Methods and materials for immunizing mammals using adenoviral vectors

Abstract

This document provides methods and materials relating to the use of adenoviral vectors to immunize mammals (e.g., humans). For example, methods and materials are provided for intranasally administering adenoviral vectors (e.g., single-cycle adenoviral vectors) as a heterologous vaccination booster to induce IgG, IgA, and T cell immune responses in mammals (e.g., humans) that have been vaccinated with at least one prior vaccine (e.g., one or more prior mRNA-based vaccines) against a targeted pathogen.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 407,984, filed September 19, 2022. The disclosure of the prior application is deemed to be part of, and is incorporated by reference into, the disclosure of this application.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically with the XML filename "07039-2163WO1_SL.xml." The size of the XML file, created on September 15, 2023, is 38,000 bytes. The material in the XML file is incorporated herein by reference in its entirety.

[0003] Technical Field This document relates to methods and materials for immunizing a mammal using adenoviral vectors. For example, adenoviral vectors can be used to intranasally deliver one or more immunogens (e.g., one or more immunogens associated with an infection-causing pathogen) to cells within a mammal (e.g., a human) so that the mammal generates an effective IgG, IgA, and T cell immune response to the immunogen(s). [Background technology]

[0004] Background information The infectious disease caused by the coronavirus known as COVID-19 was first reported to the World Health Organization (WHO) Regional Office in China on December 31, 2019. As of June 3, 2020, approximately 612,200,000 confirmed cases of COVID-19, including 6,500,000 deaths, have been reported to WHO (covid19.who.int / ). Summary of the Invention [Means for solving the problem]

[0005] overview This document provides methods and materials for immunizing a mammal using an adenoviral vector. For example, this document provides methods and materials for intranasally delivering nucleic acid to cells in a mammal (e.g., a human) using an adenoviral vector. This document also provides methods and materials for inducing IgG, IgA, and T cell immune responses in a mammal (e.g., a human) using an adenoviral vector. The adenoviral vector can be used as a vaccination booster for a prior vaccine in a manner that elicits IgG, IgA, and T cell immune responses in the recipient. In some cases, the adenoviral vectors described herein (e.g., single-cycle adenoviral (SC-Ad) vectors) can be used as heterologous vaccination boosters to intranasally deliver nucleic acids encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) immunogens in a manner that elicits IgG, IgA, and T cell immune responses in a mammal (e.g., a human) that has been vaccinated with at least one prior vaccine (e.g., one or more prior mRNA vaccines) against a targeted pathogen.

[0006] As demonstrated herein, mammals (e.g., humans) vaccinated with a prior mRNA- or DNA-based SARS-CoV-2 vaccine (e.g., a Pfizer® vaccine such as Comirnaty® or an AstraZeneca® vaccine such as Vaxzevria®) can be intranasally administered SC-Ads engineered to express one or more immunogens from a coronavirus (e.g., SARS-CoV-2) to induce effective IgG, IgA, and T cell immune responses against the coronavirus. For example, intranasal administration of an SC-Ad designed to express a SARS-CoV-2 polypeptide, such as a spike polypeptide (or a fragment thereof), as a heterologous boost to a human who has received a prior mRNA- or DNA-based SARS-CoV-2 vaccine (e.g., a Pfizer® vaccine such as Comirnaty® or an AstraZeneca® vaccine such as Vaxzevria®) may trigger the human immune system to produce IgG antibodies capable of binding to and neutralizing coronavirus targets, IgA antibodies capable of binding to and neutralizing coronavirus targets, and / or induce a targeted Th1 T cell immune response against coronavirus targets.

[0007] Heterologous vaccination boosts have the ability to effectively generate an immune response against viral and / or bacterial pathogens (e.g., coronaviruses) in mammals (e.g., humans), thereby improving survival and minimizing the impact of infection. Adenoviral vectors encoding one or more immunogens can be used to confer long-lasting, long-lasting immunity to infectious pathogens (e.g., coronaviruses) in mammals. For example, adenoviral vectors encoding one or more immunogens associated with COVID-19 (e.g., one or more immunogens from SARS-CoV-2) can be used as potent heterologous vaccination boosts during the COVID-19 pandemic to generate humoral immunity against infection.

[0008] In general, one aspect of this document features a method for providing a heterologous vaccination boost to a human who has received a prior coronavirus vaccine. The method can include, or consist essentially of, intranasal administration of SC-Ad to a human, wherein the SC-Ad comprises a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprises an adenoviral polypeptide, the SC-Ad comprises a nucleic acid sequence encoding a coronavirus immunogen, and the prior coronavirus vaccine did not comprise SC-Ad. This aspect of the invention also provides an SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide for use in a method for providing a heterologous vaccination boost to a human who has received a prior coronavirus vaccine that did not comprise SC-Ad, the method including intranasal administration of SC-Ad to a human, the SC-Ad comprising an adenoviral polypeptide and a nucleic acid sequence encoding a coronavirus immunogen. The adenoviral polypeptide can be a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, or a pIIIa polypeptide. The immunogen may comprise a coronavirus spike polypeptide or an immunogenic fragment thereof. The immunogen may consist of, or consist essentially of, the amino acid sequence set forth in any one of SEQ ID NOS: 1-4 of WO 2022 / 040204. The immunogen may consist of, or consist essentially of, the amino acid sequence set forth in any one of SEQ ID NOS: 12, 42, 43, 44, 47, and 48 of WO 2022 / 040204. The SC-Ad may also comprise a nucleic acid sequence encoding an adjuvant polypeptide.The adjuvant polypeptide can be a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide, an interleukin-4 (IL-4) polypeptide, an interleukin-21 (IL-21) polypeptide, a CD40 ligand (CD40L) polypeptide, a 4-1BB ligand (4-1BBL) polypeptide, a transforming growth factor beta (TGF-β) polypeptide, a Clostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide, or a biologically active fragment thereof. A coronavirus spike polypeptide can be fused to the adjuvant polypeptide. The coronavirus spike polypeptide fused to the adjuvant polypeptide can consist essentially of, or can consist of, the amino acid sequence set forth in SEQ ID NO:5 of WO 2022 / 040204. The SC-Ad can also contain a nucleic acid sequence encoding a chaff polypeptide. The chaff polypeptide can be a fragment of an ACE2 polypeptide. The fragment of the ACE2 polypeptide may include the extracellular region of the ACE2 polypeptide and may lack the transmembrane domain. The chaff polypeptide may consist essentially of, or may consist of, the amino acid sequence set forth in SEQ ID NO: 8 of WO 2022 / 040204 or SEQ ID NO: 9 of WO 2022 / 040204. The coronavirus spike polypeptide may be fused to the chaff polypeptide. The coronavirus may be a β-coronavirus. The β-coronavirus may be SARS-CoV-2. The human may have received a prior coronavirus vaccine at least 90 days prior to its intranasal administration. The human may have received a prior coronavirus vaccine at least 150 days prior to its intranasal administration. The human may have received a prior coronavirus vaccine at least 180 days prior to its intranasal administration. The prior coronavirus vaccine may be an mRNA-based vaccine. The SC-Ad may affect human cells, and expression of the immunogen in the cells may result in an increase in the amount of serum IgG antibodies that bind to the immunogen. SC-Ad can affect human cells, and expression of the immunogen in the cells can result in increased amounts of mucosal IgA antibodies that bind to the immunogen.SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the amount of neutralizing antibodies in the human that bind to the immunogen. SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the number of Th1 T cells in the human that target the immunogen.

