EA043316B1 - METHODS AND COMPOSITIONS FOR HETEROLOGICAL repRNA IMMUNIZATIONS - Google Patents

Description

Link to the list of sequences presented in electronic form

This application contains a sequence listing, which is presented electronically via EFS-Web as an ASCII formatted sequence listing with the file name sequence_listing, creation date July 26, 2017, and which is approximately 23,045 bytes in size. The sequence listing provided concurrently via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Field to which the invention relates

The invention relates to methods and compositions for inducing an immune response in a human subject. Specifically, an induced immune response is generated by administering a heterologous prime-boost combination of in vitro transcribed (IVT) self-replicating RNA (repRNA) and an adenoviral vector. Such methods and compositions provide potent induction of humoral and cellular immune responses against an immunogen in a human subject, which can be used to provide effective treatment and/or protection against a disease, such as a tumor or infectious disease, in a human subject.

Prerequisites for creating the invention

Vaccines can be used to provide immune protection against pathogens such as viruses, bacteria, fungi or protozoa, as well as against cancer.

Infectious diseases are the second leading cause of death worldwide after cardiovascular disease, but are the leading cause of death in infants and children (Lee and Nguyen, 2015, Immune Network, 15 (2): 51-7). Vaccination is the most effective means of preventing various infectious diseases. The goal of vaccination is to generate a pathogen-specific immune response that provides long-term protection against infection. Despite significant vaccine success, the development of safe and potent vaccines remains necessary due to the emergence of new pathogens, the re-emergence of old pathogens, and the suboptimal protection provided by existing vaccines. Significant diseases emerging or re-emerging recently include the following: severe acute respiratory syndrome (SARS) in 2003, the H1N1 influenza pandemic in 2009, and Ebola virus in 2014. As a result, there is a need to develop new and effective vaccines against emerging diseases.

Cancer is one of the leading causes of death in the Western world, with lung, breast, prostate and colorectal cancers being the most common (Butterfield, 2015, BMJ, 350: h988). There are several clinical approaches to treating cancer, including surgery, chemotherapy, radiation therapy, and treatment with small molecule signaling pathway inhibitors. Each of these standard approaches has been shown to modulate antitumor immunity by increasing the expression of tumor antigens in the tumor or by causing the release of antigens from dying tumor cells and by stimulating antitumor immunity for therapeutic effect. Immunotherapy is a promising field that offers alternative treatments for cancer. Cancer vaccines are designed to stimulate tumor-specific immune responses, especially cytotoxic CD8+ T cells specific for tumor antigens. Clinical efficacy must be improved for cancer vaccines to become viable alternatives or complements to conventional cancer treatments. To date, significant efforts have been made to better understand the basic requirements for clinically effective cancer vaccines. Recent data highlight that important requirements include, among others, (1) the use of multiepitope immunogens, possibly derived from different tumor antigens; (2) selection of effective adjuvants; (3) association of cancer vaccines with agents capable of counteracting the regulatory milieu present in the tumor microenvironment; and (4) the need to select the final vaccine composition and regimen after rigorous preliminary trials comparing different antigen compositions (Fenoglio et al., 2013, Hum Vaccin Immunother, (12): 2543-7). To meet these demands, a new generation of cancer vaccines with both immunological and clinical efficacy is needed.

The potential of nucleic acid-based vaccines has been explored for many years (Vogel and Sarver N, 1995, Clin Microbiol Rev., 8(3):406-10). Clinical trials using DNA-based vaccines or mRNA-based vaccines are currently underway, including clinical trials of mRNA-based vaccines against viral targets such as rabies virus (see, for example, world wide web at clinicaltrials.gov; Alberer et al., The Lancet, published online July 2017, world wide web at dx.doi.org/10.1016/S0140-6736(17)31665-3; DeFrancesco et al., Nature Biotechnology, 35: 193-197).

The next generation of mRNA vaccines uses self-replicating RNA (repRNA), which is based on the self-replicating mechanism of positive-sense RNA viruses such as alpha viruses (see, for example, Bogers et al., 2015, J Infect Dis., 211 (6):947-55). Such repRNAs induce transient high expression of antigen in a wide range of host tissues and are capable of acting in both dividing and non-dividing cells.

Alpha viruses belong to the togavirus family and have linear, single-stranded RNA

- 1 043316 genomes with positive polarity, including two functional segments, each with its own promoter. The first segment, located at the 5' end of the genome, encodes nonstructural proteins that constitute a self-assembling replicase that synthesizes a negative polarity RNA genome, a positive polarity RNA genome, and subgenomic RNA. The second segment encodes structural envelope and capsid proteins. Self-replicating RNAs (repRNAs), also called alpha viral replicons, are nucleic acids derived from a full-length virus in which the genes encoding structural proteins have been deleted so that the replicon is able to replicate in a cell but is not able to replicate as a virus. In the case of repRNA-based antigen expression systems, genes encoding antigens can be inserted downstream of a subgenomic promoter instead of genes encoding structural proteins. RepRNAs can be delivered into the cell as a DNA molecule from which the repRNA starts, packaged in a viral replicon particle (VRP) or as a naked modified or unmodified RNA molecule

Research into repRNA vaccines is still in the preclinical stages, but clinical trials are expected to begin within a couple of years. Studies have shown that combining repRNA, launched from DNA or packaged in viral replicon particles (VRP), with adenoviral vectors in heterologous prime-boost vaccination stimulates immune responses (see, for example, WO2005046621; Zhao et al., 2009, Vet Immunol Immunopathol ., 131: 158-166 and Naslund et al., 2007, J Immunol, 178: 6761-6769). In addition, it has been shown that humoral responses induced by a repRNA vaccine can be enhanced by a protein vaccine (Bogers et al., Id.).

However, heterologous vaccination using an adenoviral vector in combination with a DNA plasmid from which the repRNA is launched has several problems. For example, DNA plasmids are associated with safety concerns such as contamination by bacterial products, risk of DNA integration into the host genome, and lack of self-limiting expression of antigens. On the other hand, heterologous vaccination using an adenoviral vector in combination with VRP-driven repRNA requires a packaging cell line to produce VRP, which is an expensive and time-consuming process with complex manufacturing issues. The use of a protein-based vaccine in combination with repRNA has the limitation that protein-based vaccines require expensive and time-consuming protein production by cells with the risk of contamination. Additionally, most protein-based vaccines have stability issues and require a cold chain, and generally protein-based vaccines are limited to stimulating humoral immune responses.

Accordingly, there is a need in the field for improved repRNA technology-based vaccines that can be used to induce protective humoral and cellular immunity against immunogens, especially when a rapid response is required in the event of a pandemic outbreak. Such vaccines must be cost-effective to produce and have minimal side effects. In addition, they should preferably be effective against a wide range of antigens.

The essence of the invention

The invention addresses this need by providing heterologous prime-boost immunization regimens using two different vaccine platforms: (i) in vitro transcribed (IVT) self-replicating mRNA (repRNA) and (ii) an adenoviral vector vaccine.

It has been found that the problems associated with the use of DNA-based vaccines, VRP-packaged vaccines and protein-based vaccines, some of which are discussed above, can be circumvented by the use of in vitro repRNA products, which is a DNA-free product that can be prepared using a rapid, versatile and cell-free production method (Kallen and Thess, 2014, Ther Adv Vaccines, 2(1) 10-31). The present inventors have unexpectedly discovered that combining IVT repRNA with an adenoviral vector in a heterologous prime-boost immunization regimen results in induced humoral and cellular immune responses that are stronger than those obtained from separate or homologous prime-boost immunization with an adenoviral vector or IVT repRNA . The invention can therefore be used to produce highly effective vaccines against a wide range of targets.

In one general aspect, the invention relates to a method of inducing an immune response in a human subject by administering a heterologous prime-boost immunization comprising a combination of IVT repRNA and an adenoviral vector.

In some embodiments, heterologous prime-boost combinations of IVT repRNA and adenoviral vector generate an induced immune response to an antigenic protein or immunogenic polypeptide thereof in a human subject. An antigenic protein or an immunogenic polypeptide thereof may be any antigenic protein or an immunogenic polypeptide thereof. For example, the antigenic protein or immunogenic polypeptide thereof may be derived from a pathogen, such as a virus, bacterium, fungus, protozoan, or cancer, such as a tumor.

Accordingly, one general aspect of the invention relates to a method of inducing an immune response in a human subject in need thereof, comprising:

a. administering to a human subject a first composition comprising an immunologically effective

- 2 043316 amount of in vitro transcribed (IVT) self-replicating RNA (repRNA), comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein one of the compositions is a priming composition and the other composition is a booster composition, thereby achieving induced immune response in a human subject, wherein the first and second antigenic proteins have at least a common antigenic determinant.

Another general aspect of the invention relates to a combination for inducing an immune response in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein one of the compositions is administered to a human subject to prime an immune response and the other composition is administered to a human subject to enhancing an immune response, wherein the first and second antigenic proteins have at least one common antigenic determinant.

Another general aspect of the invention relates to the use of a combination in accordance with an embodiment of the invention to induce an immune response in a human subject.

In preferred embodiments of the invention, the composition comprising the IVT repRNA is a priming composition and the composition comprising the adenoviral vector is a booster composition.

