutralization assay

Ad26 Nab titers in serum were assessed using a luciferase-based VNA based on the method previously described (Sprangers et al., J. Clin. Microbiol. 41 :5046-52 (2003)). Briefly, A549 human lung carcinoma cells (ATCC® CCL-185™) were plated at a density of l x l0 ⁴ cells/well in 96-well black-and-white isoplates (Wallac, Turku, Finland). E1ZE3- deleted Ad26-luciferase reporter constructs were then added at a multiplicity of infection of 500-1000, together with 2-fold serial dilutions of individual heat-inactivated cynomolgus macaque serum samples starting at a 1 :32, 1 :64 or 1 : 128 dilution for Ad26 (depending on the study, see figure legends for the exact start dilution). After incubation for 20-24 h at 37°C and 10% CO2, luciferase activity was measured using the Neo-Lite Luciferase Assay System (Perkin Elmer; Waltham, MA) and a BioTek Synergy Neo luminescence counter (BioTek; Winooski, VT) or EnVision multimode plate reader (Perkin Elmer). A 90% neutralization titers (IC90) was defined as the maximum serum dilution that neutralized 90% of luciferase activity. Each serum sample was analyzed in duplicate.

IFN -ELISpot

Antigen-specific, IFNy-secreting T-cells were enumerated in isolated PBMCs using an ELISpot kit specific for monkey IFNy (Monkey IFNy ELISpot ^PRO , MabTech). Plates precoated with an NHP IFNy-specific capture antibody (clones GZ-4 or MT126L) were washed 4 times with sterile D-PBS (180pl/well) and blocked with RPMI-10 (200 pl/well) for 30 minutes at 37°C and 5% CO2. After removal of the blocking buffer, PBMCs in RPMI-10 were seeded at 2-5xl0 ⁵ cells/well and stimulated with peptide pools reconstituted in dimethylsufoxide (DMSO), consisting of 15-mers overlapping by 11 amino acids at a final concentration of 2 pg/ml for 18-20 hours at 37°C and 5% CO2, in a final volume of 200 pl.

For study 1, two peptide pools covering the ZEBOV GP protein N-terminal and C- terminal sequence were used to limit the number of peptides per pool (43 to 58 peptides/pool). The results of the N- and C-terminal pools for ZEBOV GP were pooled for reporting purposes.

In addition, 5 peptide pools containing shared and specific peptides for Ad26 and Ad35 hexon protein were used. Responses are reported as either Ad26-hexon specific (sum of two Ad26-specific peptide pools) or total hexon response (sum of the responses to the five (5) peptide pools).

For study 2, two peptide pools covering the SUDV Gulu GP protein N-terminal and C-terminal sequence were used to limit the number of peptides per pool (43 to 58 peptides/pool). Peptides that overlapped with more than 9 consecutive amino acids within the EBOV Mayinga, SUDV Gulu and TAFV Ebola strains or MARV Angola and Ravn strains were combined into a consensus pool SUDVcon (-100 peptides/pool). The responses given in the figures are a sum of the background subtracted responses induced to the 3 peptide pools (N- and C-terminal pools and SUDVcon for SUDV GP).

For study 3, individual peptide pools covering the Envelope (Env-1, Env-2 and Env- 3), the Group-specific antigen (Gagl- and Gag-2) the Polymerase protein (Pol-A, Pol-B and Pol-C) were used (Coughlan, Front. Immunol. 11 :909 (2020)). The total Env response given in the figures is a sum of the background subtracted response induced to the 3 individual sub- Env pools (Env-1, Env-2 and Env3). Likewise, the total Gag and total Pol response is a sum of the response elicited to the individual sub-Gag pools and sub-Pol pools after background subtraction, respectively

RPML10 supplemented with 0.005-0.33 % DMSO served as a medium control and a 1/1000 dilution of a-CD3 antibody or 5.5 mg/ml PHA in RPMI-10 as a positive control. After removal of the cell suspension, wells were washed 5 times with PBS + at RT and subsequently incubated with INFy detector antibody conjugated to alkaline phosphatase (clone 7-B6-1-ALP, 1 :200 in PBS + 0.5% FBS) for 2 hours at RT. Plates were washed as described before and spots developed for 15 minutes in the dark at RT using a 5-bromo-4- chl oro-3 '-indolyphosphate p-toluidine/nitro-blue tetrazolium chloride solution filtered through a 0.45 pm filter. The development was stopped by washing extensively with tap water. Plates were air dried for at least 24 hours before spots were counted on an ImmunoSpot S5 ELISpot plate reader (C.T.L. Europe GmBH, Bonn, Germany), or A EL VIS ELISpot plate reader, and counting was done with Eli- Analyse ELISpot Image Analysis software (A EL VIS GmBH Europe, Hannover). All samples were analyzed in either duplicate or triplicate. Mean spot-forming units (SFU) per 10 ⁶ cells were calculated from the replicate measurements, followed by individual background subtraction of the mean medium control values from the mean peptide-stimulated values. For antigens covered by more than 1 peptide pool, background subtracted mean peptide-stimulated values were summed per animal per time point. Based on historical data the background/ threshold was empirically set at 50 SFU/10 ⁶ PBMC. Values below the threshold of 50 SFU/10 ⁶ PBMC were set at half that threshold (25 SFU/10 ⁶ PBMC) for the purpose of graphical representation. For calculation of the fold change, values below the threshold of 50 SFU/10 ⁶ PBMC were set at this threshold. To calculate the fold change from pre-dose 1 to peak response post-dose 1, the peak response post-dose 1 per animal was divided by the response measured pre-dosing (all studies week -2). Similarly, to determine the fold change from pre-dose 2 to peak response post-dose 2, the peak response post-dose 2 per animal was divided by the response measured pre-dosing (study 1 : week 8; study 2: week 4; study 3: week 12).