[0009] In another aspect, this document features a method for providing a vaccination boost to a human previously vaccinated against a virus that causes respiratory disease, the method comprising intranasal administration of an SC-Ad to the human, the SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide, and the SC-Ad comprising a nucleic acid sequence encoding an immunogen of the virus that causes respiratory disease. This aspect of the invention also provides an SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide and the nucleic acid sequence encoding an immunogen of the virus that causes respiratory disease, for use in a method for providing a vaccination boost to a human previously vaccinated against a virus that causes respiratory disease, the method comprising intranasally administering the SC-Ad to the human. The SC-Ad comprises an SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide and the nucleic acid sequence encoding an immunogen of the virus that causes respiratory disease. Except where the context specifically requires, the SC-Ad used in such methods or medical uses may be substantially or entirely described in connection with other aspects of the invention, or any embodiment thereof. The advantages of such methods and medical uses may be described elsewhere in this disclosure. The virus causing respiratory disease may be a coronavirus; influenza virus; respiratory syncytial virus (RSV); metapneumovirus (MPV); rhinovirus; bocavirus; or parainfluenza virus (PIV). The immunogen from a virus causing respiratory disease may be a coronavirus spike polypeptide or an immunogenic fragment thereof; an influenza hemagglutinin polypeptide; or an immunogenic fragment thereof; an RSV fusion (F) protein polypeptide or an immunogenic fragment thereof; an MPV fusion (F) protein polypeptide or an immunogenic fragment thereof; a rhinovirus VP1 protein polypeptide or an immunogenic fragment thereof; or a PIV fusion (F) protein polypeptide or an immunogenic fragment thereof. The immunogen may comprise a coronavirus spike polypeptide or an immunogenic fragment thereof. The immunogen may consist of, or consist essentially of, the amino acid sequence set forth in any one of SEQ ID NOS: 1-4 of WO 2022 / 040204.The immunogen can consist of, or consist essentially of, the amino acid sequence set forth in any one of SEQ ID NOS: 12, 42, 43, 44, 47, and 48 of WO 2022 / 040204. The SC-Ad can comprise a nucleic acid sequence encoding an adjuvant polypeptide. The adjuvant polypeptide can be a GM-CSF polypeptide, an IL-4 polypeptide, an IL-21 polypeptide, a CD40L polypeptide, a 4-1BBL polypeptide, a TGF-β polypeptide, a Clostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide, or a biologically active fragment thereof. The immunogen can be fused to the adjuvant polypeptide. The immunogen fused to the adjuvant polypeptide can consist essentially of, or consist of, the amino acid sequence set forth in SEQ ID NOS: 5 of WO 2022 / 040204. The SC-Ad can further comprise a nucleic acid sequence encoding a chaff polypeptide. The chaff polypeptide may be a fragment of an ACE2 polypeptide. The fragment of an ACE2 polypeptide may include the extracellular region of the ACE2 polypeptide and may lack the transmembrane domain. The chaff polypeptide may consist essentially of, or consist of, the amino acid sequence set forth in SEQ ID NO: 8 of WO 2022 / 040204 or SEQ ID NO: 9 of WO 2022 / 040204. An immunogen may be fused to the chaff polypeptide. The human may have received a prior coronavirus vaccine at least 90 days prior to intranasal administration. The human may have received a prior coronavirus vaccine at least 120 days prior to intranasal administration. The human may have received a prior coronavirus vaccine at least 150 days prior to intranasal administration. The human may have received a prior coronavirus vaccine at least 180 days prior to intranasal administration. The prior vaccine may be an mRNA-based vaccine. SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the amount of serum IgG antibodies that bind to the immunogen.SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the amount of mucosal IgA antibodies that bind to the immunogen.SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the amount of neutralizing antibodies in the human that bind to the immunogen. SC-Ad can affect human cells, and expression of the immunogen on the cells can result in an increase in the number of Th1 T cells in the human that target the immunogen.

[0010] In another aspect, this document features a method for providing a vaccination boost against a pathogen to a mammal previously vaccinated against the pathogen, the method comprising intranasally administering an SC-Ad to the mammal, the SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide, and the SC-Ad comprising a nucleic acid sequence encoding an immunogen for the pathogen. This aspect of the invention also provides an SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide for use in a method for providing a vaccination boost against a pathogen to a mammal previously vaccinated against the pathogen, the SC-Ad comprising the adenoviral polypeptide and the nucleic acid sequence encoding an immunogen for the pathogen. Except where the context specifically requires, the SC-Ad used in this aspect of the invention may be substantially or entirely described in connection with other aspects of the invention, or any embodiment thereof. The advantages of such methods and medical uses may be described elsewhere in this disclosure. The mammal may be a human. The pathogen may cause respiratory disease. The pathogen causing the respiratory disease can be a coronavirus; influenza virus; RSV; MPV; rhinovirus; bocavirus; or PIV. The adenovirus polypeptide can be a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, or a pIIIa polypeptide. The mammal can be a mammal that has received a prior coronavirus vaccine at least 90 days prior to intranasal administration. The mammal can be a mammal that has received a prior coronavirus vaccine at least 120 days prior to intranasal administration. The mammal can be a mammal that has received a prior coronavirus vaccine at least 150 days prior to intranasal administration. The mammal can be a mammal that has received a prior coronavirus vaccine at least 180 days prior to intranasal administration. The prior vaccine can be an mRNA-based vaccine. The SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the amount of serum IgG antibodies that bind to the immunogen.SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the amount of mucosal IgA antibodies that bind to the immunogen. SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the amount of neutralizing antibodies in the mammal that bind to the immunogen. SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the number of Th1 T cells in the mammal that target the immunogen.

[0011] Another aspect features a method for providing a heterologous vaccination boost against a pathogen to a mammal previously vaccinated against the pathogen. The method can include, or can consist essentially of, intranasal administration of SC-Ad to the mammal, wherein the SC-Ad comprises a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprises an adenoviral polypeptide, the SC-Ad comprises a nucleic acid sequence encoding an immunogen for the pathogen, and the prior vaccine did not comprise SC-Ad. This aspect of the invention also provides an SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising an adenoviral polypeptide and a nucleic acid sequence encoding an immunogen for the pathogen, for use in a method for providing a heterologous vaccination boost against a pathogen to a mammal previously vaccinated against a pathogen that did not comprise an SC-Ad. The adenoviral polypeptide can be a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, or a pIIIa polypeptide. The pathogen can be a coronavirus. The coronavirus can be a β-coronavirus. The beta coronavirus can be SARS-CoV-2. The immunogen can include a coronavirus spike polypeptide or an immunogenic fragment thereof. The mammal can be a mammal that has been vaccinated with a prior coronavirus vaccine at least 90 days prior to intranasal administration. The mammal can be a mammal that has been vaccinated with a prior coronavirus vaccine at least 120 days prior to intranasal administration. The mammal can be a mammal that has been vaccinated with a prior coronavirus vaccine at least 150 days prior to intranasal administration. The mammal can be a mammal that has been vaccinated with a prior coronavirus vaccine at least 180 days prior to intranasal administration. The prior vaccine can be an mRNA-based vaccine. The SC-Ad can affect mammalian cells, such that expression of the immunogen in the cells can result in an increase in the amount of serum IgG antibodies that bind to the immunogen. The SC-Ad can affect mammalian cells, such that expression of the immunogen in the cells can result in an increase in the amount of mucosal IgA antibodies that bind to the immunogen.SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the amount of neutralizing antibodies in the mammal that bind to the immunogen. SC-Ad can affect mammalian cells, and expression of the immunogen in the cells can result in an increase in the number of Th1 T cells in the mammal that target the immunogen.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting.

[0013] The details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0014] DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 is a diagram of the dose administration and follow-up assessment schedule. [Figure 2] 1 is a table showing the number of human subjects and the number of days since the last indicated COVID-19 vaccination before intranasal administration of the heterologous SC-Ad6-1 booster. [Figure 3] 1 is a bar graph plotting the fold increase in anti-coronavirus spike protein IgG antibodies detected in serum from humans at the indicated days after intranasal administration of a heterologous SC-Ad6-1 boost compared to a prior COVID-19 vaccine. A strong anti-spike IgG immune response induced by the intranasal heterologous boost was detected. [Figure 4]1 is a bar graph plotting the fold increase in anti-coronavirus spike protein IgA antibodies detected in nasal mucosal secretions from humans at the indicated days after intranasal administration of a heterologous SC-Ad6-1 boost. A strong anti-spike IgA immune response induced by the intranasal heterologous boost was detected. [Figure 5] Figure 1 is a bar graph plotting the fold increase in neutralization of live SARS-CoV-2 by antibodies in serum from humans at the indicated days after intranasal administration of a heterologous SC-Ad6-1 boost. A strong anti-SARS-CoV-2 neutralizing immune response induced by the intranasal heterologous boost was detected. [Figure 6] 1 is a bar graph plotting spot-forming units (SFU) of IFN-γ per million peripheral blood mononuclear cells (PBMCs) from humans at the indicated days after intranasal administration of a heterologous SC-Ad6-1 boost. The T cell immune response induced by the intranasal heterologous boost was strongly dominated by Th1 (important for pathogen immunity). [Figure 7] 1 is a bar graph plotting SFU of IL-5 per million PBMCs from humans at the indicated days after intranasal administration of a heterologous SC-Ad6-1 boost. T cell immune responses induced by the intranasal heterologous boost were predominantly Th1 (IFN-γ) rather than Th2 (IL-5). [Figure 8] FIG. 1 is a schematic representation of the genome map of SC-Ad6-1. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description This document provides methods and materials for immunizing mammals using adenoviral vectors. In some cases, the adenoviral vectors provided herein can be used to intranasally deliver nucleic acids encoding one or more immunogens (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) to a mammal (e.g., a human) so that the mammal generates an effective immune response (IgG, IgA, and T cell immune response) to the immunogen(s). In some embodiments, adenoviral vectors can be used to provide a vaccination boost. For example, the adenoviral vectors described herein (e.g., single-cycle adenoviral (SC-Ad) vectors) can be used as heterologous vaccination boosters to intranasally deliver nucleic acids encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) immunogens in a manner that elicits IgG, IgA, and T cell immune responses in a mammal (e.g., a human) that has been vaccinated with at least one prior vaccine (e.g., one or more prior mRNA-based and / or DNA-based vaccines) against a targeted pathogen.

