An improved Adenovirus based Malaria vaccine encoding and displaying a Malaria antigen

FIELD OF THE INVENTION

The invention relates to the field of medicine. More in particular, the invention relates to the use of a recombinantly produced viral vector as a carrier of an antigenic determinant for the development of a vaccine against malaria infections.

BACKGROUND OF THE INVENTION

The necessity of an efficacious vaccine against the Malaria causing agent Plasmodium falciparum is apparent from the 800 000 deaths and over 250 million clinical cases recorded each year (WHO 2009). To reduce the disease burden and achieve complete malaria eradication in endemic countries an effective vaccine is necessary. Currently the most promising vaccine candidate, a P. falciparum circumsporozoite protein (CSP) based malaria vaccine termed RTS,

S/AS01B/AS02A, showed promising but mixed results in clinical and field trials (1-5, 7-9). The immune correlates associated with RTS, S induced protection show a strong association between vaccine efficacy and the magnitude of the humoral and cellular responses (14).

Alternatively, Human Adenovirus (HAdV) vector expressing a

circumsporozoite (CS) protein in the development of a Malaria vaccine induced high cellular responses but lower antibody responses against the CS protein (10, 11, 13). The combination of priming with HAdV35.CS and boosting with RTS,S/AS01B significantly improved immunogenicity to P. falciparum CS compared to either vaccine alone (15). This significant improvement of immunogenicity demonstrates the benefit of combining the HAdV vector with a strong B-cell inducer component.

Hence there is a need for recombinant adenoviral vectors that induce both a strong cellular response and a strong humoral response.

DESCRIPTION OF THE FIGURES

Fig. 1: Direct fusion of the CS short antigen to HAdV protein IX (pIX) (A) Sequence alignment of the Plasmodium falciparum Circumsporozoite (CS) central region with the B-cell epitope (27)NANP -repeat in the CS _s h ₀ rt antigen encoded and displayed on the HAdV vectors via protein IX (pIX), directly compared to the CS transgene (TG) (12) encoded in the El region. (B) Schematic drawing of the genetic design of HAdV vectors with an expression cassette in El alone and with or without encoded pIX- CSshort and pIX-CS modification. The transgene only vectors encode CS, CS _s h ₀ rt or no- transgene and contain native pIX ('Empty'). pIX-CS _s h _0r t andpIX-CS modified vectors are either Empty or contain the CS transgene in El . (C) Western Blot of viral particles to compare the capsid incorporation of the pIX-CS _s h ₀ rt modified vectors with or without the CS transgene and a non-modified pIX control, stained with anti-CS specific (2A10) antibody and anti- fiber (4D2) as a loading control. A serial dilution of 1.5, 0.5 and 0.17xl0 ¹⁰ viral particles (VP) per well are loaded and compared for the pIX-CSshort content. Expected size of the CS transgene is -42 kDa and pIX-CS _s h ₀ rt protein ~32 kDa, however both proteins migrate higher in the gel due to the NANP- repeat. (D) In the left graph the depicted ELISA (EU/ml log) titers show total CS- specific (IgG) antibody responses (humoral response, B-cell response) in mice 4 weeks post immunization with lxlO ¹⁰ VP/animal of HAdV35.empty.pIX-CS _s h ₀ rt, HAdV35.CS.pIX-CSshort directly compared to 5μg of CS protein and HAdV35. empty. In the right graph B-cell responses 4 weeks after immunization with 5μg CS protein in PBS, mixed with ISCOM-based adjuvant or mixed with lxlO ¹⁰ VP/animal of empty Ad-vectors HAdV35, HAdV26, or HAdV5. Mice injected with only an adjuvant served as a control. The assay lower limit of detection (LLoC) is delineated with a dashed line. Statistical significance was determined using a two-way ANOVA test with Dunnett correction for multiple comparisons on log-transformed data. Black horizontal bars p < 0.05.

Fig. 2: Direct fusion of Influenza A Hemagglutinin (HA) antigen to pIX (A)

Schematic representation of different Influenza A HA antigen designs depicting the HA (512 amino acids (a. a.)) molecule with the corresponding N- & C-termini, signal peptide, membrane domain and the ectodomain (in grey). The middle bar shows the mHA (303 a. a.) protein consisting of the N- & C-termini portions of the HA protein and the ectodomain. The mHA-GCN(ecto) molecule (248 a. a.) lacks the N-terminus signal peptide and the ectodomain of the native HA is replaced with the GCN trimerization domain. (B) Diagram of the HAdV vectors encoding the different HA designs in the El region and/or fused to the pIX capsid protein and all the

combinations thereof.

Fig. 3 : CSshort antigen fusion to HAdV pIX via an alpha- helix spacer (A) The schematically depicted HAdV vectors designed with CS _s h ₀ rt fused to the pIX via 45A alpha- helix spacer (16), Gly- linker and Gly-45A. The pIX modified vectors are made both with and without the CS transgene in the El-cassette. (B) To compare the incorporation efficiency of the vectors with and without the CS transgene and the different pIX-CS _s h ₀ rt modifications in the capsid, Western Blots are shown with a serial dilution of 1.5, 0.5 and 0.17 xlO ¹⁰ VP/per well of the HAdV35.Empty/CS.pIX- 45A-pIX-CSshort, HAdV35.Empty/CS.pIX-Gly45A-pIX-CSshort and

HAdV35.Empty/CS.pIX-GlypIX-CSshort stained with anti-CS specific (2A10) antibody and anti- fiber (4D2) as a loading control. As a non-modified pIX control a HAdV35. empty vector is taken along. Expected size of the CS transgene is ~42 kDa and pIX-CS _s h ₀ rt protein variants ~35 kDa, however both proteins migrate higher in the gel due to the NANP -repeat. (C) The CS-specific antibody (EU/ml log) responses 8 weeks post immunization with lxlO ⁸ , lxlO ⁹ , or lxlO ¹⁰ VP/animal of

HAdV35.empty.pIX-45-CSshort, HAdV35.CS.pIX-45-CS _sh ort, a mix of

HAdV35.empty.pIX-45-CSshort and HAdV35.CS, HAdV35.CS alone, or

H AdV35. empty ( 10 ⁹ VP/ animal only) in mice are depicted in the graphs. (D) The two graphs show the ΙΚΝγ ELISPOT responses post immunization against the entire P.falciparum CS protein sequence or the H-2Kd restricted immunodominant HAdV35 hexon epitope KYTPSNVTL. Statistical significance was determined using a two- way ANOVA test with Dunnett correction for multiple comparisons on log- transformed data by time point and dose. Black horizontal bars p < 0.05.

Fig. 4: Human ApoE4 based alpha- helix spacer replacement (A) Schematic representation of empty vectors encoding only CS _s h ₀ rt fused to pIX via four different viral-origin spacers (SP1-4) namely, alpha helixes from Respiratory Syncytial Virus (RSV) fusion protein (F-protein), Influenza A HA, Mumps F-protein and HAdV35 pIXmod compared to the previously described human ApoE4 based alpha-helix 45 A- spacer (16). (B and C) Western blots showing the capsid incorporation efficiency of the CSshort linked via different spacers (SP1-4). 1.5, 0.75 and 0.37xl0 ¹⁰ VP/well of each spacer vector HAdV35.empty.pIX-SPl-CS _sh ort, HAdV35.empty.pIX-SP2-CS _sh ort, HAdV35.empty.pIX-SP3-CS _s hort and HAdV35.empty.pIX-SP4-CS _sh ort directly compared to equal quantity of the native-pIX HAdV35. empty vector and the

HAdV35.empty.pIX-45A-CSshort vector. The blots are stained with anti-pIX (6740) monoclonal antibody and anti- fiber (4D2) as a loading control. (D) Shows a Western blot with 1.5, 0.5 and 0.16xl0 ¹⁰ VP/well of HAdV26.empty.pIX-SPl-CS _sh ort,

HAdV26.empty.pIX-45A-CS _short and HAdV26.empty stained with anti-CS (2A10) to show the capsid incorporation of the pIX-SPl-CS _s h ₀ rt compared to the pIX-45A-

CS short. Fig. 5: Manufacturability of pIX- CS _short modified vectors (A) Replication kinetics and the relative VP/ml increase (i.e. 'productivity') in time. 10 ml of suspension

PER.C6® (sPER.C6®) cell culture is infected with HAdV26 vector at 70 and 900 VP/cell after which the culture is sampled in time (t=0, 1, 2, 3 and 4 days post infection (DPI)) (n=3) and analyzed with viral particles QPCR (VP-QPCR). The graphs show the kinetics and VP/ml increase of pIX-modified vectors

(HAdV26.CS.pIX-CSshort & HAdV26.empty.pIX-CS _s hort) compared to control vectors with high (HAdV26.TGl) and lower (HAdV26.TG2) yields in sPER.C6®. (B) Genetic stability assessment after extended passaging of pIX-modified batches in PER.C6® cells. The generation of batches entails upscaling in PER.C6® resulting in 6-7 viral passages (VPN7). To ensure genetically stable vectors, the purified batches are further passaged under controlled conditions, 70 or 900 VP/cell in 10 ml

PER.C6® 3-4 passages (VPN 13-14) beyond the envisioned commercial scale (depending on the details of the process). The viral DNA is subsequently isolated and analyzed in pIX-PCR to determine genetic stability. (C) Representative Agarose gel pictures are shown of HAdV26.CS/Empty.pIX-CS _s hort and HAdV35.CS/empty.pIX- CS _short pIX-PCR at VPN 7 and VPN 14 (passaged n=2). The positive (+) controls are the respective plasmid DNA constructs and the non-modified (-) pIX-native plasmid DNA along with PCR water (H ₂ 0) control and the DNA marker (M). DESCRIPTION OF THE INVENTION

The present invention relates to a recombinant adenoviral vector comprising a capsid comprising a fusion protein consisting of a protein IX and a fragment of a

circumsporozoite (CS) protein of Plasmodium falciparum, wherein said fragment comprises the amino acid sequence of SEQ ID NO: l . Preferably, said fusion protein comprises a linker located in between the pIX protein and the CS protein fragment. Preferably said linker comprises an amino acid sequence having for example 2 to 15 consecutive glycine and/or serine residues.

In another embodiment according to the present invention, said fusion protein comprises a spacer, located in between the pIX protein and the CS protein fragment. Preferably, said spacer comprises the amino acid sequence of SEQ ID NO:2.(SPl). In another preferered embodiment, said spacer comprises the amino acid sequence of SEQ ID NO:3.(SP2).