Determination of ZEBOV GP specific IgG in serum by ELISA

Total serum IgG targeting GP of ZEBOV was assessed by an ELISA qualified and validated for human sera as described previously (Callendret et al., PLoS One 13:e0192312 (2018)). Briefly, Maxisorp™ 96-well plates (Nunc-Immuno) were coated over night at 4°C with Galanthus Nivalis Lectin (GNA, SIGMA Aldrich; St. Louis, MO) diluted in PBS at 10 pg/ml. Remaining lectin solution was removed and 200 pl PBS/10% FBS added for 90 minutes at RT. The plates were washed as described with PBS/0.2% Tween20 (Sigma- Aldrich) (PBS-T), coated with supernatant containing recombinant filovirus GP for 90 minutes at RT, and then washed again. Serum from NHPs was serially diluted (starting dilution 1 :50) in sample buffer (PBS/0.2% Tween/1% FBS). 100 pl of diluted sample was transferred to the coated Maxisorp 96-well ELISA plates, incubated for 90 minutes at RT and washed as described. Bound IgG was detected with goat-anti-human IgG (H+L) conjugated to HRP (Millipore USA). Reactions were stopped and measured at 492 nm. IC50 values were calculated by 4-parameter curve-fit and compared against a filovirus GP strain-specific reference serum. Results were expressed as ELISA units (EU) /ml.

Determination of SUDV GP specific IgG in serum by ELISA

Total serum IgG targeting GP of SUDV Gulu was determined by ELISA. Maxisorp™ 96-well plates were coated over night at 4°C with purified SUDV GP protein diluted in 20mM Tris-HCl solution at a concentration of 0.25 pg/ml. After washing 3 times with 200ul PBS-T at RT, plates were blocked with 180 pl PBS/10% FBS at RT for 90 minutes. The plates were washed 3 times as indicated above with PBS-T. NHP serum was serially diluted (3-fold steps) in sample buffer starting at a dilution of 1 :45 (PBS/0.2% Tween/1% FBS, sample buffer) in round-bottom polypropylene plates (Nunc Cat#267245).

100 pl of diluted sample was transferred to Maxisorp 96-well ELISA plate and incubated at RT for 60 minutes. Plates were washed 3 times with PBS-T as indicated above. Bound IgG was detected with goat-anti-human IgG (H+L) conjugated to HRP (Cayman), diluted 1 : 5000 in sample buffer and incubated for 1 hour at RT. Plates were washed 3 times with 200 pl PBS-T. lOOul of LumiGlo was added and incubated in the dark for 30 minutes at RT. The reaction was measured at 492 nm. Endpoint titers were compared against a filovirus GP strain-specific reference serum and expressed as ELISA units (EU) /ml. Determination of Env Clade C and Mosl specific IgG in serum by ELISA Antibody binding to the clade C gpl40 and mosaic gpl40 antigens was determined by ELISA as described previously (Pollard et al, Lancet Infect. Dis. 21 :493-506 (2021)). Briefly, antigen (HIV_Env_C_C97ZA and HIV Env Mosl) (Barouch et al., Lancet 392:232-43 (2018)) was coated at Ipg/mL in PBS and serum samples were tested undiluted resulting in 1/10 serum dilution in the final ELISA plate and incubated on plates. Binding antibody was determined using horseradish peroxidase-conjugated detection antibody mouse-anti-human IgG (Jackson Cat#209-035-011, 1 :20,000) and SureBlue TMB (SeraCare 5120-0047). The final concentration of each sample was calculated using Gen5 software. The concentration is equivalent to the back-calculated concentration of the measured OD450-value onto the 4PL curve-fit of the standard curve as described previously (Barouch et al., Lancet 392:232-43 (2018)).

Ebola Pseudovirus neutralization assays

The filovirus pseudovirion neutralization assay (psVNA) was performed exactly as described previously (Roozendaal et al., NPJ Vaccines 5: 112 (2020)). Pseudovirus preparations were generated by co-transfection of human embryonic kidney (HEK) 293 cell cultures with a replication defective retroviral vector containing a luciferase gene along with an expression vector containing EBOV Makona GP sequence. Pseudovirus stocks were generated and characterized for suitability to assess EBOV-specific neutralization. Pseudoviruses were incubated with serial dilutions of serum samples and used to infect HEK293 cell cultures. Each serum sample was serially diluted 10 times (four-fold), starting from a dilution of 1 :40. The ability of serum to neutralize EBOV pseudovirus infectivity was assessed by measuring luciferase activity ~72 hours post-viral inoculation versus a control infection using a murine leukemia virus envelope (aMLV) pseudotyped virus. Neutralization titers were expressed as the reciprocal of the serum dilution that inhibited the virus infection by 50%.