In preferred embodiments of the invention, the induced immune response includes induced humoral, or antibody response, immune response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject. Such a response, for example, may be characterized by a large percentage of patients responding, eg, greater than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested, as determined by an enzyme-linked immunosorbent assay (ELISA).

In preferred embodiments of the invention, the induced immune response includes an induced cellular immune response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject.

In one embodiment of the invention, the enhanced immune response generated by this method includes an enhanced CD8+ T cell response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject. Such a response, for example, may be characterized by a large percentage of CD8+ responding patients, such as greater than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested, as determined by an enzyme-linked immunosorbent spot assay (ELISPOT) or intracellular cytokine staining assay. (ICS). In another embodiment, the enhanced CD8+ T cell response generated by this method includes the expansion or induction of polyfunctional CD8+ T cells specific for at least one antigenic determinant common to the first and second antigenic proteins. Such polyfunctional CD8+ T cells express more than one cytokine, for example two or more of IFN-gamma, IL-2 and TNF-alpha.

In one embodiment of the invention, the enhanced immune response generated by this method includes an enhanced CD4+ T cell response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject. Such a response, for example, may be characterized by a large percentage of CD4+ responding patients, eg, greater than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested, as determined by the ELISPOT method or the ICS method. In another embodiment of the invention, the enhanced CD4+ T cell response generated by this method includes the expansion or induction of polyfunctional CD4+ T cells specific for at least one antigenic determinant common to the first and second antigenic proteins. Such polyfunctional CD4+ T cells express more than one cytokine, for example two or more of IFN-gamma, IL-2 and TNF-alpha.

In preferred embodiments, the induced immune response includes an induced antibody response, an induced CD4+ T cell response, and an induced CD8+ T cell response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject.

In preferred embodiments, the IVT repRNA is a repRNA based

- 3 043316 Venezuelan equine encephalitis virus (VEE). In preferred embodiments, the IVT repRNA framework is one of those described by Frolov et al. (1999, J Virol., 73(5):385465). In preferred embodiments, the IVT repRNA framework is the framework sequence of SEQ ID NO: 3 without the RSV pre-F protein insert.

In preferred embodiments, the adenoviral vector is a recombinant human adenovirus serotype 26 (Ad26) vector or a recombinant human adenovirus serotype 35 (Ad35) vector.

In embodiments of the invention, the booster composition is administered 1-52 weeks after administration of the priming composition. In one embodiment of the invention, the booster composition is administered 2-52 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4-52 weeks after administration of the priming composition. In one embodiment of the invention, the booster composition is administered 1 week after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 2 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 8 weeks after administration of the priming composition. In other embodiments, the booster composition is administered 6, 10, 12, 14, 16, 20, 24, or more weeks after administration of the primer composition.

In one embodiment of the invention, the first or second antigenic protein or immunogenic polypeptide thereof is derived from a pathogen such as a virus, bacterium, fungus or protozoan. In preferred embodiments, the first or second antigenic protein or immunogenic polypeptide thereof is derived from a virus. In another embodiment of the invention, the antigenic protein is derived from cancer. In preferred embodiments, the first or second antigenic protein or immunogenic polypeptide thereof is tumor derived.

In one embodiment of the invention, the first polynucleotide and the second polynucleotide encode the same antigenic protein or immunogenic polypeptide thereof. In another embodiment of the invention, the first polynucleotide and the second polynucleotide encode different immunogenic polypeptides or epitopes of the same antigenic protein. In yet another embodiment, the first polynucleotide and the second polynucleotide encode different, but related, antigenic proteins or an immunogenic polypeptide thereof. For example, related antigenic proteins may be substantially similar proteins derived from the same antigenic protein, or different antigenic proteins derived from the same pathogen or tumor.

In preferred embodiments, the first and second antigenic proteins are identical or substantially identical.

In one embodiment, the method of the invention provides protective immunity to a human subject against a disease associated with an antigenic protein, such as a tumor or infectious disease. In one preferred embodiment, the prime-boost combination of IVT repRNA and adenoviral vector induces a protective immune response against a tumor in a human subject. In another preferred embodiment, the prime-boost combination of IVT repRNA and adenoviral vector induces an immune response against a pathogen in a human subject.

In preferred embodiments, the first or second antigenic protein is derived from the prefusion F protein from respiratory syncytial virus (RSV-preF), and the first and second antigenic proteins are identical or substantially identical.

In preferred embodiments, the first and second antigenic proteins each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, immunogenic polypeptides thereof, and combinations thereof.

In preferred embodiments, the IVT repRNA includes a nucleic acid encoding an antigenic protein derived from the RSV-preF protein. Preferably, the IVT repRNA includes a nucleic acid encoding an antigenic protein having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or an immunogenic polypeptide or antigenic determinant thereof. Most preferably, the IVT repRNA comprises the nucleic acid sequence of SEQ ID NO: 3.

Brief description of drawings

The above brief description, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the exact embodiments shown in the drawings.

On the drawings:

fig. 1 is a schematic representation of a VEE virus-based repRNA encoding the RSVpreF protein;

fig. 2 shows an experimental design for a homologous IVT repRNA immunization study in which the analysis was performed by ELISA;

fig. 3 represents RSV-preF protein-specific humoral immune responses from a homologous IVT repRNA immunization study determined by ELISA;

fig. 4 presents an experimental design for a homologous IVT repRNA immunization study in which analysis was performed by the ELISPOT method;

fig. 5 represents RSV-preF protein-specific T cell responses from a homologous IVT repRNA immunization study determined by IFN-γ ELISPOT assay using PBMC and CTL activating peptide KYKNAVTEL from RSV-F;

fig. 6 presents an experimental design for a heterologous immunization study using IVT repRNA and an adenoviral vector, in which the analysis was carried out by ELISA and ELISPOT;

fig. 7 represents RSV-preF protein-specific humoral immune responses from a heterologous immunization study using IVT repRNA and adenoviral vector determined by ELISA; and fig. 8 presents RSV-preF protein-specific T cell responses from a heterologous immunization study using IVT repRNA and adenoviral vector determined by IFN-γ ELISPOT using splenocytes.

Detailed Description of the Invention

Various publications, articles and patents are cited or described in the prior art and description; each of these referenced documents is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, or the like that are included in this specification is presented for the purpose of providing context for the invention. Such discussion is not an admission that any or all of the foregoing is part of the prior art with respect to any inventions disclosed or claimed.

Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which the present invention relates. Otherwise, certain terms used in this application have the meanings specified in the specification. All patents, published patent applications and publications cited in this application are incorporated by reference as if set forth in their entirety herein. It should be noted that as used in this application and in the accompanying claims, the singular number includes the plural unless the context clearly dictates otherwise.

Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each 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 described herein. Such equivalents are intended to be covered by the invention.

In the present specification and in the claims that follow, unless the context otherwise requires, it is to be understood that the word include and variations such as includes and including include the inclusion of a whole or step or group of wholes or steps, but not the exclusion of any another single whole or stage or group of single wholes or stages. As used herein, the term including may be replaced by the term containing or including, or sometimes, when used herein, by the term having. In the context of this application, consisting of excludes any element, step or ingredient not specified in the element of the claims. As used herein, substantially consisting of does not exclude materials or steps that do not materially affect the essential and novel features of the claims. In each case in this application, any of the terms including, consisting essentially of, and consisting of may be replaced by either of the other two terms.

In the context of this application, union and/or between multiple enumerated elements is understood to include both individual and combined options. For example, when two elements are connected and/or, the first option refers to the applicability of the first element without the second. The second option refers to the applicability of the second element without the first. The third option concerns the applicability of the first and second elements together. It is assumed that any of these options corresponds to the meaning and therefore satisfies the requirement of the term and/or as used in this application. It should also be clear that the simultaneous applicability of more than one of the options is consistent with the meaning and therefore satisfies the requirement of the term and/or.

In the context of this application, the term subject means any animal, preferably a mammal, most preferably a human, which will be or has been treated by a method in accordance with an embodiment of the invention. As used herein, the term mammal includes 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 humans.

Protective immunity depends on both innate and adaptive immune responses. As used herein, the term immune response refers to the development in a subject of a humoral and/or cellular immunological response to an antigen that has been administered to the subject by the methods of the invention. Humoral immune responses refer to the production of antibodies by B cells, and cellular immune responses refer to the cytotoxic activity of CD8+ effector T cells and CD4+ T cells, also known as helper T cells. CD4+ T cells play a key role in both humoral and cellular immune responses.

As used herein, the term inducing or stimulating and variations thereof refer to any measurable increase in cellular activity. Induction of an immune response may include, for example, activation, proliferation, or maturation of a population of immune cells, increased production of cytokines, and/or other indication of increased immune function. In some embodiments, induction of an immune response may include increased B cell proliferation, production of antigen-specific antibodies, increased proliferation of antigen-specific T cells, improved antigen presentation by dendritic cells, and/or increased expression of certain cytokines, chemokines, and costimulatory markers.

As used herein, the term induced antibody response or induced humoral immune response refers to an antibody response in a human subject administered a prime boost combination of a repRNA and an adenoviral vector of the invention that is enhanced by at least 1.5, 2, 2.5 or more times compared to the corresponding immune response observed in a person who received a homologous prime-boost immunization with either repRNA alone or an adenoviral vector alone at comparable doses using the same prime-boost schedule.