In a preferred embodiment, the recombinant adenoviral vector according to the invention further comprises a nucleic acid encoding a heterologous protein.

Preferably, said heterologous protein is a Malaria P '.falciparum Circumsporozoite protein. Even more preferably, said Malaria P. falciparum Circumsporozoite protein comprises sequence SEQ ID NO:8.

In a preferred embodiment, the recombinant adenoviral vector according to the invention is derived from a serotype selected from the group consisting of: HAdV4,

HAdV5, HAdVl l, HAdV26, HAdV35, HAdV48, HAdV49 and HAdV50. Other possible types of recombinant adenoviral vectors suited for the present invention are included but not limited to: canine adenoviruses, chimp adenoviruses, gorilla adenoviruses and chimeric adenoviruses.

Preferably, said serotype is selected from the group of HAdV26, HAdV35,

HAdV4, HAdVl l, HAdV48 and HAdV49.

In a preferred embodiment, the recombinant adenoviral vector according to the invention is replication-defective.

The invention also relates to vaccine compositions comprising the

recombinant adenoviral vector according to the invention, and a pharmaceutically acceptable carrier, further comprising preferably an adjuvant. Furthermore, the invention relates to the use of a vaccine composition according to the invention in the therapeutic, prophylactic or diagnostic treatment of malaria. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a replication-defective recombinant adenoviral vector comprising a capsid comprising a fusion protein consisting of a protein IX and a fragment of a circumsporozoite (CS) protein of Plasmodium falciparum. Said fragment comprises the amino acid sequence of SEQ ID NO: 1. The Malaria P '.falciparum Circumsporozoite (CS) protein is a mammalian- codon CS protein optimized protein (based on EMBL CAH04007 sequence) previously described by Radosevic et al. 2011 and Rodriguez et al. 2009 (12, 13).

Two forms of the CS protein are used in the present invention. The so called "transgene" form and the so called "CS short" form (or fragment), which is a truncated form of the CS protein (Fig. 1 A). The CS short comprises the amino acid sequence SEQ ID NO: l .

The transgene CS is similar to the native CS protein (3D7 clone) except for the final 14 amino acids in the C-terminus which are truncated. The transgene (TG) is inserted and encoded in the El region of the adenoviral vector. The repeat region of the CS protein consist of 27 consecutive NANP -repeats which are present in both transgene (TG) and the truncated form (CS short) which is fused to protein IX. The transgene form of the CS protein comprises the amino acid sequence SEQ ID NO:8.

The term Protein IX (pIX) refers to a protein with the main function of stabilizing the icosahedral Adenovirus capsid (Rose-Calatrava et al. 2001).

The pIX is adenovirus serotype specific, i.e. the pIX in a serotype 35

Adenovirus (HAdV35) comprises the amino acid sequence SEQ ID NO:6; the pIX in a serotype 26 Adenovirus (HAdV26) comprises the amino acid sequence SEQ ID NO:7.

The term linker refers to short peptide sequences that can be placed between protein domains. Linkers are composed of flexible residues such as glycine (gly) and/ or serine in different size ranges, ensuring free movement of different domains relative to one another. Examples of linkers include but are not limited to 3 -Gly (Gly- Gly-Gly) and middle linker (Gly-Gly-Ser-Gly)x2. In one preferred embodiment according to the present invention, the Glycine linker comprises an amino acid sequence having 2 to 15 consecutive glycine and/or Serine residues.

The term spacer refers to short peptide sequences that can be placed between protein domains. Several spacers are disclosed herein and can be used in the present invention, i.e. the ApoE4 protein alpha-helical 45A spacer (16) that is from human ApoE4 protein origin; the spacer 1 (SP1), a RSV Fusion protein; the spacer 2 (SP2), an Influenza A HA; the spacer 3 (SP3), a Mumps Fusion protein and finally spacer 4 (SP4), a HAdV35.pIX fragment with point mutations. The SP1-SP4 spacers are from viral origin and comprise the amino sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:5 respectively. The term 'recombinant' for an adenovirus, as used herein implicates that it has been modified by the hand of man, e.g. it has altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e. it is not a naturally occurring wild type adenovirus.

Sequences herein are provided from 5' to 3' direction, as custom in the art.

An "adenovirus capsid protein" refers to a protein on the capsid of an adenovirus that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. An adenovirus of (or 'based upon') a certain serotype according to the invention typically comprises fiber, penton and/or hexon proteins of that certain serotype, and preferably comprises fiber, penton and hexon protein of that certain serotype. These proteins are typically encoded by the genome of the recombinant adenovirus. A recombinant adenovirus of a certain serotype may optionally comprise and/or encode other proteins from other adenovirus serotypes. Thus, as non-limiting example, a recombinant adenovirus that comprises hexon, penton and fiber of HAdV35 is considered a recombinant adenovirus based upon HAdV35.

A recombinant adenovirus is 'based upon' an adenovirus as used herein, by derivation from the wild type, at least in sequence. This can be accomplished by molecular cloning, using the wild type genome or parts thereof as starting material. It is also possible to use the known sequence of a wild type adenovirus genome to generate (parts of) the genome de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, Invitrogen, GenScripts, Eurofms).

The recombinant adenovirus of the present invention is preferably based upon an adenovirus from a serotype selected from the group consisting of: HAdV4,

HAdV5, HAdVl l, HAdV26, HAdV35, HAdV48, HAdV49 and Ad50. Other possible types of adenoviruses suited for the present invention are included but not limited to: canine adenoviruses, chimp adenoviruses, gorilla adenoviruses and chimeric adenoviruses. Preferably, said serotype is selected from the group of HAdV4, HAdV26, HAdV35, HAdV48, HAdV49 and HAdV50.

The replication-defective recombinant adenoviral vector further comprises a heterologous nucleic acid encoding a heterologous antigen (or polypeptide).

It is understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described there to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

In one particular aspect of the invention the recombinant viral vector according to the invention, comprises a nucleic acid encoding an antigenic determinant that is the circumsporozoite (CS) protein, or an immunogenic part thereof. Preferably, said heterologous nucleic acid is codon-optimized for elevated expression in a mammal, preferably a human. Codon-optimization is based on the required amino acid content, the general optimal codon usage in the mammal of interest and a number of provisions of aspects that should be avoided to ensure proper expression. Such aspects may be splice donor or -acceptor sites, stop codons, Chi- sites, poly(A) stretches, GC- and AT -rich sequences, internal TATA boxes, etcetera.

In a preferred embodiment, the invention relates to a replication-defective recombinant viral vector according to the invention, wherein the adenine plus thymine content in said heterologous nucleic acid, as compared to the cytosine plus guanine content, is less than 87%, preferably less than 80%, more preferably less than 59% and most preferably equal to approximately 45%.

The person skilled in the art will also appreciate that changes can be made to a protein, e.g. by amino acid substitutions, deletions, additions, etc, e.g. using routine molecular biology procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of a polypeptide. This can easily be checked according to routine procedures well known to the skilled person.

The term "vaccine" refers to an agent or composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a certain pathogen or disease. In the present invention, the vaccine comprises an effective amount of a recombinant adenovirus that encodes a heterologous protein, or an antigenic fragment thereof, which results in an immune response against the heterologous protein. The term "vaccine" according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises other components that induce an immune response, e.g. against other proteins and/or against other infectious agents.

The vectors of the present invention are recombinant adenoviruses, also referred to as recombinant adenoviral vectors. The preparation of recombinant adenoviral vectors is well known in the art.

In certain embodiments, an adenoviral vector according to the invention is deficient in at least one essential gene function of the El region, e.g. the Ela region and/or the Elb region, of the adenoviral genome that is required for viral replication. In certain embodiments, an adenoviral vector according to the invention is deficient in at least part of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential gene function of the El region and at least part of the non-essential E3 region. The adenoviral vector can be "multiply deficient", meaning that the adenoviral vector is deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome. For example, the aforementioned El -deficient or E1-, E3 -deficient adenoviral vectors can be further deficient in at least one essential gene of the E4 region and/or at least one essential gene of the E2 region (e.g., the E2A region and/or E2B region).

Adenoviral vectors, methods for construction thereof and methods for propagating thereof, are well known in the art and are described in, for example, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128,

5,965,541, 5,981,225, 6,040,174, 6,020,191, 6,113,913, and 8,932,607 and Thomas Shenk, "Adenoviridae and their Replication", M. S. Horwitz, "Adenoviruses", Chapters 67 and 68, respectively, in Virology, B. N. Fields et al, eds., 3d ed., Raven

Press, Ltd., New York (1996), and other references mentioned herein. Typically, construction of adenoviral vectors involves the use of standard molecular biological techniques, such as those described in, for example, Sambrook et al, Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al, Recombinant DNA, 2d ed., Scientific American Books

(1992), and Ausubel et al, Current Protocols in Molecular Biology, Wiley

Interscience Publishers, NY (1995), and other references mentioned herein.

According to the invention, an adenovirus is a human adenovirus of the serotype 35. The vaccines according to the invention based on this serotype as well as those based on HAdV26 surprisingly appear more potent than the ones described in the prior art that were based on HAdV5. The serotype of the invention further generally has a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Recombinant adenoviral vectors of this serotype and of HAdV26 with different transgenes are evaluated in clinical trials, and thus far show to have an excellent safety profile. Preparation of HAdV26 vectors is described, for example, in WO 2007/104792 and in Abbink et al, (2007) Virol 81(9): 4654-63. Exemplary genome sequences of HAdV26 are found in GenBank Accession EF 153474 and in SEQ ID NO: l of WO 2007/104792. Preparation of HAdV35 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. Exemplary genome sequences of HAdV35 are found in GenBank Accession AC 000019 and in Fig. 6 of WO 00/70071. A recombinant adenovirus according to the invention may be replication-competent or replication-deficient.

In certain embodiments, the adenovirus is replication deficient, e.g. because it contains a deletion in the El region of the genome. As known to the skilled person, in case of deletions of essential regions from the adenovirus genome, the functions encoded by these regions have to be provided in trans, preferably by the producer cell, i.e. when parts or whole of El, E2 and/or E4 regions are deleted from the adenovirus, these have to be present in the producer cell, for instance integrated in the genome thereof, or in the form of so-called helper adenovirus or helper plasmids. The adenovirus may also have a deletion in the E3 region, which is dispensable for replication, and hence such a deletion does not have to be complemented.