[0016] Except where the context specifically requires, the adenoviral vectors described herein may be used in any of the aspects of the invention.

[0017] The adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can be derived from any adenovirus. The adenovirus used to generate the adenoviral vectors provided herein can be of any suitable serotype (e.g., Ad1 through Ad57). In some cases, the adenovirus can be a replication competent adenovirus. In some cases, the adenovirus can be a replication-deficient adenovirus. In some cases, the adenovirus can be capable of infecting human cells (e.g., a human adenovirus). In some cases, the adenovirus can be capable of infecting non-human cells, such as chimpanzee cells (e.g., a non-human adenovirus). Examples of adenoviruses that can be used to generate the adenoviral vectors provided herein include, but are not limited to, Ad5 adenovirus, Ad6 adenovirus, ChAdOx1, and ChAdOx2.

[0018] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can be SC-Ads. SC-Ads can have a genome lacking all or a portion of at least one of the following adenoviral nucleic acid sequences: a fiber protein-encoding sequence, a V protein-encoding sequence, a hexon-encoding sequence, a penton base-encoding sequence (also referred to as a pill-encoding sequence), a VA RNA-encoding sequence, a pilla protein-encoding sequence (also referred to as a minor capsid protein-encoding sequence), or other early or late gene product-encoding sequence. Examples of nucleic acid sequences encoding adenoviral polypeptides include, but are not limited to, the sequences set forth in GenBank gi numbers 209842, 58478, or 2935210 and / or the sequences annotated under GenBank accession numbers M73260, X17016, or AF03015. In some cases, deletions of all or part of the nucleic acid encoding one or more of the following polypeptides can be engineered into the nucleic acid encoding an adenovirus so that the adenoviral vector does not encode the full-length adenoviral polypeptide or a fully functional version of the adenoviral polypeptide. Such deletions can be of any length, resulting in the deletion of one or more encoded amino acids and reducing or eliminating the normal function of the polypeptide. For example, a portion of the adenoviral nucleic acid sequence can be removed so that the otherwise encoded polypeptide lacks 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more amino acid residues and lacks its normal activity. The deleted portion(s) can be removed anywhere along the entire length of the sequence.For example, a portion of the adenoviral nucleic acid sequence can be removed at the 5' end, 3' end, or an internal region of the adenoviral nucleic acid can be removed, such as the fiber protein coding sequence, V protein coding sequence, hexon coding sequence, penton base coding sequence, VA RNA coding sequence, pIIIa protein coding sequence, or other early or late gene product coding sequence. In some cases, the SC-Ad can be an SC-Ad described elsewhere (see, e.g., Matchett et al., J. Virol., 93(10):e02016-18 (2019); WO 2009 / 111738; or WO 2022 / 040204).

[0019] The adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can include a nucleic acid sequence encoding any suitable immunogen (e.g., a nucleic acid that drives expression of any suitable immunogen). In some cases, the immunogen can be an antigen. The immunogen can be a full-length immunogenic polypeptide or a portion thereof (e.g., can be derived from an immunogenic polypeptide).

[0020] When the immunogenic polypeptide is derived from a pathogen, the immunogenic polypeptide can be a polypeptide derived from any type of pathogen (e.g., a virus, a bacterium, a protozoan, a prion, a viroid, or a fungus). In some cases, the immunogenic polypeptide can be a polypeptide expressed by a virus (e.g., a viral polypeptide). For example, the immunogenic polypeptide can be a polypeptide expressed by a coronavirus (e.g., a β-coronavirus). Examples of viruses capable of expressing immunogenic polypeptides include, but are not limited to, SARS-CoV, HCoVNL63, HKU1, MERS-CoV, SARS-CoV-2, HIV-1, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, influenza, Ebola virus, chikungunya virus, Zika virus, cytomegalovirus, West Nile virus, respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus, bocavirus, parainfluenza virus (PIV), and the viruses listed in Table 3-1 of Learning from SARS: Preparing for the Next Disease Outbreak: Workshop Summary. Institute of Medicine (US) Forum on Microbial Threats; Knobler S, Mahmoud A, Lemon S, et al., editors. Washington (DC): National Academies Press (US); 2004. In some cases, the immunogen may be derived from a polypeptide expressed by bacteria (e.g., a bacterial polypeptide).Examples of bacteria expressing polypeptides from which immunogens can be derived include, but are not limited to, Clostridium (e.g., C. difficile), Staphylococcus aureus (e.g., methicillin-resistant S. aureus), Campylobacter (e.g., Campylobacter jejuni), Mycobacterium (e.g., M. tuberculosis), and Borrelia (B. burgdorferi). Examples of immunogenic polypeptides that may be expressed by pathogens include, but are not limited to, a C. difficile toxin (TcdA) polypeptide, a C. difficile toxin B (TcdB) polypeptide, a coronavirus spike polypeptide, the amino acid sequence set forth in SEQ ID NO: 1 of WO 2022 / 040204, a coronavirus nucleoprotein, a coronavirus membrane protein, a coronavirus envelope protein, a coronavirus nonstructural protein (e.g., coronavirus nonstructural proteins 1-16), an influenza hemagglutinin polypeptide, an RSV fusion (F) protein polypeptide, an MPV F protein polypeptide, a rhinovirus VP1 protein polypeptide, and a PIV F protein polypeptide. For example, an immunogenic polypeptide associated with a pathogen can have or be encoded by a sequence set forth in, e.g., National Center for Biotechnology Information (NCBI) Accession Numbers: MN938384 and AY772062.

[0021] When the immunogenic polypeptide is derived from an allergen, the immunogenic polypeptide can be a polypeptide derived from any type of allergen (e.g., a substance capable of eliciting an immune response that results in an allergic reaction). Examples of allergens from which immunogenic polypeptides can be expressed and / or shed include, but are not limited to, Fel d 7, Can f1, β-lactoglobulin, prolamin, parvalbumin, gliadin, Fel d1, chitinase, glutenin, cupin, prolamin, profilin, porcalcin, bet v-1-related protein, 2S albumin, vicilin, legumin, nsLTP, and Aed a2. For example, adenoviral vectors encoding one or more immunogens described herein can be used to intranasally deliver immunogens to a mammal (e.g., a human) so that the mammal produces antibodies (e.g., IgG and IgA antibodies) against the allergens associated with those immunogens. For example, immunogens can be delivered intranasally to a mammal (e.g., a human) using a nucleic acid molecule capable of encoding an adenoviral vector encoding one or more immunogens, such that the mammal produces antibodies (e.g., IgG and IgA antibodies) against the allergens associated with those immunogens. For example, immunogenic polypeptides associated with allergens can have or be encoded by sequences set forth in, e.g., NCBI Accession Numbers: NP_001363134.1, AAD56719, NP_001363136.1, NP_001363139.1, P27762.1, P10414.2, P15494.2, P43176.2, NP_001191706.1, NP_001003190.1, XP_030099003.1, or XP_001657779.1.

[0022] When the immunogenic polypeptide is a polypeptide derived from a cancer cell (e.g., a cancer cell in a mammal with cancer), the immunogenic polypeptide can be expressed by any cancer cell. For example, the immunogenic polypeptide expressed by a cancer cell can be a tumor antigen. In some cases, the immunogenic polypeptide expressed by a cancer cell can be a cell surface tumor antigen. In some cases, the immunogenic polypeptide expressed by a cancer cell can be a tumor-associated antigen (TAA; for example, an antigen such as an abnormal protein present on tumor cells). In some cases, the immunogenic polypeptide can be a tumor-specific antigen (TSA; for example, an antigen present only on tumor cells). Examples of immunogenic polypeptides that can be expressed by cancer cells and used as described herein include, but are not limited to, folate receptor alpha, mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), epidermal growth factor receptor (EGFR), folate receptor alpha, mesothelin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), antigens produced by Epstein-Barr virus, and antigens produced by human papillomavirus. For example, adenoviral vectors encoding one or more immunogens described herein can be used to intranasally deliver immunogens to a mammal (e.g., a human) so that the mammal produces antibodies (e.g., IgG and IgA antibodies) against allergens associated with those immunogens. For example, a nucleic acid molecule capable of encoding an adenoviral vector encoding one or more immunogens described herein can be used to intranasally deliver immunogens to a mammal (e.g., a human) such that the mammal produces antibodies (e.g., IgG and IgA antibodies) against allergens associated with those immunogens. For example, an immunogenic polypeptide associated with a cancer cell can have or be encoded by a sequence set forth, for example, in NCBI Accession Numbers: XP_002754883.1, AAA03229.1, Q02496.2, AAD33253.1, CEQ32409.1, YP_401631.1, YP_401632.1, or QAR15051.1.