As used herein, the term induced cellular immune response refers to the cellular immune response in a human being administered a prime-boost combination of repRNA and adenoviral vector in accordance with the invention, which is increased by at least 1.5-, 2-, 2.5-fold or more with the corresponding immune response observed in a person given a homologous prime-boost immunization with either repRNA alone or an adenoviral vector alone at comparable doses using the same prime-boost schedule.

As used herein, the term protective immunity or protective immune response means that the vaccinated subject is able to control the infection or disease associated with the antigenic protein or immunogenic polypeptide thereof against which the vaccination was given. Typically, a subject who has developed a protective immune response will develop only mild or moderate clinical symptoms, or no symptoms at all. Typically, a subject who has a protective immune response or protective immunity against a particular antigenic protein does not die as a result of an infection or disease associated with the antigenic protein.

As used herein, the term immunologically effective amount refers to an amount of an active ingredient or component that produces a desired biological or medical response in a subject. The immunologically effective amount can be determined empirically and routinely depending on the stated purpose. For example, in vitro assays may not necessarily be used to determine optimal dose ranges. The selection of a specific effective dose can be made (for example, through clinical trials) by one skilled in the art, taking into account several factors, including the disease being treated or prevented, symptoms associated with the disease, the body weight of the patient, the immune status of the patient, and other factors known specialists in this field. The exact dosage to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be selected in accordance with the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

As used herein, the term in vitro transcribed refers to a method in which RNA is enzymatically synthesized in vitro in a cell-free manner, for example, using cell extracts or isolated enzymes.

As used herein, the term self-replicating RNA, self-replicating RNAreplicon or repRNA or RNA replicon refers to an RNA molecule expressing alpha virus non-structural protein genes such that it can direct its own replication amplification in a cell without producing viral progeny. For example, the repRNA may include 5' and 3' alpha virus replication recognition sequences, coding sequences for alpha virus nonstructural proteins, a heterologous gene encoding an antigen and means for expressing the antigen, and a polyadenylation pathway. The repRNA of the invention may contain one or more mutations, such as attenuating mutations or mutations that improve functionality. The repRNA of the invention may contain modified nucleic acid bases, such as those described in US 2011/0300205, the corresponding contents of which are incorporated herein by reference. For example, repRNAs of the invention may contain modified nucleosides, including, but not limited to, m1G (1-methylguanosine), m2G (K2-methylguanosine), m7G (7-methylguanosine), Gm (2'-O-methylguanosine), m22G ( N2,N2-dimethylguanosine), m2Gm (N2,2'-Odimethylguanosine) and m22Gm (N2,N2,2'-O-trimethylguanosine).

- 6 043316

As used herein, the term antigenic protein refers to a protein that is capable of stimulating an immune response in a vertebrate. As used herein, the term immunogenic polypeptide or immunogenic fragment thereof refers to a fragment of an antigenic protein that retains the ability to stimulate an immune response. As used herein, the term antigenic determinant or epitope refers to a region of an antigenic protein that specifically interacts with an antibody.

As used herein, the term carrier refers to any excipient, diluent, excipient, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation or other material well known in the art for use in pharmaceutical applications. compositions. It should 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 substance that does not interfere with the effectiveness of the composition of the invention or the biological activity of the composition of the invention. In certain embodiments, in light of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an IVT repRNA-based or adenoviral vector-based pharmaceutical composition can be used in the invention. Suitable excipients include, but are not limited to, sterile water, saline, dextrose, glycerol, ethanol, or the like. and combinations thereof, as well as stabilizers, for example, human serum albumin (HSA) or other suitable proteins and reducing sugars.

As used herein, the term priming composition, priming immunization or prime immunization refers to primary stimulation with an antigen using a first composition of the invention. In particular, the term priming or potentiation of an immune response, as used herein, refers to a first immunization with an antigen that induces an immune response to a desired antigen and induces a higher level of immune response to the desired antigen upon subsequent booster immunization with the same antigen. As used herein, the term booster composition, booster immunization, or booster immunization refers to an additional immunization administered to, or effective in a mammal, after a primary immunization. In particular, the term enhancement of the immune response, as used herein, refers to the administration of a composition delivering the same antigen that is encoded in the primary immunization.

As used herein, the term pathogen refers to an infectious agent, such as a virus, bacterium, fungus, parasite or prion, that causes disease in a host.

The terms identical or percentage identity, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a certain percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum consistency , as measured using sequence comparison algorithms that are known and standard to those skilled in the art.

As used herein, the term substantially identical, with respect to an antigen polypeptide sequence, refers to an antigen polypeptide having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identity sequences with a reference polypeptide sequence. The term substantially identical, with respect to a nucleic acid sequence, refers to a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with a reference nucleic acid sequence.

Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules hybridize to each other under stringent conditions.

The invention has found that heterologous prime-boost combinations, in particular combinations of IVT repRNA and adenoviral vector, are surprisingly effective in generating protective immune responses in humans.

Antigenic proteins.

Any DNA of interest can be inserted into the repRNA constructs and adenoviral vectors described herein for heterologous expression from the repRNAs and vectors. Foreign genes for insertion into the genome of the virus in an expressed form can be obtained using

- 7 043316 using traditional methods of isolating the desired gene, subject to the present disclosure. For organisms that contain a DNA genome, genes encoding the antigen of interest can be isolated from the genomic DNA; for organisms with RNA genomes, the desired gene can be isolated from cDNA copies of the genome. The antigenic protein may also be encoded by recombinant DNA that is modified based on the natural sequence, for example, to optimize antigenic response, gene expression, etc.

In some embodiments, prime-boost combinations of repRNA and adenovirus generate an induced immune response to an antigenic protein or immunogenic polypeptide thereof in a human subject. The antigenic protein can be any antigenic protein associated with infection or disease.

In accordance with embodiments of the invention, the antigenic protein or immunogenic polypeptide thereof may be isolated from or may be derived from a pathogen such as a virus (e.g., filovirus, adenovirus, arbovirus, astrovirus, coronavirus, coxsackievirus, cytomegalovirus, dengue virus, Epstein-Barr virus virus, hepatitis virus, herpes virus, 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, virus rabies, respiratory syncytial virus, rhinovirus, rotavirus, rubella virus, smallpox virus, varicella zoster virus, West Nile virus, zika virus, etc.), bacteria (e.g. Campylobacter jejuni, Escherichia coli, Helicobacter pylori, Micobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc.), fungus (e.g. Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, Aspergillus species, etc.), protozoa (e.g. Plasmodium, Leishmania, Trypanosome, cryptosporidiums, isospora, Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinri, etc.), or cancer (for example, bladder cancer, colon and rectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, etc.).

In accordance with embodiments of the invention, an antigenic protein or an immunogenic polypeptide thereof can be isolated from, or derived from, a tumor, such as cancer.

In some embodiments, the nucleic acids express antigenic domains rather than the entire antigenic protein. These fragments can be of any length sufficient to be immunogenic or antigenic. The fragments may be at least four amino acids long, preferably 8-20 amino acids, but may be longer, such as 100, 200, 660, 800, 1000, 1200, 1600, 2000 or more amino acids long, or any length in between.

One skilled in the art will appreciate that nucleic acid molecules encoding an antigenic protein may be modified, for example, the nucleic acid molecules described herein may be mutated, provided that the modified expressed protein elicits an immune response against the pathogen or disease. Thus, as used herein, the term antigenic protein refers to a protein that includes at least one pathogen or tumor antigenic determinant described above. The term antigenic proteins also covers antigenic proteins that are substantially similar.

IVT repRNA.

RepRNAs useful in the invention are derived from alpha viruses, which are single-stranded RNA viruses with positive polarity. In one embodiment, the RepRNA that can be used in the invention contains a 7-methylguanosine cap, a 5' untranslated region, an RNA-dependent RNA polymerase (RdRp), a P1234 polyprotein (i.e., non-structural proteins, nsPs), a subgenomic promoter element representing of interest is the variable region from which the antigenic protein is expressed, the 3' untranslated region and the poly(A) tail.

RepRNAs useful in the invention may come from any self-replicating virus containing plus-strand RNA, for example, repRNAs may come from the Togaviridae or Arteriviridae virus families, such as Aura virus, Babanchi virus, Barmah Forest virus, Bebaru virus, Buggy Creek virus , Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, FortMorgan virus, Geta virus, Highland J virus, Kizilagac virus, Mayaro virus, Middleburg virus, Mukambo virus, Ndumu virus, O'Nyong-Nyong virus, Pixuna virus, Ross River virus , SA AR86, Sagiyama virus, Semliki forest virus, Sindbis virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Wataroa virus, African pouched rat arterivirus, DeBrazza monkey arterivirus, equine arteritis virus, Kibale red colobus virus, red-tailed virus Kibale monkeys, lactate dehydrogenase increasing virus, Mikumi baboon virus 1, Pebjah virus, porcine reproductive and respiratory syndrome virus. In preferred embodiments, the repRNAs of the invention are derived from the Venezuelan equine encephalitis (VEE) virus. Examples of preferred repRNA scaffolds of the invention include those described by Frolov et al., Id. and the framework sequence of SEQ ID NO: 3 without the RSV pre-F protein insert.

The production of in vitro transcribed (IVT) RNA is well known in the art, and standard IVT and purification procedures can be used to produce IVT repRNA useful in the invention in light of the present invention.