A producer cell (sometimes also referred to in the art and herein as 'packaging cell' or 'complementing cell' or 'host cell') that can be used can be any producer cell wherein a desired adenovirus can be propagated. For example, the propagation of recombinant adenovirus vectors is done in producer cells that complement

deficiencies in the adenovirus. Such producer cells preferably have in their genome at least an adenovirus El sequence, and thereby are capable of complementing recombinant adenoviruses with a deletion in the El region. Any El -complementing producer cell can be used, such as human retina cells immortalized by El, e.g. 911 or PER.C6 cells (see US patent 5,994,128), El -transformed amniocytes (See EP patent 1230354), El -transformed A549 cells (see e.g. WO 98/39411, US patent 5,891,690), GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11 : 213-219), 293, and the like. In certain embodiments, the producer cells are for instance HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells, and the like.

For non- subgroup C El -deficient adenoviruses such as HAdV35 (subgroup B) or HAdV26 (subgroup D), it is preferred to exchange the E4-orf6 coding sequence of these non-subgroup C adenoviruses with the E4-orf6 of an adenovirus of subgroup C such as HAdV5. 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 or PER.C6 cells (see, e.g. Havenga et al, 2006, J. Gen. Virol. 87: 2135-2143; WO 03/104467, incorporated in its entirety by reference herein). In certain

embodiments, the adenovirus in the vaccine composition is a human adenovirus of serotype 35, with a deletion in the El region into which the nucleic acid encoding a CS protein antigen has been cloned, and with an E4 orf6 region of HAdV5. In certain embodiments, an adenovirus in that can be used is a human adenovirus of serotype 26, with a deletion in the El region into which the nucleic acid encoding a CS protein antigen has been cloned, and with an E4 orf6 region of HAdV5.

In alternative embodiments, there is no need to place a heterologous E4orf6 region (e.g. of HAdV5) in the adenoviral vector, but instead the El -deficient non- subgroup C vector is propagated in a cell line that expresses both El and a compatible E4orf6, e.g. the 293-ORF6 cell line that expresses both El and E4orf6 from Ad5 (see e.g. Brough et al, 1996, J Virol 70: 6497-501 describing the generation of the 293- ORF6 cells; Abrahamsen et al, 1997, J Virol 71 : 8946-51 and Nan et al, 2003, Gene Therapy 10: 326-36 each describing generation of El deleted non-subgroup C adenoviral vectors using such a cell line).

Alternatively, a complementing cell that expresses El from the serotype that is to be propagated can be used (see e.g. WO 00/70071, WO 02/40665).

For subgroup B adenoviruses, such as HAdV35, having a deletion in the El region, it is preferred 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 in the HAdV35 genome), 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, J. Gen.

Virol. 87: 2135-2143; WO 2004/001032, incorporated by reference herein). In certain embodiments, the recombinant HAdV26 or HAdV35 vectors of the invention comprise as the 5' terminal nucleotides the nucleotide sequence:

CTATCTAT. These embodiments are advantageous because such vectors display improved replication in production processes, resulting in batches of adenovirus with improved homogeneity, as compared to vectors having the original 5' terminal sequences (generally CAT CATC A) (see also patent application nos.

PCT/EP2013/054846 and US 13/794,318, entitled 'Batches of recombinant adenovirus with altered terminal ends' filed on 12 March 2012 in the name of Crucell Holland B.V.), incorporated in its entirety by reference herein. The invention thus also provides batches of recombinant adenovirus encoding a CS protein or a part thereof, wherein the adenovirus is a human adenovirus serotype 35, and wherein essentially all (e.g. at least 90%) of the adenoviruses in the batch comprise a genome with terminal nucleotide sequence CTATCTAT.

The term 'about' for numerical values as used in the present disclosure means the value ± 10%.

In certain embodiments, the invention provides methods for making a vaccine against Malaria or an other infections diseases antigen, i.e. Tuberculosis, comprising providing a recombinant adenoviral vector comprising a capsid comprising a fusion protein consisting of a protein IX and a fragment of a circumsporozoite (CS) protein of Plasmodium falciparum, wherein said fragment comprises the amino acid sequence of SEQ ID NO: l, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition.

Recombinant adenovirus can be prepared and propagated in host cells, according to well known methods, which entail cell culture of the host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.

Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up.

Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable (see e.g. WO 2010/060719, and WO 2011/098592, both incorporated by reference herein, which describe suitable methods for obtaining and purifying large amounts of recombinant adenoviruses). Producer cells are cultured to increase cell and virus numbers and/or virus titers. Culturing a cell is done to enable it to metabolize, and/or grow and/or divide and/or produce virus of interest according to the invention. This can be accomplished by methods as such well known to persons skilled in the art, and includes but is not limited to providing nutrients for the cell, for instance in the appropriate culture media. Suitable culture media are well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889- 9)·

Typically, the adenovirus will be exposed to the appropriate producer cell in a culture, permitting uptake of the virus. Usually, the optimal agitation is between about 50 and 300 rpm, typically about 100-200, e.g. about 150, typical DO is 20-60%, e.g.40%, the optimal pH is between 6.7 and 7.7, the optimal temperature between 30 and 39°C, e.g. 34-37°C, and the optimal MOI between 5 and 1000, e.g. about 50-300. Typically, adenovirus infects producer cells spontaneously, and bringing the producer cells into contact with recombinant adeviral particles is sufficient for infection of the cells. Generally, an adenovirus seed stock is added to the culture to initiate infection, and subsequently the adenovirus propagates in the producer cells. This is all routine for the person skilled in the art.

After infection of an adenovirus, the virus replicates inside the cell and is thereby amplified, a process referred to herein as propagation of adenovirus.

Adenovirus infection results finally in the lysis of the cells being infected. The lytic characteristics of adenovirus therefore permits two different modes of virus production. The first mode is harvesting virus prior to cell lysis, employing external factors to lyse the cells. The second mode is harvesting virus supernatant after (almost) complete cell lysis by the produced virus (see e.g. US patent 6,485,958, describing the harvesting of adenovirus without lysis of the host cells by an external factor). It is preferred to employ external factors to actively lyse the cells for harvesting the adenovirus. Methods that can be used for active cell lysis are known to the person skilled in the art, and have for instance been discussed in WO 98/22588, p. 28-35. Useful methods in this respect are for example, freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, combinations of the above, and the like. In one embodiment of the invention, the cells are lysed using at least one detergent. Use of a detergent for lysis has the advantage that it is an easy method, and that it is easily scalable.

Detergents that can be used, and the way they are employed, are generally known to the person skilled in the art. Several examples are for instance discussed in WO 98/22588, p. 29-33. Detergents can include anionic, cationic, zwitterionic, and nonionic detergents. The concentration of the detergent may be varied, for instance within the range of about 0.1%-5% (w/w). In one embodiment, the detergent used is Triton X-100.

Nuclease may be employed to remove contaminating, i.e. mostly from the producer cell, nucleic acids. Exemplary nucleases suitable for use in the present invention include Benzonase®, Pulmozyme®, or any other DNase and/or RNase commonly used within the art. In preferred embodiments, the nuclease is

Benzonase®, which rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiester bonds between specific nucleotides, thereby reducing the viscosity of the cell lysate. Benzonase® can be commercially obtained from Merck KGaA (code W214950). The concentration in which the nuclease is employed is preferably within the range of 1-100 units/ml. Alternatively, or in addition to nuclease treatment, it is also possible to selectively precipitate host cell DNA away from adenovirus preparations during adenovirus purification, using selective precipitating agents such as domiphen bromide (see e.g. US 7,326,555; Goerke et al, 2005, Biotechnology and bioengineering, Vol. 91 : 12-21; WO 2011/045378; WO 2011/045381).

Methods for harvesting adenovirus from cultures of producer cells have been extensively described in WO 2005/080556.

In certain embodiments, the harvested adenovirus is further purified. Purification of the adenovirus can be performed in several steps comprising clarification, ultrafiltration, diafiltration or separation with chromatography as described in for instance WO 05/080556, incorporated by reference herein. Clarification may be done by a filtration step, removing cell debris and other impurities from the cell lysate.

Ultrafiltration is used to concentrate the virus solution. Diafiltration, or buffer exchange, using ultrafilters is a way for removal and exchange of salts, sugars and the like. The person skilled in the art knows how to find the optimal conditions for each purification step. Also WO 98/22588, incorporated in its entirety by reference herein, describes methods for the production and purification of adenoviral vectors. The methods comprise growing host cells, infecting the host cells with adenovirus, harvesting and lysing the host cells, concentrating the crude lysate, exchanging the buffer of the crude lysate, treating the lysate with nuclease, and further purifying the virus using

chromatography.

Preferably, purification employs at least one chromatography step, as for instance discussed in WO 98/22588, p. 61-70. Many processes have been described for the further purification of adenoviruses, wherein chromatography steps are included in the process. The person skilled in the art will be aware of these processes, and can vary the exact way of employing chromatographic steps to optimize the process. It is for instance possible to purify adenoviruses by anion exchange chromatography steps, see for instance WO 2005/080556 and Konz et al, 2005, Hum Gene Ther 16: 1346-1353. Many other adenovirus purification methods have been described and are within the reach of the skilled person. Further methods for producing and purifying adenoviruses are disclosed in for example (WO 00/32754; WO 04/020971; US 5,837,520; US 6,261,823; WO 2006/108707; Konz et al, 2008, Methods Mol Biol 434: 13-23; Altaras et al, 2005, Adv Biochem Eng Biotechnol 99: 193-260), all incorporated by reference herein.

For administering to humans, the invention may employ pharmaceutical compositions comprising the Adenovirus and a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified Ad preferably is formulated and administered as a sterile solution although it is also possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to 7.5. The Ad typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution of Ad may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, Ad may be formulated into an injectable preparation. These

formulations contain effective amounts of Ad, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients. An adenovirus vaccine can also be aerosolized for intranasal administration (see e.g. WO 2009/117134).

For instance, adenovirus may be stored in the buffer that is also used for the Adenovirus World Standard (Hoganson et al, Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgC12, 25 mM NaCl, sucrose 10% w/v, polysorbate- 80 0.02%) w/v. Obviously, many other buffers can be used, and several examples of suitable formulations for the storage and for pharmaceutical administration of purified (adeno)virus preparations can for instance be found in European patent no. 0853660, US patent 6,225 ,289 and in international patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

In certain embodiments a composition comprising the adenovirus further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and suitable adjuvants are for instance disclosed in WO 2007/110409, incorporated by reference herein. The terms "adjuvant" and "immune stimulant" are used interchangeably herein, and are defined as 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 adenovirus vectors of the invention.

Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water

compositions), including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG- motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like. It is also possible to use vector-encoded adjuvant, e.g. by using heterologous nucleic acid that encodes a fusion of the oligomerization domain of C4-binding protein (C4bp) to the antigen of interest (e.g. Solabomi et al, 2008, Infect Immun 76: 3817-23). In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.

In other embodiments, the compositions do not comprise adjuvants. It is also possible according to the invention to administer further active components, in combination with the vaccines according to the invention. Such further active components may comprise e.g. other Malaria antigens or vectors comprising nucleic acid encoding these. Such vectors may be non-adeno viral or adenoviral, of which the latter can be of any serotype. An example of other Malaria antigens includes Malaria proteins or immunologically active parts thereof. Further active components may also comprise non-Malaria antigens, e.g. from other pathogens such as viruses, bacteria, parasites, and the like. The administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention and the further active components. In certain embodiments, further non-adenoviral antigens, may be encoded in the vectors of the invention. In certain embodiments, it may thus be desired to express more than one protein from a single adenovirus, and in such cases more coding sequences for instance may be linked to form a single transcript from a single expression cassette or may be present in two separate expression cassettes cloned in different parts of the adenoviral genome.

Adenovirus compositions may be administered to a subject, e.g. a human subject. The total dose of the adenovirus provided to a subject during one

administration can be varied as is known to the skilled practitioner, and is generally between lxlO ⁷ viral particles (VP) and lxlO ¹² VP, preferably between lxlO ⁸ VP and lxlO ¹¹ VP, for instance between 3xl0 ⁸ and 5xl0 ¹⁰ VP, for instance between 10 ⁹ and 3xl0 ¹⁰ VP.

Administration of adenovirus compositions can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as by injection e.g. intradermal, intramuscular, etc, or subcutaneous,

transcutaneous, or mucosal administration, e.g. intranasal, oral, and the like. It is preferred according to the present invention to administer the vaccine intramuscularly. The advantage of intramuscular administration is that it is simple and well- established, and does not carry the safety concerns for intranasal application in infants younger than 6 months. In one embodiment a composition is administered by intramuscular injection, e.g. into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh. The skilled person knows the various possibilities to administer a composition, e.g. a vaccine in order to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject. The subject can be of any age, e.g. from about 1 month to 100 years old, e.g. from about 2 months to about 80 years old, e.g. from about 1 month to about 3 years old, from about 3 years to about 50 years old, from about 50 years to about 75 years old, etc.

It is also possible to provide one or more booster administrations of one or more adenovirus vaccines of the invention. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a moment between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as 'priming vaccination'). In alternative boosting regimens, it is also possible to administer different vectors, e.g. one or more adenoviruses of different serotype, or other vectors such as MVA, or DNA, or protein, to the subject after the priming vaccination. It is for instance possible to administer to the subject a recombinant adenoviral vector according to the invention as a prime, and boosting with a composition comprising a malaria protein.

In certain embodiments, the administration comprises a priming and at least one booster administration. In certain embodiments thereof, the priming

administration is with a HAdV35 comprising nucleic acid encoding a Malaria protein according to the invention and the booster administration is with a HAdV26 comprising nucleic acid encoding said Malaria protein. In other embodiments thereof, the priming administration is with HAdV26 and the booster administration is with HAdV35. In other embodiments, both the priming and booster administration are with HAdV35. In certain embodiments, the priming administration is with HAdV35 and the booster administration is with a Malaria protein. In all these embodiments, it is possible to provide further booster administrations with the same or other vectors or protein.

In certain embodiments, the administration comprises a single administration of a recombinant adenovirus according to the invention, without further (booster) administrations. Such embodiments are advantageous in view of the reduced complexity and costs of a single administration regimen as compared to a prime-boost regimen. Complete protection is already observed after single administration of the recombinant adenoviral vectors of the invention without booster administrations in the cotton rat model in the examples herein.

The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention. EXAMPLES

Example 1: Generation and characterization of the pIX-CS _S hort modified

HAdV35 and HAdV26 vectors.

Generation of El pIX-CS _S hort modified vectors with or without the Malaria P.falciparum CS protein Transgene (TG)

Replication deficient Human Adenovirus vector (HAdVv) generation

For the generation of replication deficient human Adenovirus 35 (HAdV35) and Human Adenovirus 26 (HAdV26) vectors encoding the CS or CS _s h ₀ rt transgene and/or pIX-CS or CSshort, modifications are primarily inserted into the left part of the HAdV genome, namely pAdapt35.Bsu/pShuttle26 plasmids. The rescue of the HAdV35 vectors was subsequently performed by a three plasmid system where the right end of the genome pBr.HAdV35 PRdE3 5E4 orf6/7 (digested with Pad and Notl), and pWE.HAdV35.dpLX.EcoRV, (digested with Notl and EcoRV) and the left end of the genome pAdapt35.Bsu.pIX-mod, (digested with Pad) were transfected into PER.C6® cells using Lipofectamine (Invitrogen) according to the manufacturer's recommendations. The viruses were plaque purified and further propagated on PER.C6 ^® cells supplemented with 10% of fetal bovine serum (FBS) (Life

Technologies Inc.) and 10 mM MgCl ₂ , as previously described by Havenga et al. 2006 (6).

The HAdV26 vectors were generated by allowing homologous recombination (HR) between the left (pShuttle26.pIX-mod, digested with Pmel and Sbfl) and the right end cosmid (pWE.HAdV26.dE3.5orf6, digested with Pad) in BJ5183 E.coli cells as previously described (Chartier et al. 1996). The Pad linearized HAdV26 genome is transfected into PER.C6® cells using Lipofectamine (Invitrogen) according to the manufacturer's recommendations and further amplified in adherent PER.C6® cells at 37°C/10%> C0 ₂ in Dulbecco's modified Eagle's medium supplemented with 10%> of fetal bovine serum (Life Technologies Inc.) and 10 mM MgCl ₂ .

The replication deficient virus was purified by standard two-step CsCl-gAdient and dialyzed in formulation buffer 10 mM Tris (pH 7.4), 1 mM MgC12, 75 mM NaCl, 5% sucrose, 0.02% PS-80, 0.1 mM EDTA, 10 mM Histidine, and 0.5% ETOH). The virus concentration viral particles per ml (VP/ml) and infectivity unit per ml (IU/ml) titers were determined by optical density in the presence of SDS (Maizel et all 968) and infectivity titrated by TCID ₅₀ assay. As it can be observed in Table 1 the pIX- modified vectors did not differ from the pIX-native control vectors (Ad.empty) in terms of the VP/ml titers (range lxlO ¹¹ - 5xl0 ¹² ) and/or IU/ml titers (range lxlO ¹⁰ - 5x10 ¹¹ ). In addition, the VP/IU (range of 0-30, with three exceptions) ratios and the productivity in PER.C6® cells (VP/cm ² , range 5xl0 ⁷ - 5xl0 ⁹ ) were determined (Table 1). The VP/ml, IU/ml, VP/IU ratio and the productivity in PER.C6® cells of the pIX- modified vectors is comparable to the control (native pIX) vectors, hence this suggests that the plX-modification did not have a detrimental effect on the titers and productivity in PER.C6® cells (Table 1).

The "transgene only" vectors: HAdV35. Empty (no transgene), HAdV35.CS _sh0rt and HAdV35.CS were used as a control for the "pIX modified" vectors with or without the transgene, HAdV35.Empty.pIX-CS _sh ort, HAdV35.CS.pIX-CS _sh ort,

HAdV35.Empty.pIX-CS and HAdV35.CS.pIX-CS (Figure IB). Table 1 Characteristics pIX-modified HAdV26 and HAdV35 vectors

Vector TG plX Batch VP/ml ^T CID, ₀ VP/IU ratio VP/cm2

HAdV26 12

AdVac© Empty NA 1.7x10 1.2x10 14 1.8x10

12

Empty short 2.0x10 2.2x10 94 2.9x10

12

CS short 1.3X10 ¹ 2.6x10 50 1.9xl0 ^:

12

Empty 45-CS sh , ort . 1.9x1ο ¹ 8.0x10 23 9.5xl0 ^:

12

Empty SP1-CS sh, ort . 1.5x1ο ¹ 5.3x10 34 2.3χ1θ'

12

Empty SP3-CS sh , ort . l .lxlO ¹ 5.3x10 21 6.8xl0 ^:

12

Empty SP4-CS sh , ort . 2.1x1ο ¹ 8.0x10 27 1.7χ1θ'

HAdV35 12

AdVac© Empty NA 1.0x10 3.3x10 6.1x10

12

CS GlyCS sh , ort . 2.8x10 2.0x10 13 NA

12

Empty GlyCS sh , ort . 3.3x1ο ¹ 2.9x10 11 NA Luc GlyCS sh , ort . 4.8x1ο ¹ 4.9x10 10 NA

12

Empty ^ short 2.6x1ο ¹ 4.2x10 6 NA

12

CS ^ short 1.5x1ο ¹ 2.0x10 8 8.8xl0 ^:

12

Empty 45-CS short 1.4x1ο ¹ 1.8x10 8 2.5xl0 ^:

CS 45-CS short 1 2.8x1ο ¹ 1.7x10 17 1.3xl0 ^:

CS 45-CS sh , ort . 2 5.9x1ο ¹ 1.5x10 4 2.9xl0 ^:

12

Empty Gly45-CS sh , ort . l .lxlO ¹ 6.0x10 19 6.3xl0 ^:

12

CS Gly45-CS sh , ort . 1.9x1ο ¹ 1.8x10 11 1.8xl0'

Empty CSfull* 1 2.5x1ο ¹ 3.7x10 7 5.9x10

12

Empty CSfull* 2 Ι .δχΙΟ ¹ 1.4x10 13 1.7xl0 ^: CS CSfull* 1 4.5x1ο ¹ 3.6x10 12 NA CS CSfull* 2 6.8x1ο ¹ 5.6x10 12 3.0xl0 ^:

12

Empty SP1-CS short 2.2x1ο ¹ 3.3x10 7 3.8xl0'

12

Empty SP2-CS short 3.4x1ο ¹ 2.2x10 15 l . lxlO'

12

Empty SP3-CS short 3.3x1ο ¹ 4.9x10 7 7.5xl0'

12

Empty SP4-CS short 2. 4x10 3.3x10 7 4.1χ1θ'