[0023] When the immunogenic polypeptide is a polypeptide derived from a cancer cell (e.g., a cancer cell in a mammal having cancer), the immunogenic polypeptide can be expressed by any type of cancer cell, including, but not limited to, lung cancer, breast cancer, prostate cancer, liver cancer, kidney cancer, brain cancer, B-cell cancer, T-cell cancer, ovarian cancer, and skin cancer.

[0024] The immunogen can be a full-length immunogenic polypeptide or a portion thereof (e.g., derived from the immunogenic polypeptide). For example, a nucleic acid sequence encoding an immunogenic polypeptide can be modified to remove a portion of the nucleic acid so that the encoded polypeptide lacks any number of amino acids (e.g., 5, 10, 15, 20, 30, or all amino acids of the immunogenic polypeptide). In some cases, the nucleic acid sequence encoding the immunogenic polypeptide can be removed anywhere along the length of the sequence. For example, a portion of the nucleic acid sequence can be removed at the 5' end, 3' end, or an internal region of the target nucleic acid can be removed. In some cases, the immunogen can be designed to be secreted from cells infected with an adenoviral vector encoding the immunogen. For example, a nucleic acid sequence encoding an ER retention sequence can be removed from the nucleic acid sequence encoding the immunogen (e.g., so that the encoded immunogen lacks the ER retention sequence). In some cases, the immunogen can be designed to extend into the extracellular space from cells infected with an adenoviral vector encoding the immunogen. For example, the immunogen can include the ectodomain of an immunogenic polypeptide. In some cases, the immunogen can bind to (e.g., be designed to bind to) a viral receptor (e.g., an ACE2 polypeptide). For example, the immunogen can include a receptor-binding domain of an immunogenic polypeptide.

[0025] In some cases, the immunogen may comprise two or more immunogenic polypeptides described herein (e.g., a fusion polypeptide of two or more immunogenic polypeptides). For example, the immunogen may comprise a first immunogenic polypeptide and a second immunogenic polypeptide. In some cases, the first immunogenic polypeptide and the second immunogenic polypeptide may be different polypeptides. For example, the immunogen may comprise a tcdA polypeptide and a tcdB polypeptide (e.g., a tcdA / B fusion polypeptide). In some cases, the first immunogenic polypeptide and the second immunogenic polypeptide may be the same polypeptide. For example, the immunogen may comprise a tcdB polypeptide and a tcdB polypeptide (e.g., a tcdB / B fusion polypeptide). When the immunogen comprises a first immunogenic polypeptide and a second immunogenic polypeptide, the first immunogenic polypeptide may be derived from a first pathogen, and the second immunogenic polypeptide may be derived from a second pathogen. The first pathogen and the second pathogen may be the same pathogen or different pathogens. When the first and second pathogens are the same pathogen, the first and second immunogenic polypeptides can be derived from different strains of that pathogen, for example, the first and second immunogenic polypeptides can be derived from different strains of the same bacterium (e.g., C. difficile).

[0026] Examples of immunogens derived from immunogenic polypeptides that may be used as described herein include, but are not limited to, the amino acid sequence set forth in SEQ ID NO: 2 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 3 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 4 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 11 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 12 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 13 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 14 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 15 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 16 of WO 2022 / 040204, The amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 17, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 18, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 19, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 42, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 34, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 44, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 45, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 46, the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 47, and the amino acid sequence of WO 2022 / 040204 is SEQ ID NO: 48.

[0027] In some cases, the immunogens described herein may be variants of wild-type immunogens. For example, variants of coronavirus spike polypeptides (e.g., SARS-CoV-2 spike polypeptides) may comprise or consist essentially of the amino acid sequences set forth in SEQ ID NOS: 1-4 of WO 2022 / 040204, with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid deletions, additions, substitutions, or combinations thereof. Examples of amino acid deletions that may be present in variants of coronavirus spike polypeptides (e.g., as listed in any one of SEQ ID NOS: 1-4 of WO 2022 / 040204) include, but are not limited to, deletion of residues 69-70, deletion of residue 144, deletion of residues 156-157, deletion of residues 241-243, and deletion of residues 246-252.Examples of amino acid substitutions that may be present in variants of coronavirus spike polypeptides (e.g., those listed in any one of SEQ ID NOS: 1 to 4 in WO 2022 / 040204) include, but are not limited to, an L5F amino acid substitution, an S13I amino acid substitution, an L18F amino acid substitution, a T19R amino acid substitution, a T20N amino acid substitution, a P26S amino acid substitution, a G75I amino acid substitution, an A67V amino acid substitution, a V70F amino acid substitution, a T76I amino acid substitution, a D80 amino acid substitution, a D80G amino acid substitution, a T95I amino acid substitution, a D138Y amino acid substitution, a G142D amino acid substitution, a W152C amino acid substitution, an E154K amino acid substitution, an F157S amino acid substitution, an R158G amino acid substitution, an R190S amino acid substitution, a D215G amino acid substitution, an A222V amino acid substitution, a D253G amino acid substitution, a W258L amino acid substitution, a K417N amino acid substitution, a K418N amino acid substitution, a K419 ... 17T amino acid substitution, L452R amino acid substitution, L452Q amino acid substitution, Y453F amino acid substitution, S477N amino acid substitution, T478K amino acid substitution, E484Q amino acid substitution, E484K amino acid substitution, F490S amino acid substitution, E484K amino acid substitution, S494P amino acid substitution, N501Y amino acid substitution, A570D amino acid substitution, D614G amino acid substitution, H655Y amino acid substitution, Q677H amino acid substitution, These include the P681H amino acid substitution, the P681R amino acid substitution, the A701V amino acid substitution, the T716I amino acid substitution, the T859N amino acid substitution, the F888L amino acid substitution, the D950N amino acid substitution, the Q957R amino acid substitution, the S982 amino acid substitution, the K986P amino acid substitution, the V987P amino acid substitution, the T1027I amino acid substitution, the Q1071H amino acid substitution, the D1118H amino acid substitution, and the K1191N amino acid substitution. For example, a coronavirus spike polypeptide variant may include a K986P amino acid substitution and a V987P amino acid substitution (e.g., a PP substitution). In some cases, a coronavirus spike polypeptide variant may be a gamma mink variant of a coronavirus spike polypeptide. In some cases, a coronavirus spike polypeptide variant may be a delta variant of a coronavirus spike polypeptide.In some cases, the variant of the coronavirus spike polypeptide can be a lambda variant of the coronavirus spike polypeptide. In some cases, the variant of the coronavirus spike polypeptide can be an epsilon variant of the coronavirus spike polypeptide. In some cases, the variant of the coronavirus spike polypeptide can be a delta plus variant of the coronavirus spike polypeptide.

[0028] In some cases, the immunogens described herein may have an amino acid sequence having at least 85% sequence identity (e.g., at least 88% sequence identity, at least 90% sequence identity, at least 93% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 of WO 2022 / 040204.

[0029] The percent sequence identity between a particular amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows: The amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ, which contains BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ is available online at fr.com / blast or ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, options are set as follows: -i is set to the file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default settings. For example, the following command can be used to create an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt-j c:\seq2.txt-p blastn-o c:\output.txt-q-1 -r2. To compare two amino acid sequences, the Bl2seq options are set as follows: -i is set to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt), and all other options are left at their default settings.For example, the following commands can be used to create an output file containing a comparison between two amino acid sequences: C:\Bl2seq-i c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two sequences being compared share homology, then the specified output file will present those regions of homology as aligned sequences. If the two sequences being compared do not share homology, then the specified output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue occurs in both sequences. A matched position refers to a position where an identical amino acid occurs at the same position in the aligned sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO: 2 of WO 2022 / 040204, SEQ ID NO: 3 of WO 2022 / 040204, or SEQ ID NO: 4 of WO 2022 / 040204), and then multiplying the resulting value by 100. For example, an amino acid sequence that has 220 matches when aligned with the sequence set forth in SEQ ID NO: 2 of WO 2022 / 040204 is 93.2 percent identical to the sequence set forth in SEQ ID NO: 2 of WO 2022 / 040204 (i.e., 220 ÷ 236 × 100 = 93.2). Note that percent sequence identity values ​​are rounded to one decimal place. For example, 75.1, 75.2, 75.3, and 75.4 will round down to 75, and 75.5, 75.6, 75.7, 75.8, and 75.9 will round up to 76. Note also that length values ​​are always integers.