The preparation of IVT repRNA is described, for example, in US2011/0300205 and US2013/0195968, the corresponding contents of which are incorporated herein by reference. For example, repRNA molecules can be produced by IVT of a DNA molecule that encodes a self-replicating RNA molecule using a suitable DNA-dependent RNA polymerase such as phage T7 RNA polymerase, phage SP6 RNA polymerase, phage T3 RNA polymerase, etc. d. The IVT method can use a cDNA template created and propagated in a plasmid from bacteria, or created synthetically, for example, by gene synthesis and/or PNR methods. If necessary, appropriate capping-addition reactions can be used, and the poly-A can be encoded into the DNA template or can be added using a poly-A reaction. Suitable synthetic methods can be used alone or in combination with one or more other methods (eg, using recombinant DNA or RNA technology) to produce the IVT repRNA molecule of the invention. Suitable de novo synthesis methods are well known in the art and can be adapted for specific applications.

Typically, IVT repRNA useful in the invention is prepared using a DNA molecule from which the repRNA can be transcribed. Thus, the invention also provides isolated nucleic acid molecules that encode the repRNA of the invention. The nucleic acid molecules of the invention may be in the form of RNA or in the form of DNA, obtained by cloning or synthetically produced. DNA can be double-stranded or single-stranded.

IVT repRNAs useful in the invention can be included in suitable delivery systems for administration. In preferred embodiments, the 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 respective contents of which are incorporated herein by reference. For example, useful delivery systems include liposomes, polymer particles, non-toxic and biodegradable microparticles, electroporation, naked RNA injection, and cationic submicron oil-in-water emulsions. In preferred embodiments, IVT repRNAs useful in the invention are formulated in a lipid nanoparticle (LNP) composition (see, e.g., Semple et al., 2010, Nat Biotechnol. 28 (2): 172-176, the relevant contents of which are incorporated herein application via link).

As used herein, the term lipid nanoparticle or LNP refers to any lipid composition that can be used to deliver a therapeutic product, including, but not limited to, liposomes or vesicles, where the aqueous volume is encapsulated by amphipathic lipid bilayers, or where the lipids coat an interior that includes a therapeutic product, or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained in a relatively disordered mixture of lipids.

In specific embodiments, the LNPs include a cationic lipid to encapsulate and/or enhance delivery of IVT repRNA to a target cell. A cationic lipid can be any type of lipid that carries a net positive charge at a selected pH, such as physiological pH. Lipid nanoparticles can be prepared by incorporating multicomponent lipid mixtures with different ratios using one or more cationic lipids, non-cationic lipids and PEG-modified lipids. Several cationic lipids are described in the literature, many of which are commercially available. For example, suitable cationic lipids for use in the compositions and methods of the invention include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

LNP compositions may include anionic lipids. Anionic lipids can be any type of lipid that carries a net negative charge at a selected pH, such as physiological pH. Anionic lipids in combination with cationic lipids are used to reduce the overall surface charge of LNPs and to introduce pH-dependent disruption of the LNP bilayer structure to promote nucleotide release. Several anionic lipids are described in the literature, many of which are commercially available. For example, suitable anionic lipids for use in the compositions and methods of the invention include 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE).

LNP can be prepared using methods well known in the art, subject to the present disclosure. For example, LNPs can be produced using ethanol injection or ethanol dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation. In preferred embodiments, the LNPs used in the invention are prepared by dilution with ethanol.

Adenoviruses.

The adenovirus of the invention belongs to the family Adenoviridae and preferably belongs to the genus Mastadenovirus. It may be a human adenovirus, but also an adenovirus that infects other species, including, but not limited to, bovine adenovirus (e.g. bovine adenovirus 3,

- 9 043316

BAdV3), canine adenovirus (eg CAdV2), porcine adenovirus (eg PAdV3 or 5) or simian adenovirus (which includes simian adenovirus and primate adenovirus such as chimpanzee adenovirus or gorilla adenovirus). Preferably the adenovirus is a human adenovirus (HAdV or AdHu); in the invention, a human adenovirus is meant if it refers to Ad without specifying the species, for example, the short designation Ad5 means the same as HAdV5, which means human adenovirus serotype 5), or simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV). In the present invention, human adenovirus means Ad without specifying the species, for example, the short designation Ad26 means the same as HadV26, which means human adenovirus serotype 26. Also in this application, the designation rAd means recombinant adenovirus, for example, rAd26 refers to recombinant human adenovirus 26.

Most advanced research has been carried out using human adenoviruses, and human adenoviruses are preferred in accordance with some aspects of the invention. In some preferred embodiments, the recombinant adenovirus of the invention is based on a human adenovirus. In preferred embodiments, the recombinant adenovirus is based on a human adenovirus of serotype 5, 11, 26, 34, 35, 48, 49, or 50. In a particularly preferred embodiment of the invention, the adenovirus is a human adenovirus of one of serotypes 26 or 35.

These serotypes have the advantage of low seroprevalence and/or low titers of pre-existing neutralizing antibodies in the human population and experience with human use in clinical trials.

Simian adenoviruses also generally have low seroprevalence and/or low titers of pre-existing neutralizing antibodies in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g. US 6083716; 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 Mittal, 2006, Vaccine 24: 849-62; review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Therefore, in other preferred embodiments, the recombinant adenovirus according to the invention is based on a simian adenovirus, for example a chimpanzee adenovirus. In some embodiments, the recombinant adenovirus is based on 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 rAd26 and rAd35.

In a preferred embodiment of the invention, the adenoviral vectors include capsid proteins from two rare serotypes: Ad26 and Ad35. In typical embodiments, the vector is a rAd26 or rAd35 virus.

Thus, vectors that can be used in the invention include capsid protein Ad26 or Ad35 (eg, fiber, penton or hexon protein). Those skilled in the art will appreciate that the vectors of the invention do not necessarily need to use the entire Ad26 or Ad35 capsid protein. Thus, chimeric capsid proteins that include at least a portion of the Ad26 or Ad35 capsid protein can be used in the vectors of the invention. The vectors of the invention may also include capsid proteins, wherein the fiber, penton and hexon proteins are each derived from a different serotype, provided that 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 each from Ad35.

Those skilled in the art will appreciate that elements originating from multiple serotypes can be combined into a single recombinant adenoviral vector. In this way, a chimeric adenovirus can be produced that combines desirable properties from different serotypes. Thus, in some embodiments, a chimeric adenovirus of the invention may combine the absence of pre-existing immunity of serotypes Ad26 and Ad35 with characteristics such as temperature stability, assembly, anchoring, yield, redirected or enhanced infection, DNA stability in the target cell, etc. .P.

In some embodiments, the recombinant adenoviral vector useful in the invention is derived primarily or entirely from Ad35 or Ad26 (ie, the vector is rAd35 or rAd26). In some embodiments, the adenovirus is replication deficient, for example, because it contains a deletion in the E1 region of the genome. For adenoviruses of the invention derived from Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of a human subgroup C adenovirus such as Ad5. This makes it possible for such adenoviruses to propagate in well-known complementary cell lines that express Ad5 E1 genes, such as, for example, 293 cells, PER.C6 cells, and the like. (see eg Havenga et al, 2006, J Gen Virol 87: 2135-43; WO 03/104467). In some embodiments, the adenovirus is a human adenovirus serotype 35 with a deletion in the E1 region in which

- 10 043316 la cloned nucleic acid encoding the antigen, and with the E4-orf6 region of Ad5. In some embodiments, the adenovirus is a human adenovirus serotype 26 with a deletion in the E1 region into which the antigen-encoding nucleic acid was cloned and with the E4-orf6 region of Ad5. Regarding adenovirus Ad35, the 3′ end of the E1B 55K open reading frame in the adenovirus is usually retained, e.g., 166 bp. immediately upstream of the pIX open reading frame, or a fragment including it, such as a 243 bp fragment. immediately before the start codon pIX, marked at the 5' end with the restriction site Bsu36I, because this increases the stability of the adenovirus since the promoter of the pIX gene is partially located in this region (see, for example, Havenga et al, 2006, supra; WO 2004/001032).

The production of recombinant adenoviral vectors is well known in the art.

The preparation of rAd26 vectors is described, for example, in WO 2007/104792 and Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary Ad26 genomic sequences can be found in GenBank Accession EF 153474 and SEQ ID NO: 1 WO 2007/104792. The preparation of rAd35 vectors is described, for example, in US Pat. No. 7,270,811 and Vogels et al., (2003) J Virol 77(15): 8263-71. An exemplary genomic sequence of Ad35 can be found in GenBank Accession AC_000019.

In one embodiment, vectors useful for the invention include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Typically, an adenoviral vector useful in the invention is prepared using a nucleic acid comprising a complete recombinant adenoviral genome (eg, 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 obtained by cloning or synthetically. DNA can be double-stranded or single-stranded.

Adenoviral vectors used in the invention are typically replication defective. In these embodiments, the virus is made replication defective by deletion or inactivation of regions critical for viral replication, such as the E1 region. Regions can be substantially deleted or inactivated, for example, by insertion of a gene of interest (usually associated with a promoter). In some embodiments, the vectors of the invention may contain deletions in other regions, such as E2, E3 or E4 regions, or insertions of heterologous promoter-associated genes. For E2 and/or E4 mutated adenoviruses, E2 and/or E4 complementary cell lines are typically used to produce recombinant adenoviruses. Mutations in the E3 region of an adenovirus should not be introduced into the cell line because E3 is not required for replication.