Statistical analyses

Immunological parameters (i.e., ELISA, VNA, ELISpot) were log-transformed. Two types of analysis were performed: group comparisons per time point per study using ANOVA for potentially censored values (Tobit model). The fold changes in response after the A and B series vaccination (i.e., pre-dose 1 vs post-dose 1, and pre-dose 2 vs post-dose 2) for all immunological parameters (i.e., ELISA, VNA, ELISpot) were calculated per animal and then log-transformed. For study 3, in addition to the comparisons per antigen, the average fold changes over the Mosl and Clade C antigens (ELISA), and over the Gag, Pol, Env antigens (ELISpot) were calculated and analyzed. Vaccine regimens were subsequently compared using ANOVA, both per study and pooled across studies. P-values < 0.05 were considered statistically significant and a Bonferroni correction for 2 comparisons was applied for the analysis of study 1.

Results

Three independent studies were conducted in cynomolgus macaques. Ad26-vector based vaccines were administered as 2- or 3-dose homologous regimens or as a part of heterologous regimens in combination with Ad35 and MVA-vector based vaccines (FIG. 1 and FIG. 2A). Animals first received a homologous or heterologous vaccine-regimen (referred to as A series), then 26 to 57 weeks later received the same vector-regiment but with vectors encoding different transgenes (B series), to mimic a clinical situation where individuals would receive multiple different Ad26-vectored vaccine regimens during their lifetime. Animals dosed in A series are further referred to as “pre-exposed,” whereas control animals only dosed in B series are referred to as “unexposed.” The annotation after the vaccine regimen referred to “repeated dosing,” e.g., animals dosed in both A and B series.

Peripheral blood mononuclear cells (PBMC) and serum were collected from animals at defined time points during the A and B series to longitudinally assess the cellular and humoral immune responses directed towards the vector-backbone and the antigen.

Ad26 vaccine-elicited anti-vector neutralizing antibody and T-cell responses

First, the level of anti-Ad26 Nabs in the serum of animals after the A series of the 2- dose homologous Ad26-Ad26 or heterologous Ad26-Ad35 vaccine vector regimen was determined. Ad26 Nabs were elicited in all animals after the first dose, increased after a second dose with Ad26 but not Ad35, diminished and remained detectable at lower levels for more than 50 weeks post-last Ad26 vaccine dose of the A series (FIG. 3 A). Administration of the first, but not the second dose of the Ad26 vaccine encoding ZEBOV GP of the B series further amplified the Ad26 Nab titers. Ad26 Nab titers elicited in B series by the 2 doses of Ad26. ZEBOV GP in unexposed animals were in the same range as those induced during the A series of Ad26 RSV-FA2.

Heterologous vaccination with Ad26-Ad35 or Ad26/MVA regimens elicited comparable levels of the anti-Ad26 Nab titers to those elicited by the homologous Ad26- Ad26 vaccine-regimen after the first vaccination dose, while, higher anti-Ad26 Nab titers were seen with the Ad26-Ad26 vaccine-regimen post the second dose of Ad26 compared with post second dose of Ad35 or MVA in the A series (FIG. 3B, FIGs. 4 A and 4B).

T cell responses targeting the most abundant adenoviral structural protein, hexon, elicited by the Ad26 and Ad35 vectors were assessed in study 1 by interferon gamma (IFNg) ELISpot. 5 peptide pools containing shared and specific peptides for Ad26 and Ad35 hexon protein were used. Responses are reported as either Ad26-hexon specific (sum of two Ad26- specific peptide pools) or total hexon response (sum of the responses to the five peptide pools).

Ad26-hexon-protein specific T cell responses were elicited by Ad26 vaccination (FIGs. 3C and 3D) and reached peak IFNy ELISpot counts on an average of 456 spot forming units (SFU)/10 ⁶ cells 2 weeks post-last Ad26 dose in the A series, followed by a considerable contraction by the time of the B series 50 weeks later. In line with Ad26 Nab titers, first dosing with Ad26.ZEBOV in the B series resulted in a sharp increase in Ad26 hexon-specific IFNy ELISpot responses in pre-exposed (Ad26/Ad26rep: 466 SFU/10 ⁶ cells; Ad26/Ad35rep: 918 SFU/10 ⁶ cells), while a lower increase was seen in previously unexposed animals (Ad26/Ad26: 109 SFU/10 ⁶ cells; Ad26/Ad35: 144 SFU/10 ⁶ cells). The second dose of Ad26 or Ad35 in the B series 8 weeks later did not further increase Ad26 hexon-specific T cell responses in pre-exposed animals (Ad26/Ad26rep: 293 SFU/10 ⁶ cells; Ad26/Ad35rep: 445 SFU/10 ⁶ cells), whereas an increase was observed in the unexposed animals (Ad26/Ad26rep: 303 SFU/10 ⁶ cells; Ad26/Ad35rep: 558 SFU/10 ⁶ cells) at week 10 similar to the A series responses. A similar trend was observed for total Hexon responses (FIGs. 4C and 4D).