[0030] A coronavirus spike polypeptide variant may contain the entire amino acid sequence set forth in any one of SEQ ID NOs: 1-4 of WO 2022 / 040204, except that in some cases, the amino acid sequence contains 1 to 10 (e.g., 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof, provided that the coronavirus spike polypeptide variant has the ability to induce an immune response against coronavirus in a mammal (e.g., a human). In some cases, a coronavirus spike polypeptide variant may consist essentially of the amino acid sequence set forth in any one of SEQ ID NOS: 1-4 of WO 2022 / 040204, except that the amino acid sequence contains 1, 2, 3, 4, or 5 amino acid residues before the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1 of WO 2022 / 040204) and / or has 1, 2, 3, 4, or 5 amino acid residues after the articulated sequence of the sequence identifier (e.g., SEQ ID NO: 1 of WO 2022 / 040204), provided that the coronavirus spike polypeptide is capable of inducing an immune response against the coronavirus in a mammal (e.g., a human).

[0031] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can contain nucleic acid sequences encoding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) immunogens from different immunogenic polypeptides. For example, the adenoviral vectors provided herein can contain a nucleic acid sequence encoding an immunogen derived from a first pathogen (e.g., an immunogen derived from an immunogenic polypeptide expressed by SARS-CoV-2) and can contain a nucleic acid sequence encoding an immunogen derived from a second pathogen (e.g., an immunogen derived from an immunogenic polypeptide expressed by a pathogen other than SARS-CoV-2). In some cases, the adenoviral vectors provided herein that contain nucleic acid sequences encoding two or more immunogens derived from immunogenic polypeptides expressed by different pathogens may contain a nucleic acid sequence encoding a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence set forth in any one of SEQ ID NOS: 1-4 of WO 2022 / 040204, and may contain a nucleic acid sequence encoding a polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence set forth in any one of SEQ ID NOS: 20-21 of WO 2022 / 040204. When the adenoviral vectors provided herein contain nucleic acid sequences encoding two or more immunogens derived from immunogenic polypeptides expressed by different pathogens, the adenoviral vectors can be used to induce immune responses against two or more pathogens.

[0032] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may comprise nucleic acid sequences encoding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) immunogens derived from the same immunogenic polypeptide and / or the same pathogen. For example, the adenoviral vectors provided herein may comprise nucleic acid sequences encoding two or more immunogens derived from the same pathogen. In some cases, the adenoviral vectors provided herein that comprise nucleic acid sequences encoding two or more immunogens derived from immunogenic polypeptides expressed by influenza may comprise a nucleic acid sequence encoding a polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:20 of WO 2022 / 040204, and may comprise a nucleic acid sequence encoding a polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO:21 of WO 2022 / 040204.

[0033] In some cases, adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) that include nucleic acid sequences encoding one or more immunogens (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) can also include one or more regulatory sequences (e.g., enhancer or promoter sequences, such as constitutive, inducible, and / or tissue-specific promoter sequences) to drive transcription of the immunogen(s). Examples of enhancers and promoters that can be used to drive expression of nucleic acid sequences encoding one or more immunogens in the adenoviruses provided herein include, but are not limited to, a CMV enhancer sequence, a CMV promoter sequence, a CAG enhancer sequence, a CAG promoter sequence, an RSV enhancer sequence, an RSV promoter sequence, an Eflα enhancer sequence, an Eflα promoter sequence, a ubiquitin enhancer sequence, a ubiquitin promoter sequence, an adenovirus enhancer sequence, and an adenovirus promoter sequence. Any suitable method can be used to detect expression of the immunogen from adenoviral vector-hosting cells, for example, the presence or absence of the immunogen can be detected using an antibody that recognizes the immunogen.

[0034] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can include a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) adjuvant polypeptides (e.g., nucleic acids driving expression of one or more adjuvant polypeptides). For example, the adenoviral vector can include a nucleic acid sequence encoding one or more polypeptides that can enhance an immune response in a mammal. In some cases, the adjuvant polypeptide can be a cytokine. In some cases, the adjuvant polypeptide can be an immune stimulator. In some cases, the adjuvant polypeptide can be a toxin. In some cases, the adjuvant polypeptide can promote a systemic T cell response against a pathogen present in the mammal. In some cases, the adjuvant polypeptide can increase the concentration of antibodies against the pathogen at a site where the pathogen may enter the mammal's body. For example, the adjuvant polypeptide may increase the concentration of antibodies against a virus (eg, a coronavirus) at mucosal sites where the virus can enter the mammalian body.Examples of adjuvant polypeptides that may be encoded by the adenoviral vectors encoding one or more immunogens described herein include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides, interleukin 4 (IL-4) polypeptides, interleukin 21 (IL-21) polypeptides, CD40 ligand (CD40L) polypeptides, 4-1BB ligand (4-1BBL) polypeptides, transforming growth factor beta (TGF-β) polypeptides, C. difficile toxin polypeptides (e.g., C. difficile TcdA polypeptides (see, e.g., SEQ ID NO: 22 of WO 2022 / 040204), C. difficile toxin polypeptides (e.g., C. difficile TcdA polypeptides (see, e.g., SEQ ID NO: 22 of WO 2022 / 040204), C. difficile TcdB polypeptide (see, e.g., SEQ ID NO: 23 of WO 2022 / 040204, and / or the amino acid sequence set forth in SEQ ID NO: 10 of WO 2022 / 040204), and influenza polypeptides (e.g., N polypeptide, H polypeptide, M polypeptide, the amino acid sequence set forth in SEQ ID NO: 20 of WO 2022 / 040204, and / or the amino acid sequence set forth in SEQ ID NO: 21 of WO 2022 / 040204). In some cases, the adjuvant polypeptide (e.g., SEQ ID NO: 22 of WO 2022 / 040204) may be preceded by an AAT secretory sequence (e.g., SEQ ID NO: 28 of WO 2022 / 040204). In some cases, the nucleic acid sequence encoding the adjuvant polypeptide (e.g., SEQ ID NO: 23 of WO 2022 / 040204) may be preceded by a cleavage site such as a synthetic furin cleavage site (e.g., SEQ ID NO: 29 of WO 2022 / 040204). Examples of nucleic acid sequences that can encode the adjuvant polypeptides described herein include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 24 of WO 2022 / 040204 and the nucleic acid sequence set forth in SEQ ID NO: 25 of WO 2022 / 040204.In some cases, the nucleic acid sequence encoding the adjuvant polypeptide (e.g., SEQ ID NO: 24 of WO 2022 / 040204) can be preceded by an AAT secretion sequence (e.g., SEQ ID NO: 40 of WO 2022 / 040204). In some cases, the nucleic acid sequence encoding the adjuvant polypeptide (e.g., SEQ ID NO: 25 of WO 2022 / 040204) can be preceded by a cleavage site, such as a synthetic furin cleavage site (e.g., SEQ ID NO: 41 of WO 2022 / 040204). The adjuvant polypeptide can be a full-length polypeptide or a fragment of an adjuvant polypeptide described herein, provided that the fragment has the ability to enhance an immune response (e.g., a biologically active fragment). In some cases, the adjuvant polypeptide can be as described elsewhere (see, e.g., Matchett et al., Vaccines, 8(1):64 (2020)).

[0035] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may encode (e.g., may be designed to encode) a polypeptide comprising an immunogen fused to an adjuvant polypeptide. For example, a nucleic acid sequence encoding an immunogen may be fused to a nucleic acid sequence encoding an adjuvant polypeptide (e.g., such that the encoded immunogen is fused to the encoded adjuvant polypeptide). An example of an immunogen fused to an adjuvant polypeptide that may be encoded by an adenoviral vector encoding one or more immunogens described herein includes, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 5 of WO 2022 / 040204.