A packaging cell line is typically used to produce sufficient quantities of the adenoviral vectors of the invention. A packaging cell is a cell that includes those genes that have been deleted or inactivated in the replication-defective vector, allowing the virus to replicate within the cell. Suitable cell lines include, for example, PER.C6, 911, 293 and E1 A549.

In some embodiments, the adenoviral vector may express genes or portions of genes that encode antigenic peptides. These foreign heterologous or exogenous peptides or polypeptides may include sequences that are immunogenic, such as, for example, tumor-specific antigens (TSA), bacterial, viral, fungal and protozoal antigens.

The heterologous gene encoding the antigenic peptide may be under the control of a promoter (ie, operably linked to a promoter) derived from an adenovirus (eg, a major late promoter), or may be under the control of a heterologous promoter. Examples of suitable heterologous promoters include the CMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within the expression cassette.

Immunogenic compositions.

Immunogenic compositions are compositions containing an immunologically effective amount of repRNA or adenoviral vectors for use in the invention. The compositions can be formulated as vaccines (also called immunogenic compositions) according to methods well known in the art. Such compositions may include adjuvants to enhance immune responses. Optimal amounts of each component in the composition can be determined by methods well known to those skilled in the art in light of the present disclosure.

The preparation and use of immunogenic compositions are well known to those skilled in the art. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

The compositions of the invention may contain repRNA or adenoviral vectors that express

- 11 043316 containing one or more antigenic proteins or their immunogenic polypeptides. These antigenic peptides or polypeptides may include any sequences that are immunogenic, including, but not limited to, tumor-specific antigens (TSA), bacterial, viral, fungal and protozoal antigens. For example, the antigenic protein or immunogenic polypeptide thereof may be derived from a pathogen, such as a virus, bacterium, fungus, protozoan, or it may also be derived from a tumor. In one or more preferred aspects, the compositions of the invention include repRNA or adenoviral vectors expressing one or more antigenic proteins from a virus, such as respiratory syncytial virus (RSV, influenza virus, HIV, Ebola virus, HPV, HSV, CMV, RSV, hepatitis, Zika virus, SARS virus, Chikungunya virus, dengue virus or West Nile virus.

Antigenic proteins can be any protein from any pneumovirus containing an antigenic determinant. In a preferred embodiment, the antigenic proteins are prefusion F protein from respiratory syncytial virus (RSV-preF).

In a preferred embodiment, the antigenic proteins encoded by IVT repRNA or adenoviral vectors have the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, immunogenic polypeptides thereof, and combinations thereof.

Immunogenic compositions used in the invention may include adjuvants.

Adjuvants suitable for co-administration in accordance with the invention must be potentially safe, well tolerated and effective in humans, including QS-21, DetoxPC, MPL-SE, MoGM-CSF, TiterMax-G, CRL - 1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum and MF59.

Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines such as interferon alpha, interferon gamma, platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (gCSF), granulocyte-macrophage colony-stimulating factor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL-I, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12 or encoding nucleic acids.

The compositions of the invention may include a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substances well known to those skilled in the art. Such substances must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other substance may depend on the route of administration, for example, intramuscular, subcutaneous, oral, intravenous, dermal, intramucosal (eg, intestinal), intranasal, or intraperitoneal routes.

A way to enhance the immune response.

The invention provides an improved method of priming and enhancing the immune response to any antigenic protein or immunogenic polypeptide thereof in a human subject using IVT repRNA in combination with an adenoviral vector.

In accordance with one general aspect of the invention, a method of inducing an immune response in a human subject in need thereof comprises:

a. administering to a human subject a first composition comprising an immunologically effective amount of in vitro transcribed (IVT) self-replicating RNA (repRNA) comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby elicit an immune response in a human subject, wherein the first and second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition.

In accordance with embodiments of the invention, the induced immune response includes induced antibody response against an antigenic protein in a human subject.

Preferably, the enhanced immune response also includes an enhanced CD4+ response or an enhanced CD8+ T cell response against the antigenic protein in the human subject. The enhanced CD4+ T cell response produced by the method in accordance with an embodiment of the invention may be, for example, an increase or induction of a dominant CD4+ T cell response against an antigen protein and/or an increase or induction of multifunctional CD4+ T cells specific for an antigen protein, in a human subject. Polyfunctional CD4+ T cells express more than one cytokine, for example two or more of IFN-gamma, IL-2 and TNF-alpha. The enhanced CD8+ T cell response produced by the method in accordance with an embodiment of the invention may be, for example, an increase or induction of polyfunctional CD8+ T cells specific for an antigen protein in a human subject.

More preferably, the enhanced immune response induced by the method according to an embodiment of the invention includes an enhanced CD4+ T cell response, an enhanced antibody response, and an enhanced CD8+ T cell response against an antigenic protein in a human subject.

Assays that can be used to determine immune responses are well known in the art. Some of these tests include, for example, ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunosorbent spot assay), and ICS (cytokine intracellular staining method). ELISA tests measure, for example, the levels of secreted antibodies or cytokines. When ELISA assays are used to determine the levels of antibodies that bind to a particular antigen, an indicator of the humoral immune response, they can also reflect the activity of CD4+ T cells, since the production of high-affinity antibodies by B cells depends on the activity of CD4+ helper T cells. ELISPOT and ICS are single-cell assays that analyze, for example, T cell responses to a specific antigen. ELISPOT assays measure the secretory activity of individual cells, and ICS assays analyze intracellular cytokine levels. CD4+ specific and CD8+ specific T cell responses can be determined using ICS assays.

In one or more embodiments of the invention, the IVT repRNA is used to prime the immune response, and the Ad26 or Ad35 vector is used to enhance the immune response, in accordance with an embodiment of the invention. In other embodiments, the Ad26 or Ad35 vector is used to prime an immune response, and the IVT repRNA is used to enhance the immune response, in accordance with an embodiment of the invention.

The antigens in the priming and booster compositions need not be identical, but must have common antigenic determinants or be substantially similar to each other.

Administration of immunogenic compositions is usually carried out by the intramuscular, subcutaneous or intradermal route. However, other routes of administration can be used, such as intravenous, dermal or intranasal. Intramuscular administration of immunogenic compositions can be accomplished using a needle to inject the composition in the form of a suspension. An alternative is to use a needleless injection device to administer the composition (using, for example, a Biojector(TM) or lyophilized powder containing the composition.

For intravenous, dermal, subcutaneous or intralesional injection, the composition must be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can readily prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. The IVT repRNA of the invention can be formulated into lipid nanoparticles for administration. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included in the compositions, as needed. Slow-release dosage forms may also be used.

Typically, administration will be for the prophylactic purpose of generating an immune response against the antigen prior to infection or development of symptoms. Diseases and disorders that may be treated or prevented in accordance with the invention include those in which the immune response may play a protective or therapeutic role. In other embodiments, the IVT repRNA and adenoviral vector can be administered for prophylaxis following exposure to an infectious agent.

Immunogenic compositions containing IVT repRNA and an adenoviral vector are administered to a subject, causing an immune response in the subject. An amount of the composition sufficient to induce a detectable immune response is defined as the immunologically effective dose. As shown below, the immunogenic compositions of the invention induce a humoral as well as a cellular immune response. In a preferred embodiment, the immune response is a protective immune response.

The actual amount of compositions administered, as well as the rate and timing of administration, will depend on the nature and severity of the disease, disorder or condition being treated. Prescribing treatment, e.g. deciding on dosage, etc., is the responsibility of general practitioners and other physicians and usually takes into account the disease, disorder or condition being treated, the condition of the individual patient, the site of delivery, the route of administration and other factors known to medical practitioners. Examples of the methods and protocols listed above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

Once the IVT repRNA and adenoviral vectors are prepared and such particles are optionally formulated into a composition, the composition can be administered to an individual, particularly a human.

The therapeutically effective amount or dosage may vary depending on various factors, such as the route of administration, the target site, the physiological condition of the subject (including, for example, age, body weight, health status), whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Treatment doses are optimally titrated to optimize safety and effectiveness.

In one exemplary regimen, IVT repRNA is administered (eg, intramuscularly) in a volume of about 100 μl to about 10 ml containing a dose of <200 μg, <100 μg, <50 μg, or <10 μg IVT repRNA, but expression can be observed at significantly lower levels, such as <1 μg, <100 ng, <10 ng, or <1 ng IVT repRNA per dose. Preferably, IVT repRNA is administered in a volume of 0.25 ml to 1.0 ml. More preferably, IVT repRNA is administered in a volume of 0.5 ml.

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

In one exemplary scheme, the adenoviral vector is administered (eg, intramuscularly) in a volume of about 100 μl to about 10 ml, containing concentrations of about 10 ⁴ -10 ¹² viral particles/ml. Preferably, the adenoviral vector is administered in a volume of 0.25 ml to 1.0 ml. More preferably, the adenoviral vector is administered in a volume of 0.5 ml.