12

Empty mHA(ecto)* 1.6x10 4.0x10 4 NA

12

mHA mHA(ecto)* 1.8X10 ¹ 2.7x10 7 NA mHA.GCN(ect 12

Empty o)* 1.6x10 4.9x10 NA mHA.GC mHA.GCN(ect 12

N o)* 1.7x10 4.9x10 8 NA

12

Empty HA(ecto)* 1.9x1ο ¹ 1.2x10 16 NA

12

HA HACecto)* 1.4x1ο ¹ 1.8x10 8 NA

''plX not incorporated into the capsid, NA: not available pIX-CS and pIX-CS _S hort capsid incorporation and transgene expression assessment by Western blot

Capsid incorporation of pIX-CS and/or pIX-CS _s h ₀ rt variants was performed by Western Blot (WB) of purified viral particles at 1.5, 0.5 and 0.17 xlO ¹⁰ viral particles per well (VP/well) concentration. The reduced and denatured purified viral particles were separated on pre-cast 12% Bis/Tris Nu-PAGE gel (Invitrogen) in MOPS buffer (Invitrogen) at 175 V, 500 mA. The protein was subsequently transferred to a nitrocellulose membrane according to manufacturer's recommendations using iBlot® Transfer stacks (iBlot system; Invitrogen). The staining was performed for 1 hour with anti-CS specific antibodies (2A10) (or anti-HAdV35.pIX monoclonal) and anti- fiber (HAdV5 4D2, Abeam) as a loading control in 5% Non-fat dry milk (BioAd)/Tris buffered Saline Tween 20 (Invitrogen). Visualization of the protein of interest was achieved by staining with fluorescent ly labeled IRDye800CW ^® 1 : 10 000 goat anti- mouse and recorded on the Odyssey® (Li-Cor).

Direct comparison of the pIX-CS _s h ₀ rt (~32 kDa) in the capsid of HAdV35.Empty.pIX- CSshort, HAdV35.CS.pIX-CSshort and negative control vector HAdV35. Empty, stained with the anti-CS antibody (2A10) and anti- fiber antibody (4D2), shows a comparable content in the pIX-CS _s h ₀ rt between the CS transgene encoding and Empty vectors at equal loading (fiber protein~35 kDa) per well (Figure 1C, left WB and Table 2). The same can be observed for the HAdV26. Empty or CS transgene pIX-CS _s h ₀ rt vectors. There is no apparent difference between the capsid incorporated pIX-CS _s h ₀ rt in either HAdV26 with or without (empty) encoded CS transgene at equal loading (fiber protein ~40 kDa visible at the highest concentration VP/well) (Figure 1C, right WB and Table 3).

Table 2 Capsid incorporation plX-modifications

Capsid modification pIX fusion Capsid incorporation (n) TG pIX a.a.

HAdV35 Advac CS CSshort 151 (3)

Luc CSshort 151 (3)

Empty CSshort 151 (3)

CS Gly-CSshort 154 (2)

Empty Gly-CSshort 154 (2)

CS 45-CSshort 185 (2)

Empty 45-CSshort 185 (2)

CS Gly45-CSshort 188 (2)

Empty Gly45-CSshort 188 (2)

Empty SP1 -CSshort 183 (1)

Empty SP2-CSshort 184 ++4 (1)

Empty SP3-CSshort 181 (1)

Empty SP4-CSshort 183 + 0)

CS CSFULL 376 - (2)

Empty CSFULL 376 - (2) mHA mHA(ecto) 248 - (2)

Empty mHA(ecto) 248 - (2) mHA.GCN mHA. GCN(ecto) 305 - (2)

Empty mHA. GCN(ecto) 305 - (2)

HA HA(ecto) 512 - (2)

Empty HA(ecto) 512 - (2)

++++: very good, +++: good, ++: poor, +: barely detected and -: not detected

(n)= number of experiments, a.a.: amino acids

Table 3 Capsid incorporation plX-modifications

Capsid modification pIX fusion Capsid incorporation (n)

TG pIX a.a.

rHAdV26

Advac Empty CS short 151 (2)

CS CS short 151 (2)

Luc CS s,nort 151 (2)

Empty SPl-CS short 183 (1)

Empty SP3-CS short 181 (1)

Empty SP4-CS short 183 (1)

Empty 45-CS short 185 (1)

++++: very good, +++: good, ++: poor, +: barely detected and -: not detected

(n)= number of experiments, a.a.: amino acids In addition to the capsid pIX-CS _sh0rt (~32 kDa) in the CS transgene encoding vectors, the CS protein (-42 kDa) itself is also detected in the purified HAdV26 and HAdV35 batches, which suggests nonspecific association to the capsid or at least co- purification (Figure 1C). Assessment of capsid incorporation of pIX-CS in the HAdV35 pIX-CS modified vectors with and without CS as a transgene, by staining purified vectors with anti-CS (2A10) showed no capsid incorporation of the pIX-CS (Table 2). To ensure that the lack of capsid incorporation of pIX-CS in the

HAdV35.Empty.pIX-CS and HAdV35.CS.pIX-CS was not due to decreased or absent expression of the pIX-CS modification in the PER.C6® cells during production of the vector, PER.C6® cell lysates infected with 5000 VP/cell were tested on WB and stained with anti-HAdV35.pIX monoclonal antibody. The WB analysis shows expression of pIX-CS (-55 kDa) in PER.C6® infected with either the HAdV35.CS and Empty pIX-CS modified vectors (data not shown). The latter suggest that the lack of pIX-CS in the capsid is due to the inefficient incorporation of the CS protein fused to pIX into the viral capsid and not the lack of pIX-CS protein expression during the production of the vector in PER.C6®. It can be concluded that pIX-CS _sh0rt is efficiently incorporated in both the HAdV26 and HAdV35 viral capsids and that the encoded CS transgene in HAdV26 and HAdV35 vectors had no effect on the incorporation efficiency of pIX-CS _s h ₀ rt. In contrast the pIX-CS was not incorporated into the viral capsid which indicates that the size of the antigen (CS _sh0rt 151 a.a. vs. CS 376 a.a.) or possibly the tertiary structure might have an effect on the capsid incorporation.

Transgene expression

In order to ensure that plX-modified vectors can still express a transgene, we assessed whether pIX-capsid modification could have a possible effect on the transgene expression. The plX-modified and El transgene encoding vectors were tested in A549 cells for the expression of the transgenes under non replicating conditions. A549 cells were transduced with HAdV26.CS.pIX-CS _s h ₀ rt and HAdV26.Empty.pIX-CS _s h ₀ rt at 10000, 25000 and 50000 VP/cell or HAdV35.CS.pIX-CS _short /pIX-CS and

HAdV35.Empty.pIX-CS _short /pIX-CS at 1000, 2500 and 5000 VP/cell, incubated for 48 hours at 37°C, harvested, lysed and analyzed in WB with anti-CS (2A10) antibody. The expression of CS transgene (-42 kDa) of all the pIX-CS _sh0rt vectors encoding the CS transgene was comparable to the transgene only controls (i.e. HAdV26.CS and HAdV35.CS). In the Firefly luciferase (luc) encoding vectors the expression of luciferase was also determined (data not shown). Due to the endogenous pIX promoter and the possible enhancing effect of the upstream CMV promoter in El- cassette (Nakai et al 2007) the expression of the pIX-modification is expected and confirmed under non-replicating conditions. Surprisingly however, CS _s h ₀ rt protein as a transgene (HAdV35.CS _s h ₀ rt) was not detected in A549 cells, indicating some disregulation in protein turnover. Cumulatively these observations imply that (1) fusion of CSshort to pIX most probably stabilizes the CS _s h ₀ rt protein since on its own no viable protein is detected and (2) pIX- CS _s h ₀ rt modifications do not have an effect on the CS transgene expression in A549 cells. Taken together the CS _s h ₀ rt fused to pIX did incorporate into the viral capsid and doing so did not impair the transgene expression. pIX-HA modified vectors: capsid incorporation and transgene expression assessment by Western blot

As part of the development of an Influenza AdVac© based vaccine, pIX modified vectors were designed displaying different variants of an Influenza A protein in combination with homologous transgenes of these variants in El . Three different variants of the Influenza A Hemagglutitin (HA) were designed and fused to pIX, namely 512 amino acid HA, 303 amino acids mHA, and 248 amino acids

mHA.GCN(ecto) (ect-domain swapped with the GCN domain) with or without the respective proteins integrated as transgene in the El region of the same vector

(Figure 2). Analysis of capsid incorporation of the pIX-HA- variants by direct staining of the HAdv35.HA.pIX-HA, HAdV35.Empty.pIX-HA, HAdV35.mHA.pIX-mHA, HAdV35.Empty.pIX-mHA, HAdV35.mHA.GCN.pIX-mHA.GCN(ecto) and

HAdV35.Empty.pIX-mHA.GCN(ecto) viral particles (VP) in Western Blot with both polyclonal anti-HA and monoclonal anti-pIX antibodies showed no pIX modified proteins in the capsid (Table 2). However, the transgene expression in A549 cells was confirmed by staining A549 cell lysates transduced with the indicated vectors at 1000, 2500 and 5000 VP/cell (data not shown). The observations were identical for the CS (376 amino acids) fusion to the pIX which, as discussed, did not incorporate into the capsid but was produced during the replication and vector production in PER.C6® cells (data not shown). Overall, efficient capsid incorporation of the CS _s h ₀ rt (151 a.a.) was observed while no capsid incorporation was observed for the CS (376 a.a.) and the HA variants (248, 303, and 512 a.a.) directly fused to pIX. This indicates that it is difficult to predict which antigens will be efficiently incorporated into the capsid and which antigens will not. An overview of the capsid incorporation efficiency of all the plX-modified vectors, based solely on the qualitative observations (of band intensities) made in WB analysis by direct comparison to the control vectors is provided in Table 2 and 3.