[0036] In some cases, adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may comprise a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chaff polypeptides (e.g., a nucleic acid that drives expression of one or more chaff polypeptides). The chaff polypeptide may be a full-length polypeptide or a fragment thereof, provided that it reduces the rate of pathogen entry into cells in a mammal or inhibits pathogen entry. In some cases, the chaff polypeptide may be a soluble polypeptide. For example, a soluble chaff polypeptide may be a full-length chaff polypeptide or a fragment of a chaff polypeptide that lacks the transmembrane domain. For example, a soluble chaff polypeptide may comprise the ectodomain of a chaff polypeptide. In some cases, the chaff polypeptide may target (e.g., target and bind to) a particular pathogen (e.g., a virus such as a coronavirus) to reduce the rate of pathogen entry into cells in a mammal or inhibit pathogen entry. In some cases, a chaff polypeptide may target (e.g., target and bind to) two, three, four, five, six, or more different pathogens. In some cases, a chaff polypeptide may comprise one or more mutations (e.g., inactivating mutations). Examples of chaff polypeptides that may be encoded by adenoviral vectors encoding one or more immunogens described herein include, but are not limited to, full-length ACE2 polypeptides and fragments thereof, full-length CD13 polypeptides and fragments thereof, full-length CEACAM1 polypeptides and fragments thereof, full-length sialylated polypeptides and fragments thereof, full-length CD46 polypeptides and fragments thereof, full-length nestin polypeptides and fragments thereof, the amino acid sequence set forth in SEQ ID NO: 8 of WO 2022 / 040204, and the amino acid sequence set forth in SEQ ID NO: 9 of WO 2022 / 040204.

[0037] In some cases, adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may encode (e.g., may be designed to encode) a polypeptide that includes an immunogen fused to a chaff polypeptide. For example, a nucleic acid sequence encoding an immunogen may include a nucleic acid sequence encoding a chaff polypeptide (e.g., such that the encoded immunogen is fused to the encoded chaff polypeptide).

[0038] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can include a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) marker polypeptides (e.g., nucleic acids that drive expression of one or more marker polypeptides). Examples of marker polypeptides that can be encoded by the adenoviral vectors encoding one or more immunogens described herein include, but are not limited to, fluorescent polypeptides (e.g., GFP, RFP, CFP, and YFP), streptavidin polypeptides, Cre recombinase polypeptides, Cas polypeptides, luciferase polypeptides, β-galactosidase polypeptides, and sodium iodide symporter polypeptides.

[0039] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may include a nucleic acid sequence (e.g., a nucleic acid that drives the expression of one or more polypeptides capable of forming multimers) encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) polypeptides capable of forming multimers. Examples of polypeptides capable of forming multimers that may be encoded by the adenoviral vectors encoding one or more immunogens described herein include, but are not limited to, immunoglobulin constant region polypeptides (e.g., Ig polypeptides), streptavidin polypeptides, and sigma coil polypeptides.

[0040] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may encode (e.g., may be designed to encode) a polypeptide comprising an immunogen fused to a polypeptide capable of forming multimers. For example, a nucleic acid sequence encoding an immunogen may include a nucleic acid sequence encoding a polypeptide capable of forming multimers (e.g., such that the encoded immunogen is fused to an encoded polypeptide capable of forming multimers). Examples of immunogens fused to a polypeptide capable of forming multimers that may be encoded by the adenoviral vectors encoding one or more immunogens described herein include, but are not limited to, the amino acid sequence set forth in SEQ ID NO: 5 of WO 2022 / 040204, the amino acid sequence set forth in SEQ ID NO: 6 of WO 2022 / 040204, and the amino acid sequence set forth in SEQ ID NO: 7 of WO 2022 / 040204.

[0041] In some cases, the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) may have a genome that is at least 85% identical (e.g., at least 88% sequence identity, at least 90% sequence identity, at least 93% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to the sequence set forth in any one of SEQ ID NOs: 28-39 of WO 2022 / 040204. For example, the adenoviral vectors provided herein may have a genome that comprises, consists of, or consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 29 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 30 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 31 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 32 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 33 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 34 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 35 of WO 2022 / 040204.For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 36 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 37 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 38 of WO 2022 / 040204. For example, the adenoviral vectors provided herein can have a genome comprising, consisting of, or consisting essentially of the nucleic acid sequence set forth in SEQ ID NO: 39 of WO 2022 / 040204.

[0042] In some cases, adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) can have a genome that includes one or more of the coding regions set forth in any one of SEQ ID NOS: 28-39 of WO 2022 / 040204. For example, an SC-Ad can be designed to have a genome in which all of its encoded polypeptides have the same amino acid sequence as the polypeptide encoded by a nucleic acid set forth in any one of SEQ ID NOS: 28-39 of WO 2022 / 040204. In some cases, the nucleic acid molecule can be designed to encode an adenoviral vector provided herein (e.g., an adenoviral vector encoding one or more immunogens, such as SC-Ad described herein). As used herein, the term "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. Single-stranded nucleic acids can be the sense strand or the antisense strand. Furthermore, nucleic acids can be circular or linear.

[0043] In some cases, cells (e.g., cell lines) can be engineered to contain an adenoviral vector described herein (e.g., one or more adenoviral vectors encoding one or more immunogens, such as SC-Ad described herein). In cases where the adenoviral vector lacks all or part of at least one adenoviral sequence, the cell containing the adenoviral vector can provide a defective adenoviral polypeptide. For example, if an adenovirus is engineered to lack nucleic acid encoding an adenoviral fiber polypeptide, an adenoviral fiber polypeptide-expressing cell line can be used to generate an adenovirus that contains a fiber polypeptide (e.g., a wild-type fiber polypeptide) while simultaneously lacking nucleic acid encoding the fiber polypeptide (e.g., a wild-type fiber polypeptide). For example, if an adenovirus is engineered to lack nucleic acid encoding a V polypeptide, an adenovirus V polypeptide-expressing cell line can be used to generate an adenovirus that contains a V polypeptide (e.g., a wild-type V polypeptide) while simultaneously lacking nucleic acid encoding the V polypeptide (e.g., a wild-type V polypeptide). For example, if an adenovirus is engineered to lack a nucleic acid encoding a pIIIa polypeptide, an adenovirus-expressing cell line can be used to engineer the adenovirus so that it contains a pIIIa polypeptide (e.g., a wild-type pIIIa polypeptide) while simultaneously lacking the nucleic acid encoding the pIIIa polypeptide (e.g., a wild-type pIIIa polypeptide). In some cases, cells containing the adenoviral vectors described herein can increase the effective copy number of the virus by at least 100-fold (e.g., 100-15,000-fold, 500-10,000-fold, 5,000-10,000-fold, or 5,000-15,000-fold). The virus can be expanded until the desired concentration is achieved in standard cell culture medium (e.g., DMEM or RPMI-1640 supplemented with 5-10% fetal bovine serum at 37°C in 5% CO).Viral titers are typically assayed by inoculating culture media with cells (e.g., A549 or 293 cells) or quantifying viral genomes by optical density or real-time PCR. In some cases, cells containing the adenoviral vectors provided herein can be used to propagate the adenoviral vector (e.g., to establish an adenoviral vector stock). For example, adenoviral vector stocks can be generated by propagation in mammalian cells. In some cases, adenoviral vector stocks can be aliquoted, frozen, and stored at -70 to -80°C (e.g., at concentrations higher than the therapeutically effective dose). In some cases, adenoviral vector stocks can be stored in a stabilizing solution. Examples of stabilizing solutions include, but are not limited to, sugars (e.g., trehalose, dextrose, and glucose), amino acids, glycerol, gelatin, monosodium glutamate, and Ca. 2+ , and Mg 2+ Examples include:

[0044] In some cases, an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more immunogens) can be formulated into a composition (e.g., a pharmaceutical composition such as a vaccine composition) for nasal administration to a mammal (e.g., a human). For example, an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more immunogens) can be formulated with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents for nasal administration.Examples of pharmaceutically acceptable carriers, excipients, and diluents that may be used in the compositions described herein include, but are not limited to, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses, e.g., microcrystalline cellulose, and cellulose ethers such as hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropylmethylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinylpyrrolidone (crospovidone), carboxymethylcellulose, polyethylene-polyoxypropylene-block polymers, and cross-linked sodium carboxymethylcellulose (croscarmellose sodium), and the like. Examples of suitable pharmaceutical formulations include sodium stearate, titanium dioxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propylparaben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylate, wax, wool fat, lecithin, and corn oil. Suitable pharmaceutical formulations vary in part depending on the application and route of administration. Such forms do not prevent the composition or formulation from reaching target cells or exerting its effect.

[0045] In some cases, the adenoviral vectors described herein that encode one or more immunogens (and / or nucleic acid molecules that may encode an adenoviral vector described herein that encodes one or more immunogens) may include multiple identical adenoviral vectors designed to encode one or more immunogens (and / or multiple identical nucleic acid molecules that may encode an adenoviral vector described herein that encodes one or more immunogens).