Typically, the adenovirus is administered in an amount of from about 10 ⁹ to about 10 ¹² viral particles (vp) to a human subject per administration, more typically in an amount from about 10 ¹⁰ to about 10 ¹² vp. In a preferred embodiment, the adenoviral vector is administered in an amount of about 5 x 10 ¹⁰ vp. In another preferred embodiment, the adenoviral vector is administered in an amount of about 0.8 x 10 ¹⁰ vp. In another preferred embodiment, the adenoviral vector is administered in an amount of about 2x10 ¹⁰ vp. In another preferred embodiment, the adenoviral vector is administered in an amount of about 4 x 10 ¹⁰ vp.

The compositions of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated.

Booster compositions are administered several weeks or months after administration of the priming composition, for example, about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks , 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks or one to two years after administration of the priming composition.

Preferably, the booster composition is administered 1-52 weeks after administration of the priming composition. In a preferred embodiment of the invention, the booster composition is administered 2-52 weeks after administration of the priming composition. In another preferred embodiment of the invention, the booster composition is administered 4-52 weeks after administration of the priming composition. In another preferred embodiment of the invention, the booster composition is administered 1 week after administration of the priming composition. In another preferred embodiment of the invention, the booster composition is administered 2 weeks after administration of the priming composition. In another preferred embodiment of the invention, the booster composition is administered 4 weeks after administration of the priming composition. In another preferred embodiment of the invention, the booster composition is administered 8 weeks after administration of the priming composition.

The priming and booster compositions of the invention may each comprise one, two, three or multiple doses.

In one embodiment, the invention relates to a method of inducing an immune response against a tumor in a human subject.

The method includes:

a. administering to a human subject a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding an antigenic protein produced by a tumor cell, a substantially similar antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding an antigenic protein, a substantially similar antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby induce an immune response against a tumor in the human subject, wherein the first and the second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition.

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Preferably, the induced immune response provides protective immunity against the tumor in the human subject.

In a preferred embodiment, the composition comprising the IVT repRNA is a priming composition and the composition comprising the adenoviral vector is a booster composition.

In accordance with embodiments of the invention, the booster composition is administered 152 weeks after administration of the priming composition. The booster composition can also be administered later than 52 weeks after administration of the priming composition.

In one embodiment of the invention, the booster composition is administered 2-52 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4-52 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 1 week after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 2 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 8 weeks after administration of the priming composition.

In additional embodiments, the booster composition is administered at least 2 weeks or at least 4 weeks after administration of the priming composition. In still some embodiments, the booster composition is administered 4-12 weeks or 4-8 weeks after administration of the priming composition.

In a preferred embodiment, the IVT repRNA is a VEE virus-based repRNA.

In a preferred embodiment, the adenoviral vector is an Ad26 or Ad35 vector.

The antigenic protein produced by the tumor cell may be any tumor antigen. In a preferred embodiment, the tumor antigen is a tumor-specific antigen that is present only on tumor cells. A tumor antigen may also be a tumor-associated antigen that is present on some tumor cells as well as some normal cells.

According to yet another embodiment, the invention relates to a method for inducing an immune response against a virus in a human subject. The method includes:

a. administering to a human subject a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding an antigenic protein of a virus, a substantially similar antigenic protein, or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding an antigenic protein, a substantially similar antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby elicit an immune response against the virus in the human subject, wherein the first and the second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition.

Preferably, the enhanced immune response provides protective immunity against the virus in the human subject.

In a preferred embodiment, the composition comprising the IVT repRNA is a priming composition and the composition comprising the adenoviral vector is a booster composition.

In one embodiment, the booster composition is administered 1-52 weeks after administration of the priming composition. The booster composition can also be administered later than 52 weeks after administration of the priming composition.

In another embodiment of the invention, the booster composition is administered 2-52 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4-52 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 1 week after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 2 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 4 weeks after administration of the priming composition. In another embodiment of the invention, the booster composition is administered 8 weeks after administration of the priming composition.

In additional embodiments, the booster composition is administered at least 2 weeks or at least 4 weeks after administration of the priming composition. In still some embodiments, the booster composition is administered 4-12 weeks or 4-8 weeks after administration of the priming composition.

In a preferred embodiment, the IVT repRNA is a repRNA based

VEE virus.

In a preferred embodiment, the adenoviral vector is an Ad26 or Ad35 vector.

The antigenic protein can be any antigenic protein of a virus. In a preferred embodiment, the antigenic protein is a glycoprotein or nucleoprotein of a virus.

Embodiments

The invention also provides the following non-limiting embodiments.

Embodiment 1 is a method of inducing an immune response in a human subject in need thereof, comprising:

a. administering to a human subject a first composition comprising an immunologically effective amount of in vitro transcribed (IVT) self-replicating RNA (repRNA) comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby elicit an immune response in a human subject, wherein the first and second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition.

Embodiment 2 is the method according to Embodiment 1, wherein the composition comprising the IVT repRNA is a priming composition and the composition comprising the adenoviral vector is a booster composition.

Embodiment 3 is the method according to Embodiment 1, wherein the composition comprising the adenoviral vector is a priming composition and the composition comprising IVT repRNA is a booster composition.

Embodiment 4 is the method according to any of Embodiments 1-3, wherein the induced immune response comprises induced antibody immune response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject.

Embodiment 5 is the method according to Embodiment 4, where the induced antibody immune response is determined by ELISA.

Embodiment 6 is the method according to any of Embodiments 1-3, wherein the induced immune response comprises an induced cellular immune response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject.

Embodiment 7 is the method according to Embodiment 6, where the induced cellular immune response is determined by the ICS method or the ELISPOT method.

Embodiment 8 is a method according to any of Embodiments 1-7, wherein the induced immune response provides protective immunity to a human subject against a disease associated with at least one of the first and second antigenic proteins.

Embodiment 9 is a method according to any of Embodiments 1-8, wherein the IVT repRNA is a Venezuelan equine encephalitis (VEE) virus-based repRNA.

Embodiment 10 is the method according to any of Embodiments 1 to 9, wherein the adenoviral vector is a recombinant human adenovirus serotype 26 (Ad26) vector or a recombinant human adenovirus serotype 35 (Ad35) vector.

Embodiment 11 is the method according to any of Embodiments 1-10, wherein the booster composition is administered 1-52 weeks after administration of the primer composition.

Embodiment 12 is the method according to any of Embodiments 1-11, wherein the booster composition is administered at least 1 week after administration of the priming composition.

Embodiment 13 is a method according to any of Embodiments 1-12, wherein the first or second antigenic protein is derived from a pathogen or tumor.

- 16 043316

Embodiment 14 is a method according to any of Embodiments 1-12, wherein the first or second antigenic protein is derived from a virus.

Embodiment 15 is a method according to Embodiment

14, wherein the first or second antigenic protein is derived from a pneumovirus, filovirus, HIV, dengue virus, zika virus, influenza virus, or hepatitis B virus.

Embodiment 16 is a method according to any of Embodiments 1-15, wherein the first and second antigenic proteins are identical or substantially identical.

Embodiment 17 is a method according to any of Embodiments 1-16, wherein the first or second antigenic protein is derived from a prefusion F protein from respiratory syncytial virus (RSV-preF), and wherein the first and second antigenic proteins are identical or essentially identical.

Embodiment 18 is the method of Embodiment 17, wherein the first and second antigenic proteins each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, immunogenic polypeptides thereof, and combinations.

Embodiment 19 is the method of Embodiment 18, wherein the IVT repRNA comprises a polynucleotide encoding at least one antigenic protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and its immunogenic polypeptides.

Embodiment 20 is the method of Embodiment 21, wherein the IVT repRNA comprises a polynucleotide having the sequence of SEQ ID NO: 3.

Embodiment 21 is the method of Embodiment 20, wherein the adenoviral vector includes a polynucleotide encoding at least one antigenic protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and its immunogenic polypeptides.

Embodiment 22 is a method according to any of Embodiments 1-21, wherein the IVT repRNA is administered as a lipid nanoparticle composition in an amount of 0.1-1000 μg of IVT repRNA per dose.

Embodiment 23 is a method according to any of Embodiments 1-22, wherein the adenoviral vector is administered in an amount of ^109-1012 viral particles per dose.

Embodiment 24 is a method of inducing an immune response against at least one subtype of pneumovirus in a human subject, comprising:

a. administering to a human subject a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein of at least one pneumovirus subtype or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein of at least one pneumovirus subtype or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby achieve an immune response against at least one subtype pneumovirus in a human subject, wherein the first and second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition.

Embodiment 25 is a combination for inducing an immune response in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein one of the compositions is administered to a human subject to prime an immune response and the other composition is administered to a human subject to enhancing an immune response, wherein the first and second antigenic proteins have at least one common antigenic determinant.

Embodiment 26 is the use of a combination to produce a medicament for inducing an immune response in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

- 17 043316

b. a second composition comprising an immunologically effective amount of an adenoviral vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein one of the compositions is administered to a human subject to prime an immune response and the other composition is administered to a human subject to enhancing an immune response, wherein the first and second antigenic proteins have at least one common antigenic determinant.

Embodiment 27 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the composition comprising IVT repRNA is a priming composition and the composition comprising an adenoviral vector is booster composition.

Embodiment 28 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the composition comprising the adenoviral vector is a priming composition and the composition comprising IVT repRNA is booster composition.

Embodiment 29 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the first or second antigenic protein or immunogenic polypeptide thereof is derived from a pathogen or tumor.

Embodiment 30 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the first or second antigenic protein or immunogenic polypeptide thereof is derived from a virus.