Ad26 vector pre-exposure has no consistent impact on antigen specific responses induced by one dose of Ad26

Having established that neutralizing Ad26 antibodies and hexon-specific T cells were present after the A vaccination series, the antigen-specific immune response following the B series of Ad26 vaccines in a homologous regimen, or in a heterologous regimen with Ad35 or MVA vaccine vectors was investigated.

Across all three studies, no major impact of pre-existing vaccine-induced Ad26 immunity on antigen-specific cellular immunity, as measured by IFNy ELISpot, was detected after the first Ad26 dose in the B series (FIGs. 5A and 5C-5E). This observation held true irrespective of whether the immune response was directed against a membrane-bound antigen such as the glycoproteins of ZEBOV or SUDV, the HIV envelope protein (Env) (FIGs. 5A- 5D), or intracellular antigens such as HIV group-specific antigen (Gag) or HIV polymerase protein (Pol). The only statistically significant difference was observed in the ZEBOV response at week 4, where a higher IFNy ELISpot response was seen in Ad26/Ad35 compared to Ad26/Ad35rep dosed animals (p=0.046, pairwise comparison) (FIG. 3B, Table 1). However, no significant differences were detected in the relative fold change in antigen- specific IFNy ELISpot responses at the post-dose 1 peak response over baseline response for any of the antigen between Ad26 pre-exposed and unexposed animal (study 1 : comparison Ad26rep vs unexposed, p=1.0000; study 2: Ad26rep vs unexposed, p=0.3400; study 3: Ad26rep vs unexposed p=0.6700, ANOVA) (FIG. 5E, Table 2). Table 1 : Statistical results for the magnitude of cellular immune responses

Table 2: Comparison of the fold changes in immune responses for cellular and humoral responses per study and across studies using ANOVA

As observed for cellular responses, pre-exposure did not result in consistent decrease of absolute antigen-specific humoral immune responses induced by the first Ad26 dose in the B series as measured by IgG-ELISA, although the impact was more nuanced (FIGs. 6A-6E, Table 3). In study 1, a trend for higher Ebola binding antibody titers was seen in the Ad26- Ad26rep group compared to the Ad26-Ad26 group (FIG. 6A) (Table 3). Similarly, no impact of pre-existing immunity on Ebola Zaire binding antibodies was observed in animals which received Ad26-Ad35 in the A series (FIG. 6B). There was a transient, but inconsistent difference observed in the absolute Ebola Nab titers in pre-exposed animals (groups Ad26/Ad26rep and Ad26/Ad35rep) compared with unexposed animals (groups Ad26/Ad26 and Ad26/Ad35) (FIG. 7A-7B). There was a higher response in Ad26/Ad26 compared to Ad26/Ad26rep dosed animals (Table 3).

Table 3: Statistical results over the magnitude of humoral responses, pairwise comparison.

The impact of pre-existing Ad26 immunity on Ebola SUDV GP antibody titers (study 2) was transient with a statistically significant lower response observed at week 4 (p=0.0022, Tobit model, but not at week 2 (p=0.0977, Table 3) (FIG. 6C). By contrast, binding antibody titers to Env Clade C and Env Mosl (study 2) were transiently significantly higher in animals with pre-existing immunity at week 4 (Clade C: p=0.0443, Mosl : p=0.0323, Tobit model) but not at week 12 (Clade C: p= 0.3629, Mosl : p = 0.3895) (FIGs. 6D-6E). Analysis of the relative fold change in antibody titers showed that for study 1 and 3 there was no statistically significant difference between animals with or without pre-existing Ad26 immunity (study 1 comparison Ad26/Ad26rep vs Ad26/Ad26, p =0.5371; study 3: Ad26/MVArep vs Ad26/MVA, p= 0.2891, ANOVA), whereas for study 2, a significantly lower fold increase was observed in animals with pre-existing Ad26-vaccine-induced immunity (Ad26/Ad26rep vs Ad26/Ad26: p=0.0189, ANOVA) (FIG. 6F, Table 2). For study 1, next to binding antibody titers also Ebola Zaire Nab titers tended to be reduced in animals with pre-existing Ad26 immunity (FIG. 7A), with significant difference seen compared with the non-exposed animals at week 10 (Ad26/Ad26rep vs Ad26/Ad26, p = 0.188, ANOVA) and 12 (Ad26/Ad26rep vs Ad26/Ad26, p = 0.138, ANOVA) but not at week 4 and week 19 and also did not reach statistical significance when comparing the fold change from pre-dosing to post-dose 1 (FIGs. 7C-7D).