Immunogenicity of pIX-CS _S hort capsid modified vectors directly compared to the immunogenicity of the CS protein

Determination of CS-specific humoral immune responses by antibody-ELISA

P. falciparum CS-protein-specific total IgG, or subclass-specific IgG 1 and IgG2a responses in serum were determined by an enzyme-linked immunosorbent assay (ELISA) as previously described in (10). MaxisorpTM flat-bottomed 96-well plates (Nunc-Immuno, location) were coated over night at 4°C with 2 μg/ml of CS-specific (NANP)6C peptide (Pepscan Presto, Netherlands) in 0.05 M carbonate buffer (pH 9.6). Plates were washed in PBS/0.2% Tween20 (PBS-T) and blocked with 1 % BSA in PBS/0.02% Tween20 for 1 hour at room temperature, then washed again. Mouse serum samples were diluted in sample buffer (PBS/0.02%> T ween/0.2%> FBS) at a starting concentration of 1 : 100, plated in a 2-fold dilution series and incubated at for 2 hours at RT. Plates were washed with PBS-T. Total bound IgG was detected with biotinylated goat-anti-mouse IgG (Dako, location) and streptavidin-conjugated horseAdish peroxidase (HRP; Becton Dickinson). For the detection of IgG subclasses, duplicate plates were incubated in parallel and bound IgGl or IgG2a was detected with goat-anti-mouse IgGl or -anti IgG2a antibodies directly conjugated to HRP (Southern Biotechnology Associates, Birmingham, AL). After washing of the plates with PBS-T, o-Phenylenediamine (OPD; Pierce) was added, the plates developed for

10 minutes and the reaction stopped with 1M H ₂ SO ₄ before optical density was determined at 492 nm. Relative serum titers of total IgG (ELISA units/ml) were calculated in comparison to a P. falciparum CS-specific reference serum, using a 4- parameter curve fit model. The IgGl- and IgG2a specific measurements were used directly to calculate the ratio between IgG2a and IgGl , by dividing the reciprocal dilution at which the OD492 reached three times that of the background measurement in a naive control sample. Statistical analysis

The response variables, i.e. EU (ELISA Unit) and SFU (Spot Forming Unit) were log- transformed and group comparisons were performed using ANOVA models. In case of repeated measurements over time, a random intercept was added to the ANOVA model to properly account for correlated observations. Comparisons between groups containing values below the lower limit of quantification were analyzed using censored regression models. A correction for multiple testing was applied using the Dunnett method, since there was a fixed reference group in each analysis. Differences with a p<0.05 were considered significant. All statistical analyses were performed using S AS ^® software, version 9.2 (S AS Institute Inc . , 2011 ) .

The CS-specific humoral response (B-cell responses) induced by

HAdV35.Empty.pIX-CSshort and HAdV35.CS.pIX-CS _s h ₀ rt was tested in mice and compared to CS protein (CSP). Balb/C mice (n=7 per group) were immunized intramuscularly with lxlO ¹⁰ VP HAdV35.Empty.pIX-CS _sh ort, HAdV35.CS.pIX-CS _sh ort or 5 μg (unadjuvanted) yeast-produced CSP. Mice immunized with lxlO ¹⁰ VP HAdV35. empty vector served as a negative control. CS-specific B-cell responses were determined at week 4 post immunization. Both pIX-CS _s h ₀ rt vectors induced high CS-specific total IgG titers that highly exceeded those induced by the CSP (p<0.0001 ANOVA with Dunnett correction for multiple comparisons), indicating that the display of the CS antigen on the capsid of the viral particles is a very potent platform, even compared to the highly immunogenic CS protein (Figure ID left panel). To determine whether the relatively high immunogenicity of the pIX-CS _s h ₀ rt vectors is mediated by a potentially adjuvating effect of the vector itself, Balb/C mice were immunized intramuscularly with 5 μg CSP alone or mixed with lxlO ¹⁰ VP

HAdV35. empty, HAdV26. empty or HAdV5. empty (n=6 mice per group). 5 μg adjuvanted CSP was taken as a reference point and adjuvant only served as a negative control.

4 weeks after immunization CS-specific IgG titers in mice immunized with adjuvanted CSP exceeded those induced with CSP protein alone (p = 0.0006,

ANOVA with Dunnett correction for multiple comparisons) and reached levels similar to those observed in animals immunized with lxlO ¹⁰ VP of pIX-CS _s h ₀ rt modified HAdV35 vectors at the same time point post immunization. No statistically significant (p=0.8) increase in CS-specific IgG titers was observed in the mice immunized with HAdV. empty vectors mixed with CSP when compared to the CSP protein alone (Figure ID). Balb/C mice immunized with a dose range of lxlO ⁷ , lxlO ⁸ , lxlO ⁹ , or lxlO ¹⁰ VP HAdV35.Empty.pIX-CS _sh ort compared to the HAdV35.CS transgene only vector, were more potent over a period of 8 weeks post immunization at inducing CS-specific IgG titers (data not shown).The immunogenicity of the HAdV26.Empty.pIX-CSshort and HAdV26.CS.pIX-CS _sh ort compared to the

HAdV26.CS transgene only vector was also determined in mice. Balb/C mice were immunized with a dose range of lxlO ⁷ , lxlO ⁸ , lxlO ⁹ , or lxl0 ¹⁰ VP

HAdV26.Empty.pIX-CSshort, HAdV26.CS.pIX-CS _sh ort and HAdV26.CS. As a negative control HAdV26. empty was used at the highest dose lxlO ¹⁰ VP per animal (n=5). The results observed were similar to those with the HAdV35 pIX-CS _s h ₀ rt; in overall, 4 weeks post immunization, the plX-modified vectors were more potent at inducing CS- specific IgG titers at all doses, than the CS transgene only vector (data not shown). In sum, plX-CSshort modified vectors are more potent than a CS protein alone or a HAdV35.CS transgene vector (without the pIX-CS _s h ₀ rt fusion protein) at inducing CS- specific antibody responses. This effect cannot be achieved by mixing the protein with the HAdV. Empty vectors suggesting that the fusion of the CS _s h ₀ rt protein to the capsid is necessary for induction of superior antigen specific antibody responses.

Example 2: Generation and characterization of pIX-CS _S hort modified HAdV35 and HAdV26 vectors containing a spacer between the pIX and the CS _S hort

HAdV35 and HAdV26 vectors were modified by fusing a CS _s h ₀ rt protein to pIX via a 45 A spacer (16, Vellinga et al. 2004) only, a 45 A spacer combined with a Gly- linker for added flexibility or a Gly-linker only. The sequence of the 45A spacer can be found in Table 4. HAdV35.pIX-45-CS _sh ort, HAdV35.pIX-Gly45-CS _sh ort and

HAdV35.pIX-Gly-CSshort vectors with or without CS transgene in El were generated in PER.C6® as indicated above for the HAdV35 direct fusion (pIX-CS _s h ₀ rt) vectors (Figure 3 A and Table 1). pIX-CSshort spacer variants capsid incorporation and transgene expression assessment by Western blot

Capsid incorporation of pIX-CS _s h ₀ rt spacer variants was assessed by WB as described above. Purified viral particles 1.5, 0.5 and 0.17 xlO ¹⁰ VP/well were reduced, denatured, separated on gel and transferred to a nitrocellulose membrane. The staining of pIX-CSshort spacer variants was performed with a CS-specific antibody (2A10) and anti- fiber antibody (4D2) as a loading control, where the HAdV35. empty vector served as a negative control. To ensure that the CS transgene has no effect on the capsid incorporation of the different spacer variant pIX-CS _s h ₀ rt modifications all the vectors with and without the transgene were compared side by side. At comparable loading (fiber band -35 kDa), the WB results show no difference in capsid incorporation of pIX-CS _s h ₀ rt spacer variant, between the vectors encoding the CS transgene in El and the empty vectors. The same as with the pIX-CS _s h ₀ rt direct fusion modifications, in purified vectors encoding the CS transgene, the CS transgene seems to be co-purified with the pIX spacer vectors. An obvious difference between the 45- spacer containing vectors and either the Gly-only or direct fusion CS _s h ₀ rt is a band migrating at approximately 100 kDa in addition to the pIX- fusion protein (Figure 3B). This might be an assay artefact or an unknown effect of the 45 -spacer on the pIX and CSshort modification. Direct head to head comparison of the different pIX-CS _s h ₀ rt variants, including the direct fusion, stained with anti-CS antibody (2A10) shows comparable levels of pIX incorporated in the capsid between all the vectors. It seems that the addition of the spacer or the Gly-linker does not have a detrimental effect on the capsid incorporation efficiency. pIX-45-CS _s h ₀ rt, pIX-Gly45-CS _s h ₀ rt and pIX-Gly- CSshort are efficiently incorporated into the viral capsid in vectors with and without the CS transgene.

Spacer pIX-CS _S hort capsid modified vectors immunogenicity directly compared to the CS transgene vector

The CS-specific B-cell responses against the HAdV35.empty.pIX-Gly45-CS _s h ₀ rt, HAdV35.CS.pIX-Gly45-CSshort, HAdV35.empty.pIX-Gly-CS _sh ort, HAdV35.CS.pIX-

Gly-CSshort, HAdV35.CS.pIX-45-CSshort, HAdV35.empty.pIX-45-CS _sh ort vectors were determined in mice and directly compared to the HAdV35.CS control vector. Balb/C mice (n=5) were immunized intramuscularly at a dose range of lxlO ⁷ , lxlO ⁸ , lxlO ⁹ , lxlO ¹⁰ VP . Animals immunized with lxlO ¹⁰ VP of HAdV35. empty served as a negative control. Total CS-specific IgG responses in the serum were measured at 2- week intervals over a period of 8 weeks post immunization. At lxl 0 ⁷ VP/ mouse, none of the tested vectors mounted immune responses above the lower assay limit of quantification. At lxl 0 ⁸ all constructs displaying the CS _sh0rt induced CS-specific IgG responses by week 4 post immunization and these responses were maintained until the end of the experiment at week 8. HAdV35.CS showed comparatively lower potency, with the lowest dose to induce CS-specific IgG responses being lxlO ⁹ VP. Across all doses that induced detectable CS-specific responses, overall titers elicited by the four vectors pIX-CS _s h ₀ rt via pIX were higher than those induced by HAdV35.CS. CS _s h ₀ rt display on any of the three spacer constructs proved to further increase

immunogenicity, resulting both in earlier induction of CS-specific IgG responses compared to the pIX-CS _sh0rt modified vector and titers that were significantly higher than those induced by HAdV35.CS (lxlO ⁸ - lxlO ¹⁰ VP; mixed models analysis). Although the three different spacer constructs induced CS-specific IgG titers of comparable magnitude, HAdV35.empty.pIX-45-CS _s h ₀ rt proved to be the only vector consistently reaching responses that significantly exceeded HAdV35.CS at all three immunogenic doses (lxlO ⁸ - lxlO ¹⁰ VP) (data not shown).