Embodiment 31 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the first or second antigenic protein is derived from RSV-preF protein, and wherein the first and second antigenic proteins are identical or substantially identical.

Embodiment 32 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the first and second antigenic proteins each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, immunogenic polypeptides thereof and combinations thereof.

Embodiment 33 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the IVT repRNA includes a polynucleotide encoding at least one antigenic protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and immunogenic polypeptides thereof.

Embodiment 34 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the adenoviral vector includes a polynucleotide encoding at least one antigenic protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and immunogenic polypeptides thereof.

Embodiment 35 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the IVT repRNA is a Venezuelan equine encephalitis (VEE) virus-based repRNA.

Embodiment 36 is the method of Embodiment 24, the combination of Embodiment 25, or the use of Embodiment 26, wherein the adenoviral vector is an Ad26 vector or an Ad35 vector.

Embodiment 37 is a method of inducing an immune response in a human subject in need thereof, comprising:

a. administering to a human subject a first composition comprising an immunologically effective amount of in vitro transcribed (IVT) self-replicating RNA (repRNA) comprising a polynucleotide encoding non-structural proteins of the Venezuelan equine encephalitis (VEE) virus and another polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. administering to a subject a second composition comprising an immunologically effective amount of an Ad26 vector or an Ad35 vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, to thereby achieve an immune response in a human subject, wherein 043316 The first and second antigenic proteins have at least one common antigenic determinant, and the first composition is a priming composition and the second composition is a booster composition.

Embodiment 38 is a combination for inducing an immune response in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of in vitro transcribed (IVT) self-replicating RNA (repRNA), comprising a polynucleotide encoding non-structural proteins of the Venezuelan equine encephalitis virus (VEE), and another polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, and

b. a second composition comprising an immunologically effective amount of an Ad26 vector or an Ad35 vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein the first and second antigenic proteins have at least one common antigenic determinant, and the first composition is a priming composition, and the second composition is a booster composition.

Embodiment 39 is the use of the combination in accordance with Embodiment 38 to produce a medicament for inducing an immune response in a human subject.

Embodiment 40 is the method of Embodiment 37, the combination of Embodiment 38, or the use of Embodiment 39, wherein the first or second antigenic protein or immunogenic polypeptide thereof is derived from a pathogen or tumor.

Embodiment 41 is the method of Embodiment 38, the combination of Embodiment 39, or the use of Embodiment 40, wherein the first or second antigenic protein or immunogenic polypeptide thereof is derived from a virus.

Embodiment 42 is the method of Embodiment 37, the combination of Embodiment 38, or the use of Embodiment 39, wherein the first or second antigenic protein is derived from RSV-preF protein, and wherein the first and second antigenic proteins are identical or substantially identical.

Embodiment 43 is the method of Embodiment 37, the combination of Embodiment 38, or the use of Embodiment 39, wherein the first and second antigenic proteins each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, immunogenic polypeptides thereof and combinations thereof.

Embodiment 44 is the method of Embodiment 37, the combination of Embodiment 38, or the use of Embodiment 39, wherein the first composition comprises an immunologically effective amount of an IVT repRNA having the sequence SEQ ID NO: 3, and the second composition comprises an immunologically effective amount of an Ad26 vector comprising a polynucleotide encoding an antigenic protein having the amino acid sequence of SEQ ID NO: 1.

Embodiment 45 is a method in accordance with any of Embodiments 37-44, a combination in accordance with any of Embodiments 38-44, or a use in accordance with any of Embodiments 39-44, wherein the second composition is administered to a human subject after 2 -12 weeks, preferably 4-8 weeks, after administration of the first composition.

Embodiment 46 is a method according to any of Embodiments 38 or 41-45, a combination according to any of Embodiments 39 or 4145, or a use according to any of Embodiments 40-45, wherein the first composition is formulated as a composition lipo-nanoparticles.

Examples

The following examples are presented for illustration and not as limitation of the claimed invention.

Example 1.

An animal study was carried out to investigate the potential of homologous prime-boost immunization with repRNA-based vaccines encoding a model antigen. The model antigen used was the prefusion F protein from respiratory syncytial virus (RSV-preF). The study tested a range of doses of homologous vaccinations with an interval of 4 weeks between immunizations.

Manipulations with animals.

The studies complied with all applicable sections of the final regulations of the Animal Welfare Act (9 CFR parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Laboratory Equipment.

- 19 043316 animals - National Academy Press, Washington D.C. Eighth Edition (Manual).

A total of 30 (6-week-old) female Balb/c mice were purchased from Jackson laboratories.

Materials for vaccines.

The IVT repRNA vaccine vector that was used is shown schematically in FIG. 1. The IVT repRNA vector consisted of two open reading frames (ORFs), where the first encoded the nonstructural proteins (NSPs) of the Venezuelan equine encephalitis virus (VEE) and the second encoded the RSV-preF protein. The IVT repRNA vector was prepared using in vitro transcription (IVT) and purification methods. Briefly, linear template DNA for in vitro transcription of T7 was prepared by digestion of the replicon-carrying plasmid DNA of the 5′ T7 promoter and 3′ poly-A tail. The digested product was purified using the Zymo DNA Clean & Concentrator®-25 kit. IVT repRNA was prepared using the T7 Ambion MEGAscript™ Transcription Kit (Thermo) followed by an HPLC purification step using a GE CaptoCore 700 Hi Screen column to remove buffer components, free NTPs, and proteins. Following IVT purification, repRNA was capped using a post-transcriptional enzymatic capping reaction using a capping kit from Cellscript, and the RNA was concentrated in a centrifuge concentrator and buffer exchanged to a final concentration of 1-5 μg/μl. The composition of repRNA in lipid nanoparticles (LNP) was prepared by dilution in ethanol. Cholesterol was obtained from Sigma-Aldrich, a 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero- 3-phosphoethanol amine-N-[methoxy(πpolyethylene glycol)-2000] (DSPE-PEG) was obtained from Avanti Polar Lipids. Lipid components (in ethanol) at 48:40:10:2 molar percent cholesterol:DOTAP:DSPC:DSPE-PEG were mixed with repRNA in 10 mM citrate buffer using an 8:1 molar N:P ratio (nitrogen on DOTAP to phosphate on RNA). After emulsification for 1 hour, the particles were dialyzed against PBS. Before in vivo injections, the particles were further diluted to the required RNA concentration in PBS.

The IVT repRNA expressed VEE viral replicases and the antigenic RSV-preF protein having the amino acid sequence of SEQ ID NO: 2. The template DNA sequence used to produce the IVT repRNA had the nucleotide sequence of SEQ ID NO: 3.

Vaccination and experimental design.

Balb/c mice were vaccinated according to a homologous prime-boost immunization schedule using repRNA (for division into groups and experimental design, see Table 1 and Fig. 2). Before immunization, each mouse was anesthetized with 1–4% isoflurane in oxygen using a rodent anesthesia machine, and the animals received intramuscular (IM) injections of lipid nanoparticle (LNP)-formulated repRNA (50 μL). Priming and booster doses were administered at 4-week intervals (Fig. 2).

Whole blood without anticoagulant was processed to obtain serum.

Each serum was analyzed in an RSV preF-specific ELISA.

Table 1

Separation of animals into groups in a homologous repRNA immunization experiment to study humoral responses

Animal groups (Balb/c) Vaccine Carrier Immunizations (weeks) DOSE (mcg) Number of animals/GROUP 1-4 ρεπΡΗΚ-RSVpref LNP 0.4 5, 1, 0.4, 0.02 6 5 NO LNP 0.4 0 6

Anti-RSV-F IgG ELISA assay.

RSV-F-specific humoral responses were determined 28 and 42 days after immunization using a modified enzyme-linked immunosorbent assay (ELISA) as previously described by Krarup et al. (A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat. Commun. 6:8143).

Briefly, MaxiSorp polystyrene 96-well plates (NUNC) were coated with human anti-RSV F monoclonal antibody (Synagis) overnight at 4°C at a concentration of 1 μg/ml in PBS. The next day, plates were washed with PBST wash buffer (PBS, 0.05% Tween) and blocked in PBS with 1% bovine serum albumin, followed by incubation with stabilized prefusion F protein (0.25 μg/ml in PBST), which was captured immobilized anti-RSV F antibodies.

Serum samples were diluted in PBST with 1% bovine serum albumin and incubated with goat anti-mouse IgG-HRP conjugate from Bio-Rad (1/5000 dilution in PBST) and samples were then incubated in wells to allow detection of RSV-F specific mouse IgG . OPD substrate was used for detection. All incubations were carried out at room temperature

- 20 043316 temperature for 1 hour. After each step, the plates were washed three times with PBST. The results of the ELISA assay are shown in FIG. 3. Priming immunizations with doses of 0.4 μg or higher resulted in humoral immune responses having detectable IgG titers, and homologous boosting immunizations resulted in only a very slight increase in IgG titers (cross-dose).

Example 2.

To examine cellular responses following homologous repRNA boost immunization, an animal test was performed in which splenocytes were analyzed by ELISPOT using an RSV-F specific peptide.

Manipulations with animals.

The studies complied with all applicable sections of the final regulations of the Animal Welfare Act (9 CFR parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals - National Academy Press, Washington D.C. Eighth Edition (Manual).

Female Balb/c mice (6 weeks old) were purchased from Jackson laboratories.