Together these data suggest that pre-existing vaccine-elicited Ad26 immunity was associated with temporary and inconsistent effects on the cellular and humoral antigenspecific responses induced by the first Ad26 in series B, which did not amount to a consistent or substantial negative impact.

Vector pre-exposure has no consistent impact on vaccine-antigen-specific responses induced by a second dose of vaccine, irrespective of vector.

Next, the immune response induced after the second vaccine dose in the B series, consisting of vaccines based on either Ad26, Ad35 (Study 1) or MVA (study 2 and 3) was assessed. For study 1, higher absolute ZEBOV-specific IFNy ELISpot responses were induced in unexposed compared to pre-exposed animals receiving the heterologous Ad26/Ad35 regimen, which was most pronounced at week 10 and 12 (week 10: p=0.0016, week 12: p=0.011, Tobit model, Table 1), but not for the homologous Ad26/Ad26 regimen (FIGs. 8A-8B). There was a comparable fold increase in ZEBOV GP-specific IFNy ELISpot responses above pre-boosting levels following a second (homologous) dose of Ad26. ZEBOV at week 8 in animals with and without pre-existing Ad26 immunity (FIG. 8E, Table 2).

For the heterologous Ad26-MVA schedule (study 2) the magnitude of SUDV-specific IFNy-producing cells after MVA-BN-Filo dosage at week 9 was comparable for animals preexposed to Ad26 or not and thereafter a trend for an increase in SUDV-specific IFNy- producing cells over time in the Ad26 unexposed group was observed. A similar increase was not observed in the animals with pre-existing immunity to Ad26 and MVA, although the difference between the pre-exposed and unexposed animals did not reach statistical significance (FIG. 8C, Table 3). Furthermore, there was no clear difference in the relative fold change of the peak response between pre-exposed and unexposed animals (p=0.2793, ANOVA) (FIG. 8E, Table 2).

In study 3, in which animals were dosed with Ad26.Mos4.HIV-MVA-mBN414A (Ad26/MVA rep group), higher levels of the individual, absolute Env, Gag, and Pol-specific IFNy-ELISpot responses were induced in pre-exposed versus unexposed animals after the second dose, which reached statistical significance for the Pol-specific response (p=0.0494, Tobit model), but not for the Env or Gag responses (FIG. 8D, Table 1). On the other hand, a significant difference was detected between pre-exposed and unexposed animals in terms of the relative fold-increase in individual Env-specific responses (p=0.0107, ANOVA, Table 2), but not in the individual Gag antigen (p=0.7088, ANOVA), individual Pol antigen (p=0.00788, ANOVA), or the average fold-change of the 3 antigens (p=0.0877, ANOVA) (FIG. 8E, Table 2).

An analysis of all 3 studies pooled mirrored the results from the individual studies, showing no statistically significant difference in the fold change of the cellular immune response in animals pre-exposed to Ad26, Ad35 or MVA vectors compared to unexposed animals (p=0.4530, ANOVA).

For vaccine-induced humoral immune responses, specific antibody titers induced by the final vaccine dose in the different regimens were similar in animals with and without preexisting Ad26, Ad35 and MVA immunity at all observation timepoints (FIGs. 9A-9E).

The relative fold-change response comparing pre-dose 2 to the post-dose 2 peak response showed a significantly higher ani-SUDV-specific antibody response in animals preexposed to Ad26 and MVA than in unexposed animals (p=0.0044 ANOVA). For the other vaccination schedules, no difference in fold-change between pre-exposed and unexposed animals was observed for the humoral immune response (FIG. 9F). Similarly, pooled results across the three studies (FIG. 9F) showed no statistically significant difference when comparing the relative fold change induced in pre-exposed versus unexposed animals (Ad26- Xrep vs Ad26-X, where X refers to either Ad26, Ad35, or MVA, p = 0.5046, ANOVA).

Absolute Ebola Zaire GP Nab titers in animals pre-exposed and unexposed to Ad26 and/or Ad35 in study 1 were comparable with no statistically significant differences observed in the relative fold change (pre-dose 2 to peak response post-dose 2) (Ad26/Ad26rep vs Ad26/Ad26 p =0.2780; Ad26/Ad35rep vs Ad26/Ad35 p=0.1876, ANOVA) (FIGs. 7C-7D).

Taken together, the data from 3 studies indicate that Ad26 vaccine-induced immunity has a limited impact on immune responses induced by subsequent Ad26 vaccines either in a homologous dosing or combined with Ad35 or MVA vaccines in NHPs.

Example 2: A study to evaluate innate and pro-inflammatory responses of Ad26.RSV.preF-based vaccine, Ad26.COV2.S vaccine, and Ad26.ZEBOV vaccine.