[0046] In some cases, a composition comprising an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more immunogens) may include a population of two or more (e.g., 2, 3, 4, 5, or more) different adenoviral vectors. For example, a composition can be designed to include two populations of adenoviral vectors, a first population containing one or more coronavirus immunogens (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), and a second population containing one or more adjuvant polypeptides (e.g., a GM-CSF polypeptide, an IL-4 polypeptide, an IL-21 polypeptide, a CD40L polypeptide, a 4-1BBL polypeptide, a TGF-β polypeptide, a C. difficile TcdA polypeptide, a C. difficile TcdB polypeptide, and / or biologically active fragments thereof). For example, a composition can be designed to include two populations of adenoviral vectors, a first population containing one or more coronavirus immunogens (e.g., an amino acid sequence set forth in any one of SEQ ID NOs: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), and a second population containing a chaff polypeptide (e.g., a fragment of an ACE2 polypeptide).

[0047] In some cases, a composition comprising an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more immunogens) can be designed to include different populations of adenoviral vectors (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding a single immunogen), where each population of adenoviral vectors of the composition contains a single immunogen. For example, a composition of adenoviral vectors can be designed to include a first population of adenoviral vectors having a first coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204) and a second population of adenoviral vectors having a second coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204). For example, a composition of adenoviral vectors can be designed to include a first population of adenoviral vectors having a first coronavirus immunogen (e.g., the amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), a second population of adenoviral vectors having a second coronavirus immunogen (e.g., the amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), and a third population of adenoviral vectors having a third coronavirus immunogen (e.g., the amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204).In another example, the adenoviral vector composition can be designed to include a first population of adenoviral vectors having a first coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), a second population of adenoviral vectors having a second coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), and a third population of adenoviral vectors having an adjuvant polypeptide (e.g., a GM-CSF polypeptide, an IL-4 polypeptide, an IL-21 polypeptide, a CD40L polypeptide, a 4-1BBL polypeptide, a TGF-β polypeptide, a C. difficile TcdA polypeptide, a C. difficile TcdB polypeptide, and / or a biologically active fragment thereof). In another example, the adenoviral vector composition can be designed to include a first population of adenoviral vectors comprising a first coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), a second population of adenoviral vectors comprising a second coronavirus immunogen (e.g., an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 42-44, 47, and 48 of WO 2022 / 040204), and a third population of adenoviral vectors comprising a chaff polypeptide (e.g., a fragment of an ACE2 polypeptide).

[0048] This document also provides methods of using the adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein.

[0049] In some cases, the adenoviral vectors described herein and / or nucleic acid molecules that can encode the adenoviral vectors described herein can be administered intranasally to a mammal (e.g., a human) as a vaccination boost to increase the immune response (e.g., increase IgG and IgA antibody responses and / or increase T cell responses) against a pathogen (e.g., a bacterial or viral pathogen associated with the immunogen encoded by the adenoviral vector).

[0050] In some cases, the adenoviral vectors described herein and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein can be administered intranasally to a mammal (e.g., a human) as a heterologous vaccination boost to increase the immune response (e.g., to increase IgG and IgA antibody responses and / or to increase T cell responses) against a pathogen (e.g., a bacterial or viral pathogen associated with the immunogen encoded by the adenoviral vector). For example, an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein that encodes one or more immunogens) can be administered intranasally to a mammal (e.g., a human) as a heterologous vaccination boost to provide the mammal with an immune response (e.g., IgG, IgA, and T cell immune responses) effective to reduce the severity of an infection caused by a pathogen associated with the immunogen(s) encoded by the adenoviral vector. In some cases, an adenoviral vector described herein encoding one or more immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector encoding one or more immunogens) can be administered intranasally to a mammal described herein (e.g., a human) as a heterologous vaccination boost to provide the mammal with an immune response (e.g., IgG, IgA, and T cell immune responses) effective to prevent the mammal from exhibiting symptoms of infection caused by a pathogen associated with the immunogen(s) encoded by the adenoviral vector. In some cases, an adenoviral vector described herein and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein can be administered intranasally to a mammal (e.g., a human) as a heterologous vaccination boost to increase IgG, IgA, and T cell immune responses in the mammal.

[0051] In some cases, the adenoviral vectors described herein and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein can be administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen) to increase the number of IgG and IgA antibodies in the mammal. For example, the adenoviral vectors described herein encoding one or more immunogens (and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein encoding one or more immunogens) can be administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen) to increase the number of IgG and IgA antibodies in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95%, or more.In some cases, an adenoviral vector described herein and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein is administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen), and the effects of the adenoviral vector described herein are observed beginning at about 15 days after administration (or beginning at about 16 days after administration, or beginning at about 17 days after administration, or beginning at about 18 days after administration, or beginning at about 19 days after administration, or beginning at about 20 days after administration, or beginning at about 21 days after administration, or beginning at about 22 days after administration) and continuing up to at least about 40 days after administration (e.g., continuing up to at least about 43 days after administration, continuing up to at least about 45 days after administration, continuing up to at least about 50 days after administration, or continuing up to at least about 60 days after administration). IgG and IgA antibodies to the immunogen(s) may be produced in the mammal for at least about 55 days after administration, continuing to at least about 60 days after administration, continuing to at least about 65 days after administration, continuing to at least about 70 days after administration, continuing to at least about 75 days after administration, continuing to at least about 80 days after administration, continuing to at least about 85 days after administration, continuing to at least about 90 days after administration, continuing to at least about 95 days after administration, continuing to at least about 100 days after administration, continuing to at least about 105 days after administration, continuing to at least about 110 days after administration, continuing to at least about 115 days after administration, continuing to at least about 120 days after administration, continuing to at least about 125 days after administration, continuing to at least about 130 days after administration, or continuing to at least about 135 days after administration).The pre-vaccinated vaccine is administered intranasally to a mammal within about 21 days to about 120 months (e.g., about 21 days to about 60 months, about 21 days to about 36 months, about 21 days to about 24 months, about 21 days to about 12 months, about 21 days to about 8 months, about 21 days to about 6 months, about 21 days to about 4 months, about 21 days to about 3 months, about 21 days to about 2 months, about 1 month to about 120 months, about 3 months to about 120 months, about 6 months to about 120 months, about 12 months to about 12 months, about 1 month to about 120 ... The vaccine may be administered to a mammal (e.g., a human) about 120 months, about 18 months to about 120 months, about 24 months to about 120 months, about 36 months to about 120 months, about 48 months to about 120 months, about 1 month to about 60 months, about 2 months to about 48 months, about 3 months to about 36 months, about 4 months to about 24 months, about 6 months to about 18 months, about 1 month to about 3 months, about 3 months to about 6 months, about 6 months to about 12 months, about 12 months to about 24 months, about 24 months to about 48 months, about 36 months to about 72 months, about 48 months to about 84 months, or about 60 months to about 96 months.

[0052] In some cases, an adenoviral vector described herein and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein is administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen), and the effects of the adenoviral vector described herein are observed beginning at about 15 days after administration (or beginning at about 16 days after administration, or beginning at about 17 days after administration, or beginning at about 18 days after administration, or beginning at about 19 days after administration, or beginning at about 20 days after administration, or beginning at about 21 days after administration, or beginning at about 22 days after administration) and continuing up to at least about 40 days after administration (e.g., continuing up to at least about 43 days after administration, continuing up to at least about 45 days after administration, continuing up to at least about 50 days after administration, or continuing up to at least about 60 days after administration). IgG and IgA antibodies to the immunogen(s) may be produced in the mammal for at least about 55 days after administration, continuing to at least about 60 days after administration, continuing to at least about 65 days after administration, continuing to at least about 70 days after administration, continuing to at least about 75 days after administration, continuing to at least about 80 days after administration, continuing to at least about 85 days after administration, continuing to at least about 90 days after administration, continuing to at least about 95 days after administration, continuing to at least about 100 days after administration, continuing to at least about 105 days after administration, continuing to at least about 110 days after administration, continuing to at least about 115 days after administration, continuing to at least about 120 days after administration, continuing to at least about 125 days after administration, continuing to at least about 130 days after administration, or continuing to at least about 135 days after administration).The pre-vaccinated vaccine is administered intranasally to a mammal within about 21 days to about 120 months (e.g., about 21 days to about 60 months, about 21 days to about 36 months, about 21 days to about 24 months, about 21 days to about 12 months, about 21 days to about 8 months, about 21 days to about 6 months, about 21 days to about 4 months, about 21 days to about 3 months, about 21 days to about 2 months, about 1 month to about 120 months, about 3 months to about 120 months, about 6 months to about 120 months, about 12 months to about 12 months, about 1 month to about 120 ... The vaccine may be administered to a mammal (e.g., a human) about 120 months, about 18 months to about 120 months, about 24 months to about 120 months, about 36 months to about 120 months, about 48 months to about 120 months, about 1 month to about 60 months, about 2 months to about 48 months, about 3 months to about 36 months, about 4 months to about 24 months, about 6 months to about 18 months, about 1 month to about 3 months, about 3 months to about 6 months, about 6 months to about 12 months, about 12 months to about 24 months, about 24 months to about 48 months, about 36 months to about 72 months, about 48 months to about 84 months, or about 60 months to about 96 months.