Materials for vaccines.

IVT repRNA was prepared and formulated as described in Example 1.

Vaccination and experimental design.

Balb/c mice were vaccinated according to a homologous prime-boost immunization schedule using repRNA (for division into groups and experimental design, see Table 2 and Fig. 4). Before immunization, each mouse was anesthetized with 1–4% isoflurane in oxygen using a rodent anesthesia machine, and the animals received intramuscular (IM) injections of lipid nanoparticle (LNP)-formulated repRNA (50 μl). Priming and booster doses were administered at 4-week intervals (Fig. 4).

After 6 weeks, all mice were sacrificed to obtain splenocytes for testing in the IFN-γ ELISPOT assay using the RSV-F specific CTL peptide KYKNAVTEL assay.

table 2

Dividing animals into groups for homologous repRNA immunization (i.m.) to study cellular responses

Group Priming immunization Booster immunization 1 (4 weeks) repRNA dose (µg) Number of mice/group Vaccination weeks 1 PBS - 4 0.6f 2 repRNA - 1 10 0.6f 3 repRNA repRNA 1 10 0.4, 6f

ELISPOT IFN-g Assay.

RSV-F-specific cellular immune responses using splenocytes were determined at the time point shown in FIG. 4, interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assay using the IFNy ELISPOT kit for mouse cells from BD. Briefly, 500,000 splenocytes per well were stimulated overnight with CTL-activating RSV-F peptide (KYKNAVTEL) at a final concentration of 1 μg/ml. The stains were then developed following the manufacturer's instructions included with the kit. The results of the ELISPOT analysis are shown in FIG. 5 and they show that cellular responses were only slightly enhanced (without statistical significance) after boost immunization.

Example 3.

Due to the lack of boosting potential of IVT repRNA vaccines in a homologous prime-boost mode, animal testing was performed to investigate the potential of heterologous prime-boost immunizations with repRNA-based vaccines in combination with adenoviral-based vaccines, which both encode a model antigen. Specifically, the study tested two heterologous vaccinations comprising repRNA and Ad26-RSV-F with an interval of 4 weeks between immunizations to determine whether the heterologous prime-boost vaccination could improve humoral or cellular immune responses compared to the homologous repRNA vaccination of Examples 1 and 2.

Manipulations with animals.

The studies complied with all applicable sections of the final regulations of the Animal Welfare Act (9 CFR parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals - National Academy Press, Washington D.C. Eighth Edition (Manual).

A total of 36 female Balb/c mice (6 weeks old) were purchased from Jackson laboratories.

Materials for vaccines.

IVT repRNA was prepared and formulated as described in Example 1.

The recombinant adenoviral vector, which was a vaccine vector based on purified E1/E3-deleted replication-deficient recombinant adenovirus type 26 (Ad26), containing the RSV-F gene inserted at the E1 position, was manufactured by Janssen R&D. The vector was maintained in PER.C6™ cells, plaque purified, scaled up and then purified using

- 21 043316 two-step CsCl-coupling procedure and then formulated in a formulation buffer based on

TRIS and stored below -65°C.

The recombinant adenoviral vector expressed RSV-F derived from the RSV-A2 strain.

The expressed RSV-F protein had the amino acid sequence SEQ ID NO: 1.

Vaccine materials were stored at -80°C in a temperature-controlled freezer.

Vaccination and experimental design.

Balb/c mice were vaccinated using a heterologous prime-boost immunization scheme using repRNA and Adeno-vector (for division into groups and experimental design, see Table 3 and Fig. 6). Before immunization, each mouse was anesthetized and the animals received intramuscular (IM) injections of Adeno-vector or lipid nanoparticle (LNP) repRNA (as described in Example 1) into the upper thigh. Primer and booster doses were administered at 4-week intervals (Figure 6).

An animal study was performed to determine whether heterologous prime-boost vaccination using repRNA and Ad26-RSV-F could improve humoral or cellular immune responses (see Table 3 for mice grouping and Fig. 6 for experimental design). ) in contrast to homologous repRNA vaccination, as demonstrated by Examples 1 and 2. All immunizations were performed intramuscularly. Three control groups of mice received only priming immunizations (IM) with either PBS, LNP-formulated repRNA (1 μg), or Ad26-RSV-F (1 x 10 ⁹ vp). The potential of heterologous prime-boost immunizations was tested in two additional groups of mice. One group of 8 mice received LNP-formulated repRNA (1 μg) as a priming immunization and 4 weeks later received a boost immunization with Ad26-RSV-F (1x10 ⁹ vp). Another group of 8 mice received Ad26-RSV-F (1x10 ⁹ VP) as a primer, followed by a boost 4 weeks later with LNP-formulated repRNA (1 μg).

Table 3

Dividing animals into groups in an experiment to study heterologous immunization

Group Priming Booster- DOSE DOSE Quantity Get vaccinated A IMMUNIZATION IMMUNIZATION Adeno REPRN mice IYA OF THE WEEK Me 1 (4 WEEKS) (VP) K (mcg) /GROUP 1 PBS PBS - - 4 0.6+ 2 repRNA - - 1 8 0.6+ 3 ADENOVIRUS - 10 ^y - 8 0.6+ 4 repRNA ADENOVIRUS 10 ^y 1 8 0,4,6+ 5 ADENOVIRUS repRNA 10 ^y 1 8 0,4,6+

Anti-RSV-F IgG ELISA assay.

RSV-F-specific humoral responses were determined 42 days after immunization by an anti-RSV-F IgG ELISA as described in Example 1. The results of the ELISA assay are shown in FIG. 7. Heterologous prime-boost immunizations using repRNA and an adenoviral vector resulted in humoral immune responses having increased IgG titers compared with homologous immunizations using repRNA or an adenoviral vector.

ELISPOT IFN-γ Assay.

RSV-F-specific cellular (splenocyte) immune responses were determined 42 days after immunization using an interferon-gamma ELISPOT assay as described in Example 2. The results of the ELISPOT assay are shown in FIG. 8. Heterologous prime-boost immunizations using repRNA and adenoviral vector resulted in enhanced cellular immune responses compared with prime-boost immunizations using repRNA or adenoviral vector alone.

In summary, the study demonstrated that enhanced humoral and cellular immune responses were induced by immunization with heterologous repRNA and adenoviral vector vaccines compared with homologous immunizations with repRNA alone or adenoviral vector alone.

It will be appreciated by those skilled in the art that modifications to the embodiments described above are possible without departing from their broad inventive spirit. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined in the appended claims.

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Claims (14)

CLAIM 1. A method of inducing an immune response in a human subject in need thereof, comprising: A. administering to a human subject a first composition comprising an immunologically effective amount of in vitro transcribed (IVT) self-replicating RNA (repRNA) comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein the IVT repRNAs are formulated into non-virion particles, And b. administering to a subject a second composition comprising an immunologically effective amount of an adenoviral vector, a recombinant human adenovirus serotype 26 (Ad26) vector or a recombinant human adenovirus serotype 35 (Ad35) vector, comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof , together with a pharmaceutically acceptable carrier, to thereby achieve an immune response in a human subject, wherein the first and second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition. 2. The method according to claim 1, where the composition comprising IVT repRNA is a priming composition, and the composition comprising an adenoviral vector is a booster composition. 3. The method according to claim 1, where the composition comprising the adenoviral vector is a priming composition, and the composition comprising IVT repRNA is a booster composition. 4. The method according to any one of claims 1 to 3, wherein the induced immune response comprises an induced immune response of antibodies against at least one antigenic determinant common to the first and second antigenic proteins in a human subject. 5. The method according to any one of claims 1 to 3, wherein the induced immune response comprises an induced cellular immune response against at least one antigenic determinant common to the first and second antigenic proteins in a human subject. 6. The method according to any one of claims 1 to 5, wherein the induced immune response provides protective immunity to the human subject against a disease associated with at least one of the first and second antigenic proteins. 7. The method according to any one of claims 1 to 6, wherein the IVT repRNA is a repRNA based on the Venezuelan equine encephalitis virus (VEE). 8. The method according to any one of claims 1 to 7, wherein the booster composition is administered 1 to 52 weeks after administration of the priming composition. 9. The method according to any one of claims 1 to 8, wherein the booster composition is administered at least 1 week after administration of the priming composition. 10. Method according to any one of claims 1 to 9, wherein the first and second antigenic proteins are derived from a pathogen or tumor. 11. Method according to any one of claims 1 to 10, wherein the first or second antigenic protein is derived from a virus. 12. Method according to any one of claims 1 to 11, wherein the first and second antigenic proteins are identical or substantially identical. 13. A combination for inducing an immune response in a human subject, comprising: a. a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein the IVT repRNAs are formulated into non-virion particles, and b. a second composition comprising an immunologically effective amount of an adenoviral vector, which is a recombinant human adenovirus serotype 26 (Ad26) vector or a recombinant human adenovirus serotype 35 (Ad35) vector, comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein the first and second antigenic proteins have at least one common antigenic determinant, and one of the compositions is a priming composition and the other composition is a booster composition. 14. Use of a combination for the preparation of a medicament for inducing an immune response in a human subject, comprising: a. a first composition comprising an immunologically effective amount of an IVT repRNA comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof, together with a pharmaceutically acceptable carrier, wherein the IVT repRNAs are formulated into non-virion particles, and -