The vaccines assessed in this study are: 1) Ad26.COV2.S, a monovalent vaccine composed of a recombinant replication incompetent human Ad26 vector, constructed to encode the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus spike (S) protein, stabilized in its prefusion conformation;

2) Ad26/protein preF RSV vaccine, a respiratory syncytial virus (RSV) vaccine that is a combination of 2 vaccine components, administered as a single intramuscular (IM) injection. The 2 vaccine components are: a) Ad26.RSV.preF, a recombinant, human replication-incompetent adenovirus serotype 26 (Ad26) vector, containing a DNA transgene that encodes the prefusion conformation stabilized F protein (pre-F) derived from the RSV A2 strain; and b) RSV preF protein, a recombinant pre-F protein derived from the RSV A2 strain; and

3) Ad26.ZEBOV, a monovalent vaccine composed of recombinant, human replication incompetent Ad26 vector, constructed to encode the full-length Ebola virus (EBOV, formerly known as Zaire ebolavirus) Mayinga GP.

Participants who received Ad26.ZEBOV as the first vaccination (Group 3), receive MVA-BN-Filo, at a dose level of 1 * 10 ⁸ infectious units (Inf. U) on Day 57, to complete the vaccine regimen.

4) MVA-mBN226B (further referred to as MVA-BNR-Filo), a multivalent vaccine encoding the EBOV GP, the SUDV GP, the MARV Musoke GP, and the TAFVNP (formerly known as Cdted’Ivoire ebolavirus). The EBOV GP encoded by MVA-BN-Filo has 100% homology to the one encoded by Ad26.ZEBOV.

Note'. All participants are unblinded on the Day 29 visit. Participants who received the Ad26/protein preF RSV vaccine or Ad26.ZEBOV as the first vaccination are offered a single dose of study vaccine Ad26.COV2.S at 5* 10 ¹⁰ vp on Day 29 (primary analysis timepoint) to allow access to a COVID-19 vaccine (per local regulations). No study assessments other than serious adverse events (SAE)/adverse events of special interest (AESI) follow-up are performed for the optional vaccination with Ad26.COV2.S on Day 29.

The aim of this study is to explore innate, pro-inflammatory, and other relevant pathway responses that are different or shared between different Ad26-based vaccines and to explore whether these responses are associated with the reactogenicity and adaptive humoral and cellular immune responses induced by Ad26-based vaccines. Gene expression analysis in blood cells can provide information on the inflammatory signals and pathways triggered by Ad26.COV2.S, Ad26/protein preF RSV vaccine, and Ad26.ZEBOV.

Gene expression profiles are generated from whole blood obtained at different timepoints post vaccination to provide insights into putative and potentially unique pathways that are triggered by Ad26-based vaccines. In addition, serum samples collected at early timepoints can provide information regarding systemic cytokines and chemokines and other protein and lipid mediators of innate activation to complement the transcriptome analysis. Samples to assess cellular and humoral immune responses and reactogenicity safety data are also collected.

Table 5: Objectives and Endpoints

OVERALL DESIGN

This is a randomized, observer-blind, multicenter, interventional Phase 1 study in adult participants aged 18 to 59 years in stable health. A target of 160 participants are randomized in this study in a 2: 1 : 1 ratio to 1 of 3 groups (Group l :Group 2:Group 3) as outlined below. Group 1 will include approximately 50% SARS.CoV.2 seropositive and 50% SARS.CoV.2 seronegative participants. Guidance on SARS.CoV.2 serostatus distribution is not applicable to Groups 2 and 3.

--Group 1 will receive Ad26.COV2.S (5* IO ¹⁰ vp) on Day 1 —Group 2 will receive Ad26/protein preF RSV vaccine (1 x 10 ¹¹ vp/150 pg) on Day 1

—Group 3 will receive Ad26.ZEBOV (5 x 1O ¹⁰ vp) on Day 1 and MVA-BN-Filo on Day 57.

All participants are stratified by SARS-CoV-2 serostatus. All study vaccines are administered by intramuscular (IM) injection. All enrolled participants are unblinded at the Day 29 on-site unblinding visit (after completing all Day 29 procedures) and continue the study in an open-label fashion with follow-up until 6 months post first vaccination.

Participants who received the Ad26/protein preF RSV vaccine or Ad26.ZEBOV as the first vaccination (Groups 2 and 3) are offered a single dose of Ad26.COV2.S at 5* IO ¹⁰ vp on Day 29. Participants who received Ad26.ZEBOV as the first vaccination (Group 3), receive MVA-BN-Filo, at a dose level of 1 * 10 ⁸ infectious units (Inf. U) on Day 57, to complete the vaccine regimen. MVABN-Filo is not administered within 28 days of the optional Ad26.COV2.S vaccination on Day 29.

Table 6: Study Design

* All participants are unblinded on Day 29. If the participant received the Ad26/protein preF RSV vaccine or Ad26.ZEBOV as the first vaccination (Groups 2 and 3), the participant is offered a single dose of Ad26.COV2.S at 5 x 1010 vp on Day 29

** Group 1 includes approximately 50% SARS.CoV.2 seropositive and 50% SARS.CoV.2 seronegative participants.

After vaccination on Day 1, participants remain under observation at the study site for at least 15 minutes for presence of any acute reactions and solicited events. Any unsolicited AEs, solicited local (injection site) or systemic AEs, and vital signs (systolic and diastolic blood pressure [sitting], heart rate, respiratory rate, and body temperature) are documented by study-site personnel following this observation period. In addition, participants record solicited signs and symptoms in a diary for 7 days post-vaccination on Day 1. MVA-BN-Filo on Day 57 (Group 3) is administered per instructions in the label. Safety assessments following MVA-BN-Filo vaccination are limited to SAE and AESI follow up until end of study.