Immune responses against the CS transgene in pIX-45-CS _S hort capsid modified vectors

The induction of cellular immune responses in addition to humoral responses against the Malaria causing agent P. falciparum, is considered a desirable feature of a protective vaccine against infection at the pre-erythrocytic stage. Adenoviral vectors are characterized by strong induction of T cell responses against encoded transgenes, as evidenced by high CD4+ and CD8+ T cell responses detected in mice and nonhuman primates immunized with HAdV35.CS (Radosevic et al. 2011 , Rodriguez et al. 2009). We therefore assessed whether implementation of pIX-display of the B- cell epitope carrying CS _sh0rt antigen on HAdV35 encoding the CS transgene

(including the immune-dominant H-2KD restricted CD8+ T cell epitope

NYDNAGTNL) would maintain the induction of strong humoral immune responses as seen before, and whether this combination would impact T cell responses against the encoded transgenes.

For this purpose, the CS-specific humoral responses induced by the

HAdV35.empty.pIX-45-CSshort and HAdV35.CS.pIX-45-CS _sh ort were tested in Balb/C mice (n=5) immunized intramuscularly with a dose titration lxlO ⁸ , lxlO ⁹ and lxlO ¹⁰ VP/animal. The vectors were directly compared to the HAdV35.empty.pIX-45-CS _s h ₀ rt ₊ HAdV35.CS transgene vector mix, HAdV35.CS alone and HAdV35 was used as a negative control. To adjust the VP number given to the group receiving the mix (HAdV35.empty.pIX-45-CSshort + HAdV35.CS), the total VP number in the remaining groups was supplemented by using HAdV35. empty, which resulted in a formal total vector dose of 2x10 ⁸ - 2x10 ¹⁰ VP/mouse.

CS-specific total IgG responses in serum were measured at 8 weeks post

immunization (Figure 3C). In contrast to HAdV35.CS and similarly to

HAdV35.empty pIX-45-CS _sh ort, HAdV35.CS.pIX-45-CS _sh ort induced measurable CS- specific antibody responses at the lowest dose (lxl 0 ⁸ ), indicating higher potency of the vector compared to plX-unmodified HAdV35.CS. In addition, the titers mounted by both HAdV35.CS.pIX-45-CSshort and HAdV35.empty pIX-45-CS _sh ort at week 2 at lxlO ¹⁰ VP strongly exceeded those induced by HAdV35.CS at the same dose of lxlO ¹⁰ VP (p<0.005 for all comparisons, Figure 3C). Although the overall titers in all groups increased in magnitude over time, those induced by HAdV35.CS remained significantly lower than those induced by the two pIX-45-CS _s h ₀ rt fusion constructs at week 8 post immunization (p<0.005 for all comparisons in the groups receiving lxl 0 ⁸ and lxlO ⁹ VP, Figure 3C). Application of the HAdV35.empty.pIX-45-CS _sh ort fusion construct and HAdV35.CS in the equal mix also resulted in significantly increased CS-specific titers at all doses and time points, with the exception of the lowest dose, lxlO ⁸ , where the difference in titers did not reach significance at week 8 post immunization. This indicates that immune responses against the transgene do not interfere with the induction of humoral responses against the pIX-CS _s h ₀ rt fusion construct.

Determination of CS- and HAdV35 hexon-specific cellular immune responses by ELISPOT

Antigen-specific cellular immune responses in vaccinated mice were assessed using an IFNy enzyme-linked immune-spot assay (ELISPOT) based on the method previously described in (10). Freshly isolated splenocytes were incubated either with a pool of 15-mer peptides overlapping by 11 amino acids, spanning the entire sequence of the P falciparum CS protein, with the described H-2Kd-MHC class I-restricted, immunodominant NYDNAGTNL CS epitope, or with the immunodominant HAdV35 hexon epitope KYTPSNVTL restricted by the same MHC class I epitope. The peptide pools and single peptides were used at a final concentration of 1 ug/ml for each individual peptide. 96-well multiscreen plates (Millipore, Bedford, MA) were coated overnight with 100 μΐ/well of 10 μg/ml anti-mouse IFN-γ (BD Pharmingen, San Diego, CA) in endotoxin- free Dulbecco's phosphate-buffered saline (D-PBS). The plates were then washed three times with D-PBS containing 0.05% Tween 20 (D- PBS-Tween), blocked for 2 h with D-PBS containing 5% fetal bovine serum (FBS) at 37°C, and rinsed with RPMI 1640 containing 10% FBS. Splenocytes from individual mice were stimulated with the CS peptide pool, the CS 9-mer or the HAdV35 hexon 9-mer for 18 h at 37°C. Following incubation, the plates were washed six times with D-PBS-Tween and once with distilled water. The plates were then incubated with 2 μg/ml biotinylated anti-mouse IFN-γ (BD Pharmingen, San Diego, CA) for 2 h at room temperature, washed six times with D-PBS-Tween, and incubated for 2 h with a 1 :500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology

Associates, Birmingham, AL). Following six washes with D-PBS-Tween and one with PBS, the plates were developed with nitroblue tetrazolium-5-bromo-4-chloro-3- indolyl-phosphate chromogen (Pierce, Rockford, IL), the reaction was stopped with tap water, the plates air dried, and read using an ELISPOT reader (Aelvis GmbH). The numbers of spot-forming units (SFU) per 10 ⁶ cells were calculated.

CS-specific cellular immune responses were assessed against all four constructs by ELISPOT (peptides spanning the entire CS protein sequence) at week 8 post prime (Figure 3D). Due to the absence of the immune-dominant T cell epitope

NYDNAGTNL in the CS _sh0 rt sequence, HAdV35.empty.pIX-45-CS _sh0 rt did not induce any cellular immune responses, as expected. All other immunization regimen induced responses followed a dose-titration relationship, with increasing responses

corresponding to increasing doses. IFNy responses induced by HAdV35.CS.pIX-45- CSshort did not significantly differ from those induced by HAdV35.CS at lxlO ⁸ and lxlO ¹⁰ VP dose, but were slightly reduced at the intermediate dose of lxl 0 ⁹ VP (p=0.04). Responses induced by the equal mix of HAdV35.empty.pIX-45-CS _s h ₀ rt and HAdV35.CS did not differ from those induced by HAdV35.CS at any of the given doses. As vaccination control, IFNy responses targeting the immune-dominant H-2Kd restricted epitope KYTPSNVTL in HAdV35 hexon were measured, which, as expected, did not differ between groups immunized with the same dose (Figure 3D). These results indicate that pIX-CS _s h ₀ rt-niodified vectors may result in a slight reduction of vector potency, but not in the absolute magnitude of the cellular responses that can be reached at higher dosing of the vector. Taken together, these results indicate that CS _s h ₀ rt display on pIX can be implemented on CS transgene- expressing HAdV35 vectors, significantly increasing humoral immune responses while maintaining strong cellular immune responses.

Replacement of the alpha-helical 45A-spacer (from human origin) with the 45A spacer SPl (from viral origin)

To circumvent the potential unfavorable effects, in terms of autoimmune disease upon vaccination with the vectors containing the human origin 45 -spacer, four

biochemically similar spacers (SP1-SP4) from viral origin were designed (Table 4).

Table 4 Biochemical characteristics spacers

Spacer Sequence (a.a.) Origin Predicted secondary structure

45A ARLSKELQAAQARLGADMEDVCGRLVQYRGEDGA ApoE4 Alpha-helix (34)

SPl CNGTDAKIKLIKQELDKYK AVTELQLLMQST (32) RSV F Alpha-helix

SP2 TNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDL (33) HA Alpha-helix

SP3 QTNARAIAAMK SIQATNRAIFEVKEGTQQ (30) Mumps F Alpha-helix

SP4 SSARDDKATAALAQADSATREA WSQQALDA HAdV35p ^L IX mo _A d Alpha-helix (32)

The following four spacer proteins from viral origin were tested in vitro: spacer 1

(SPl), a RSV Fusion protein comprising the amino acid sequence of SEQ ID NO:2; spacer 2 (SP2), an Influenza A HA comprising the amino acid sequence of SEQ ID NO:3; spacer 3 (SP3), a Mumps Fusion protein comprising the amino acid sequence of SEQ ID NO:4; and finally spacer 4 (SP4), a HAdV35.pIX fragment with point mutations comprises the amino acid sequence of SEQ ID NO:5.

HAdV35. empty vectors encoding the pIX - CS _s h ₀ rt with the four different spacers, SPl -4, HAdV35.empty.pIX-SPl-CS _sh ort, HAdV35.empty.pIX-SP2-CS _sh ort, HAdV35.empty.pIX-SP3-CSshort and HAdV35.empty.pIX-SP4-CS _sh ort were generated as described above using the three plasmid system in PER.C6® cells (Figure 4A and Table 1). The CsCl-purified vectors were tested in WB to determine the capsid incorporation efficiency of the four plX proteins with the four different spacers. Three different concentrations of the SP1-4 vectors (1.5, 0.75 and 0.37xl0 ¹⁰ VP/well) were directly compared to the 45 -spacer control vector and the native plX in

HAdV35.empty (Figure 4B). Staining of the protein was performed with both, anti- pIX monoclonal and anti-CS (2A10) antibodies. Since the anti-plX antibody recognizes one specific epitope in plX HAdV35, in contrast to the 27-NANP repeat sequence for the anti-CS antibody (2A10), the anti-plX antibody was considered to be more sensitive to the differences in capsid incorporation than the anti-CS NANP- repeat specific antibody. At equal loading (fiber band -35 kDa) of the vectors, the pIX-SPl-CSshort (~32 kDa) showed to be the most abundant in the capsid, compared to pIX-45-CSshort (~32 kDa) and plX (~14 kDa). In terms of capsid incorporation, pIX-SP2-CSshort was the second best followed by pIX-SP3-CS _s h ₀ rt and finally the pIX- SP4-CSshort with the lowest capsid incorporated modified plX. To confirm the application of SPl as a functional spacer for plX fusions, the same capsid

modifications were generated in HAdV26, except for SP2. HAdV26.empty.pIX-SPl- CSshort, HAdV26.empty.pIX-SP3-CSshort and HAdV26.empty.pIX-SP4-CS _sh ort were produced, as described earlier, with the whole genome plasmids in PER.C6® cells and characterized (Table 1). The indicated vectors were analyzed in WB by comparing three different concentrations (1.5, 0.5 and 0.16xl0 ¹⁰ VP/well) of the SPl, SP3 and SP4 to the control vector 45 spacer (Figure 4D). Consistent with the results observed for the HAdV35 SP1-SP4 spacer vectors, the staining performed with anti- CS NANP -repeat antibody 2A10 showed no difference in the capsid incorporation efficiency (unlike the staining with the anti-plX antibody) (Table 3). In terms of capsid incorporation efficiency the SPl spacer is comparable to the 45-spacer in