Blood is collected from all participants to assess humoral and cellular immune responses pre-vaccination and at 7 days, 28 days, and 6 months post first vaccination. Additionally, blood samples for mRNA expression analysis (PAXgene) and innate immune responses are collected pre-vaccination and at 1 day, 3 days, 7 days, 28 days, and 6 months post first vaccination for all groups. An additional blood sample is collected 3 months post vaccination in Group 1 participants only.

Safety issues that might arise from this study are escalated to an internal Data Review Committee (DRC), as needed.

The end of the study is defined as the last participant’s last visit.

NUMBER OF PARTICIPANTS

A target of 160 participants are randomized in this study in a 2: 1 : 1 ratio to 1 of 3 groups (Group 1 : Group 2: Group 3).

VACCINATION GROUPS AND DURATION

The study duration is approximately 6 months per participant. The study comprises screening (pre-vaccination) for each participant; vaccination on Day 1 only (Group 1), vaccination on Days 1 and 29 (Day 29 vaccination optional for Group 2), or vaccination on Days 1, 29, and 57 (Day 29 vaccination optional for Group 3); and a 6-month immunogenicity and safety follow-up period following the first vaccination.

Study Vaccine Administration

The investigational medicinal products (IMPs) administered to participants in this study are Ad26.COV2.S, Ad26/protein preF RSV vaccine, Ad26.ZEBOV and MVA-BN- Filo. The Ad26.COV2.S vaccine used in this study (including optional Ad26.COV2.S vaccine on Day 29 in Groups 2 and 3) is to be administered as a single injection (0.5 mL) in the deltoid muscle. The Ad26/protein preF RSV vaccine used in this study is composed of Ad26.RSV.preF and RSV preF protein, to be administered as a single injection (1 mL) in the deltoid muscle. The Ad26.ZEBOV vaccine used in this study is to be administered as a single injection (0.5 mL) in the deltoid muscle. The MVA-BN-Filo vaccine is to be administered as a single injection (0.5 mL) in the deltoid muscle:

— Ad26.COV2.S is used at a dose level of 5 * IO ¹⁰ vp.

— Ad26/protein preF RSV vaccine composed of

1) Ad26.RSV.preF is used at a dose level of 1 x 10 ¹¹ vp; and

2) RSV preF protein is used at a dose level of 150 pg.

— Ad26.ZEBOV is used at a dose level of 5 * 10 ¹⁰ vp.

—MVA-BN-Filo is used at a dose level of 1 * 10 ⁸ Inf U. IMMUNOGENICITY EVALUATIONS

Blood samples are collected for the determination of humoral and cellular immune responses, mRNA expression analysis, and evaluation of innate immune responses. Possible immunogenicity evaluations include (but are not limited to) the assays summarized in the table below:

Table 7: Summary of Immunogenicity Assays

Ad26 = adenovirus type; ELISA = enzyme-linked immunosorbent assay; ELISpot = enzyme- linked immunospot; F = fusion; ICS = intracellular cytokine staining; IFN-y = interferon gamma; Ig = immunoglobulin; IL-2 = interleukin-2; MSD = Meso Scale Discovery; PBMC = peripheral blood mononuclear cells; S = spike; SARS-CoV-2 = severe acute respiratory syndrome coronavirus-2; Th = T-helper; TNF-a = tumor necrosis factor alpha; VNA = virus neutralization assay.

* Non-disease specific biomarker readouts SAFETY EVALUATIONS

Safety assessments include the monitoring of AEs, vital signs and clinical safety laboratory assessments.

Adverse events and special reporting situations, whether serious or non-serious, that are related to study procedures or that are related to non-investigational sponsor products are reported from the time a signed and dated informed consent form (ICF) is obtained until 6 months post Day 1 vaccination/early withdrawal (this includes AEs and special reporting situations related to optional Ad26.COV2.S vaccination on Day 29 and MVA-BN-Filo vaccination on Day 57).

Solicited AEs, collected through a diary, are recorded for each vaccination from the time of vaccination until 7 days post-vaccination on Day 1.

All other unsolicited AEs and special reporting situations, whether serious or non- serious, are reported from the time of vaccination until 28 days post-vaccination on Day 1.

All SAEs, AESIs, and adverse events leading to discontinuation from the study/vaccination (regardless of the causal relationship) are to be reported from the moment of first vaccination until completion of the participant’s last study -related procedure, which may include contact for safety follow-up.

All AEs are followed until resolution or until clinically stable.

STATISTICAL METHODS

Sample Size Determination

A sample size of 40 participants is considered sufficient for exploration of the innate immune responses and immunogenicity responses after immunization within: 1 A) the Ad26.COV2.S vaccine in SARS.CoV.2 seropositive participants, IB) Ad26.COV2.S vaccine in SARS.CoV.2 seronegative participants, 2) the Ad26/protein preF RSV vaccine, and 3) the Ad26.ZEBOV vaccine. Hence a total sample size of 160 participants is planned to be enrolled.