HAdV35 and HAdV26 vectors. Slight differences are observed with the SP2 spacer and larger differences with the SP3 and SP4 in terms of capsid incorporation. This effect of the spacers on the observed capsid incorporation efficiency shows that the choice of the spacer is important. The differences observed in capsid incorporation efficiency with the SP2, SP3 and SP4 spacers in HAdV35 suggest that the choice of the alpha-helical spacer is important for efficient capsid incorporation. Example 3: Manufacturability pIX-CS _shor t modified HAdV35 and HAdV26 vectors

Kinetics and propagation in PER. C6® cells of HAdV26 pIX modified vectors

Since many pathogens such as malaria causing parasite are often endemic to resource- limited areas, manufacturing proceses with high yields are necessary to ensure proper vaccine coverage. Based on observations that some HAdV vectors can be sensitive to certain genetic modifications such as inserted transgenes, the relative viral particle increase (VP/ml) and the kinetics of the pIX modified vectors were assessed in suspension cultures of PER.C6® cells in parallel with control vectors known for their high and low producibility (i.e. 'good producers' and 'low producers'). The growth of the adenoviral vectors on PER.C6® cells (in suspension) in a bioreactor (data not shown) was performed to assess the relative VP/ml increase and the kinetics of these vectors. The assessment of adenovirus vector propagation on PER.C6® cells entails: the infection (n=3) of PER.C6® cells (10-15 ml lxlO ⁶ viable cells (vc/ml)) at a predetermined VP/cell ratio of either 70 or 900, the harvest (100 μΐ n=3) at 0, 1, 2, 3 and 4 days post infection (DPI) and the titer (VP/ml) determination by CMV promoter based VP-QPCR.

Titer (VP/ml) determination by CMV VP-QPCR

In order to determine the VP/ml titers and the kinetics of pIX modified vectors in

PER.C6® cells, cell lysis was performed by addition of 1% Triton™ X-100 (Sigma- Aldrich) followed by a treatment with DNase I (Roche) to remove the un-capsidated viral DNA in the solvent. After lysis the samples were diluted lOOOx in H ₂ 0 (Gibco) and CMV based QPCR was performed. For the QPCR the forward primer (5 '- TGGGCGGTAGGCGTGTA-3 ') and reverse primer (5 '-

CGATCTGACGGTTCACTAAACG-3 ') were used. The reactions were performed in triplicate in a final volume of 15 μΐ of which 5 μΐ of the diluted sample was mixed with 10 μΐ reaction mix. The reaction mix contained, in addition to 10 pmol primers and 2.5 pmol probe, 2x TaqMan Gene expression master mix (Life Technologies) and 25 mM MgCl ₂ . The final reaction was performed by denaturing the samples at 95°C for 8 minutes, followed by 10 seconds at 95°C and 30 seconds at 60°C for 35 cycles in the QPCR machine (Applied Biosystems Viia7).

The results show, a VP increase of approximately 2 Log when the cells are infected with 70 VP/cell, for the 'good' producer control and 1 Log for the 'low' producer control at day 3. Further increase in VP/ml titer is observed at day 4 for both (~1 log) (Figure 5 A, upper graph). The same relative increase as observed with the 70 VP/cell infections is also observed for the 'good' producer control (~2 Log) and the 'low' producer control (~1 Log) at day 3, when the cells were infected with 900 VP/cell (Figure 5A, lower graph). The tested HAdV26.CS.pIX-CS _sh ort and rHHAdV26.empty.pIX-CSshort vectors follow the same relative VP/ml increase, from time point 0 to day 4, as the 'good' producer control. This observation leads to the conclusion that pIX-CS _s h ₀ rt modification of the HAdV26 vectors with or without CS transgene in El, does not affect the relative titer (VP/ml) increase in suspension PER.C6® cells.

Genetic stability pIX modified vectors after extended passaging in PER.C6® cells

As noted earlier, transgene products are produced during virus amplification in the El -complementing PER.C6® cell line due to the highly potent CMV promoter.

Depending on the transgene and perhaps the amount of expressed protein, this can be deleterious to virus formation. Since both the pIX-CS _s h ₀ rt and CS transgene are expressed in PER.C6® cells during virus propagation and upscaling we wanted to test whether these vectors remain genetically stable after extended passaging in PER.C6® cells. To determine the genetic stability of the pIX-CS _s h ₀ rt modified vectors with and without the CS transgene in El upon production in the producer PER.C6® cells, purified vectors were further propagated in PER.C6® up to four viral passages (VPN) beyond the envisiond commercial process (Figure 5B gives a schematic

representation of passaging adenovirus vectors in PER.C6® cells). Upon rescue and upscaling in PER.C6® cells, the vectors would have been passaged on average seven times prior to their purification with CsCl. During rescue the HAdV viral DNA is isolated to confirm the sequence of the pIX-CS _s h ₀ rt modification and El CS transgene (if applicable), by PCR and sequencing (Sanger). Further upscaling is executed with the vectors containing the correct pIX-CS _s h ₀ rt and El CS sequences. When purified, the viral DNA is isolated yet again and analyzed by PCR and confirmed by sequencing (Sanger). The subsequent passaging in PER.C6® cells (10 ml lxl 0 ⁶

VC/ml) is performed in controlled conditions by infecting the cells with either 70 or 900 VP/cell and harvesting at 3 DPI (n=2). Finally, at VPN 14 the viral DNA is isolated and analyzed by PCR. pIX and El PCR assay and viral DNA isolation

In order to assess whether the modified regions in the genome (i.e. pIX-CS _s h ₀ rt and the CS transgene) are still genetically encoded in the vector after extended passaging in PER.C6® cells, pIX and El specific PCR is performed on the viral DNA. Viral DNA from either crude lysates or purified vector batches was performed by using the

GeneClean Spin kit (BiolOl Inc.) according to the manufacturer's recommendations. The DNA was subsequently used as a template to analyze pIX region and/or El region. For the HAdV26 pIX-PCR the primers (5 '-GACATCATGAACGGGACTG- 3' and 5 '-GATCGAGATGCCGTAGAG-3 ') and for the HAdV35 pIX-PCR the primers (5 '-GGTGGGATTTTCAGATGGACAG-3 ' and 5 '-

CAGGGCATACCGTGCG-3 ') were used. The HAdV35 El - PCR was performed with primers (5 '-GGAGGTTTCGATTACCGT-3 ' and 5'-

CCCTCGATCTCGTATCATCA-3 ') and the HAdV26 El -PCR with primers (5'- TGGCGCGAAAACTGAATGAG-3 ' and 5 '-GCAGGCGGGTTGTCAAATAAG-3 '). The HAdV35 and HAdV26 pIX-PCR conditions were as follows: 1 minute at 94°C, with 30 cycles of 94°C for 30 seconds, 65°C 30 seconds and elongation at 72°C for 2 minutes. The HAdV35 and HAdV26 El -PCR conditions were: 1 minute at 94°C, 10 cycles of 94°C for 30 seconds, 62°C 30 seconds (-0.5°C/cycle) and elongation at 72°C for 2 minutes, followed by 20 cycles of 94°C for 30 seconds, 57°C 30 seconds and elongation at 72°C for 2 minutes. All the reactions were performed with TaKara LA Taq (Clontech) according to manufacturer's recommendations. PIX-PCR Agarose gel analysis shows HAdV26.CS.pIX-CS _sh ort, HAdV26.Empty.pIX-CS _sh ort,

HAdV35.CS.pIX-CSshort and HAdV35.Empty.pIX-CS _sh ort at VPN 7 (preMVS) and VPN 14 (Figure 5C). Both the HAdV35 and HAdV26 pIX-CS _sh ort modified vectors, with or without the CS transgene in El , are genetically stable after extended passaging in sPER.C6®, because only one band equivalent to the positive plasmid control is detected at VPN 7 and VPN 14. The same analysis was performed on the El -cassette with the El -PCR on these vectors; the same observations were made as with the pIX-PCR, no additional bands besides the expected band were observed

(data not shown). Taken together, this data suggests that upon production in suspension PER.C6® cells the pIX-CS _s h ₀ rt and El CS transgene in both the HAdV26 and HAdV35 vectors, remain genetically stable. In addition to the induction of superior CS-specific humoral responses, pIX-CS _s h ₀ rt modified vectors, with and without CS-transgene in El, remain genetically stable and can be produced at high VP/ml titers in the El -complementing PER.C6® cells platform. Considering that many of the pathogens which require a complex immune response are endemic to resource-limited areas, simplification of vaccine regimen and high manufacturing yields makes pIX-display technology particularly valuable to reach underserved communities most relying on vaccine coverage.

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SEQUENCE LISTING

SEQ ID NO:l (CS Short):

GNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNAN PN ANPNANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPN AN PNANPNANPNANPNANPNANKNNQGNG

SEQ ID NO:2 (SP1 Spacer):

CNGTDAKIKLIKQELDKYKNAVTELQLLMQST

SEQ ID NO:3 (SP2 Spacer):

TNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDL

SEQ ID NO:4 (SP3 Spacer):

QTNARAIAAMKNSIQATNRAIFEVKEGTQQ

SEQ ID NO:5 (SP4 Spacer) :

SSARDDKATAALAQADSATREANWSQQALDA SEQ ID NO:6 (HAdV-35 pIX) :

MSGNASFKGGVFSPYLTGRLPSWAGVRQNVMGSTVDGRPVQPANSSTLTYATLSSSPLDA AA AAAAASVAANTVLGMGYYGSIVANSTSSNNPSTLTQDKLLVLLAQLEALTQRLGELSQQV AE LRVQTESAVGTAKSK SEQ ID NO:7 (HAdV-26 pIX) :

MNGTGGAFEGGLFSPYLTTRLPGWAGVRQNVMGSTVDGRPVLPANSSTMTYATVGNSSLD ST AAAAAAAAAMTATRLASSYMPSSGSSPSVPSSIIAEEKLLALLAELEALSRQLAALTQQV SE LREQQQQQNK SEQ ID NO : 8 (P. falciparum circumsporozoite (CS) protein) :

KLATMMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEM NYYGK QENWYSLKKNSRSLGENDDGNNNNGDNGREGKDEDKRDGNNEDNEKLRKPKHKKLKQPAD GN PDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPN AN PNANPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PN ANPNANPNANPNANPNANKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKH IK EYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSV FN VVNS