Populations for Analysis Sets

The Full Analysis Set (FAS): The full analysis set includes all participants with at least one vaccine administration documented, regardless of the occurrence of protocol deviations and vaccine type. All safety and participant information analyses are based on the FAS.

The Baseline Seronegative SARS-CoV-2 Per-protocol Immunogenicity (PPI-CVN) Set includes all randomized participants who were SARS-CoV-2 seronegative at baseline (as assessed by a negative SARS.CoV.2 RT-PCR and a negative SARS-CoV-2 N-serology test), received the Ad26.COV2.S vaccination (i.e., study vaccine) on Day 1, and for whom SARS- CoV-2 immunogenicity data are available. Participants with a natural SARS-CoV-2 infection before Day 29 (as determined by SARS-CoV-2 N-serology at Day 29) are excluded from the PPI-CVN Set. Also, samples after a participant experienced a major protocol deviation expecting to impact the immunogenicity outcomes, or after a natural SARS-CoV-2 infection post Day 29 (as determined by SARS-CoV-2 N-serology at Day 183), or after receiving a CO VID-19 vaccine outside of the study before the Day 29 visit are excluded from the PPI- CVN Set.

The Baseline Seropositive SARS-CoV-2 Per-protocol Immunogenicity (PPI-CVP) Set includes all randomized participants who were SARS-CoV-2 seropositive at baseline (as assessed by a positive SARS.CoV.2 RT-PCR test and/or positive SARS-CoV-2 N-serology test), received the Ad26.COV2.S vaccination (i.e., study vaccine) on Day 1 and for whom SARS-CoV-2 immunogenicity data are available. Samples after a participant experienced a major protocol deviation expecting to impact the immunogenicity outcomes or after receiving a CO VID-19 vaccine outside of the study before the Day 29 visit are excluded from the PPI- CVP Set. Participants with a natural infection are not excluded as a natural postbaseline infection cannot be distinguished from a pre-study infection with certainty.

The RSV Per-protocol Immunogenicity (PPI-RSV) Set includes all randomized participants who received the Ad26/protein preF RSV vaccination on Day 1 and for whom RSV immunogenicity data are available. Samples taken after a participant experienced a major protocol deviation expecting to impact the immunogenicity outcomes or after receiving a CO VID-19 vaccine outside of the study before the Day 29 visit are excluded from the PPI- RSV Set.

The ZEBOV Per-protocol Immunogenicity (PPI-EBO) Set includes all randomized participants who received the Ad26.ZEBOV vaccination on Day 1 (including those who did not receive the MVA-BN-FiloR vaccine on Day 57), and for whom ZEBOV immunogenicity data are available. Samples taken after a participant experienced a major protocol deviation expecting to impact the immunogenicity outcomes or after receiving a COVID-19 vaccine outside of the study before the Day 29 visit are excluded from the PPI-EBO Set.

The Per-protocol Immunogenicity (PPI) Set includes all randomized participants who received a study vaccine on Day 1 and for whom immunogenicity data (i.e., non-disease specific biomarker readouts) are available. Samples taken after a participant experienced a major protocol deviation expecting to impact the immunogenicity outcomes or after receiving a CO VID-19 vaccine outside of the study before the Day 29 visit are excluded from the PPI Set. Also, randomized participants who were SARS-CoV.2 seronegative at baseline, received the Ad26.COV2.S vaccine on Day 1 and had a natural SARS-CoV.2 infection before Day 29 (as determined by SARS-CoV2 N-serology at Day 29) are excluded from the PPI Set.

Immunogenicity data is considered data obtained for the primary endpoints, humoral immune responses and exploratory endpoints.

The list of major protocol deviations that would lead to elimination from the immunogenicity analysis are specified in the statistical analysis plan (SAP) or major protocol violation criteria document, which will be finalized before database lock and unblinding.

The primary analysis set for analyses related to immunogenicity of RSV is the PPI- RSV Set. The primary analysis sets for analyses related to immunogenicity of SARS-CoV-2 are the PPI-CVN Set and the PPI-CVP Set. The primary analysis set for analyses related to immunogenicity of ZEBOV is the PPI-EBO Set. The primary analysis set for analyses of non-vaccine specific immunogenicity data is the PPI Set. As a sensitivity analysis, key tables may also be based on the FAS (including excluded samples as well). Depending on their occurrence, the effect of natural infections might be further explored.

Immunogenicity Analyses

No formal hypothesis on immunogenicity is tested. Continuous endpoints are summarized descriptively by group, including 95% CI and/or median and quartiles, as applicable. Descriptive statistics of the actual values and changes from baseline or fold change from baseline, as applicable, are calculated at all timepoints. Baseline is considered as the last assessment pre-vaccination. Graphical representations are made as applicable. For categorical variables, frequency tables will be presented.

Safety Analyses

No formal statistical testing of safety data is planned. All safety data are analyzed descriptively by group.