[0053] In some cases, the adenoviral vectors described herein and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein can be administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen) to increase T cell responses in the mammal. For example, the adenoviral vectors described herein encoding one or more immunogens (and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein encoding one or more immunogens) can be administered intranasally as a heterologous vaccination boost to a mammal previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen) to increase the number of activated T cells (e.g., activated Th1 T cells) in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more.

[0054] As described herein, SC-Ads genetically engineered to encode one or more immunogens (e.g., coronavirus spike polypeptides) can be administered intranasally as a heterologous vaccination boost to a mammal (e.g., a human) previously vaccinated with a vaccine targeting the same pathogen (e.g., a bacterial or viral pathogen) to induce IgG antibodies, IgA antibodies, and / or Th1 T cell responses (e.g., a predominant Th1 T cell response) in the mammal. For example, SC-Ad engineered to encode a SARS-CoV-2 spike polypeptide can be administered intranasally as a heterologous vaccination boost to humans previously vaccinated with an mRNA- or DNA-based SARS-CoV-2 vaccine (e.g., Pfizer® vaccines such as Comirnaty®, AstraZeneca® vaccines such as Vaxzevria®, Moderna® vaccines such as Spikeevax®, Novavax® vaccines such as Nuvaxovid®, and / or Sputnik) to induce IgG antibody, IgA antibody, and / or Th1 T cell responses against the coronavirus in humans.The pre-vaccinated SARS-CoV-2 vaccine may be administered intranasally to a human within about 21 days to about 120 months (e.g., about 21 days to about 60 months, about 21 days to about 36 months, about 21 days to about 24 months, about 21 days to about 12 months, about 21 days to about 8 months, about 21 days to about 6 months, about 21 days to about 4 months, about 21 days to about 3 months, about 21 days to about 2 months, about 1 month to about 120 months, about 3 months to about 120 months, about 6 ... The vaccine may be administered to a human about 120 months ago, about 12 months to about 120 months, about 18 months to about 120 months, about 24 months to about 120 months, about 36 months to about 120 months, about 48 months to about 120 months, about 1 month to about 60 months, about 2 months to about 48 months, about 3 months to about 36 months, about 4 months to about 24 months, about 6 months to about 18 months, about 1 month to about 3 months, about 3 months to about 6 months, about 6 months to about 12 months, about 12 months to about 24 months, about 24 months to about 48 months, about 36 months to about 72 months, about 48 months to about 84 months, or about 60 months to about 96 months. A pre-vaccinated SARS-CoV-2 vaccine can be a vaccine that was administered to a human at least 90 days before (e.g., at least 120 days, at least 150 days, or at least 180 days before) intranasal administration of an SC-Ad described herein (e.g., an SC-Ad genetically engineered to encode a SARS-CoV-2 spike polypeptide) to the human.

[0055] The adenoviral vectors described herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein can be intranasally administered to any suitable mammal (e.g., to increase an immune response in the mammal against a pathogen, such as a bacterial or viral pathogen, associated with the immunogen(s) encoded by the adenoviral vector). In some cases, the mammal may be a mammal that has not been previously infected with a pathogen associated with an immunogen encoded by an adenoviral vector described herein. In some cases, the mammal may be a mammal that has been previously infected with a pathogen that is closely related (e.g., genetically related) to a pathogen associated with an immunogen encoded by an adenoviral vector described herein. In some cases, the mammal may be a mammal that has been infected (e.g., has an ongoing infection) with a pathogen associated with an immunogen encoded by an adenoviral vector provided herein. Examples of mammals to which the adenoviral vectors described herein encoding one or more immunogens (and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein encoding one or more immunogens) may be administered intranasally include, but are not limited to, humans, non-humans, primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, hamsters, bats, raccoons, and ferrets. In some cases, humans may be administered one or more adenoviral vectors described herein and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein intranasally to increase an immune response against a pathogen (e.g., a bacterial or viral pathogen associated with the immunogen encoded by the adenoviral vector).

[0056] The adenoviral vectors described herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding the adenoviral vectors described herein (e.g., compositions such as vaccine compositions comprising the adenoviral vectors provided herein and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein) can be administered intranasally (e.g., administered intranasally as a heterologous vaccination boost) to a mammal (e.g., a human) in an appropriate amount (e.g., a suitable dosage). The effective amount may vary depending on the age and general health of the subject, the use of excipients, the potential for combination with other therapeutic treatments, e.g., the use of other agents, and the judgment of the treating physician. An effective amount of a composition containing an adenoviral vector encoding one or more immunogens (and / or nucleic acid molecules capable of encoding an adenoviral vector encoding one or more immunogens) can be an amount capable of inducing an immune response in a mammal described herein without causing significant toxicity to the mammal. For example, an effective amount of an adenoviral vector encoding one or more immunogens can be, for example, about 10 8 Virus particles (vp) ~ approx. 10 14 vp (e.g., about 10 8 vp~about 10 13 vp, about 10 8 vp~about 10 12 vp, about 10 8 vp~about 10 11 vp, about 10 8 vp~about 10 10 vp, about 10 8 vp~about 10 9 vp, about 10 9 vp~about 10 14 vp, about 10 10 vp~about 10 14 vp, about 10 11 vp~about 10 14 vp, about 10 12 vp~about 10 14 vp, about 10 13 vp~about 10 14 vp, about 10 9 vp~about 10 13 vp, about 10 10 vp~about 10 12vp, about 10 9 vp~about 10 11 vp, about 10 10 vp~about 10 12 vp, or about 10 1 vp~about 10 13 vp). The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can affect the actual effective amount used for a particular application. For example, the frequency of administration and / or the use of multiple therapeutic agents may require an increase or decrease in the actual effective amount administered.

[0057] The adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein (e.g., compositions such as vaccine compositions comprising the adenoviral vectors provided herein and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein) can be administered intranasally to a mammal (e.g., a human) at an appropriate frequency (e.g., administered intranasally as a heterologous vaccination boost). The administration frequency can be any frequency capable of inducing an immune response in the mammal without causing significant toxicity to the mammal. In some cases, the adenoviral vectors provided herein and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein can be administered intranasally to a mammal once (e.g., in a single dose) (e.g., administered intranasally as a heterologous vaccination boost). In some cases, the adenoviral vectors provided herein and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein can be administered intranasally to a mammal multiple times (e.g., as multiple doses) (e.g., administered intranasally as a heterologous vaccination boost). For example, the frequency of administration can be once every about 1 day to about 3 days, once every about 1 day to about 1 week, once every about 1 week to about 3 weeks, once every about 1 week to about 6 weeks, once every about 3 months to about 6 months, once every 6 months to 18 months, or once every 6 months to 1 year. As with the effective amount, various factors can affect the actual frequency of administration used for a particular application. For example, effective doses and / or the use of multiple therapeutic agents may require increased or decreased administration frequency.

[0058] In some cases, one or more adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding an adenoviral vector provided herein (e.g., a composition such as a vaccine composition comprising an adenoviral vector provided herein and / or a nucleic acid molecule capable of encoding an adenoviral vector provided herein) can be administered intranasally to a mammal (e.g., a human) as the sole active ingredient (e.g., administered intranasally as a heterologous vaccination boost) to increase the immune response (e.g., increased IgG and IgA antibody responses and / or increased Th1 responses) against a pathogen (e.g., a bacterial or viral pathogen associated with the immunogen encoded by the adenoviral vector). For example, a composition containing an adenoviral vector described herein encoding one or more coronavirus immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more coronavirus immunogens) can be administered intranasally to a mammal (e.g., a human) as the sole active ingredient (e.g., administered intranasally as a heterologous vaccination boost) to increase the immune response to the coronavirus (e.g., increase IgG and IgA antibody responses and / or increase Th1 T cell responses).

[0059] In some cases, one or more adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding an adenoviral vector provided herein (e.g., a composition such as a vaccine composition comprising an adenoviral vector provided herein and / or a nucleic acid molecule capable of encoding an adenoviral vector provided herein) can be administered intranasally to a mammal (e.g., a human) (e.g., administered intranasally as a heterologous vaccination boost) along with one or more (e.g., 1, 2, 3, 4, 5, or more) additional agents / therapies used to increase the immune response (e.g., to increase IgG and IgA antibody responses and / or increase Th1 T cells) against a pathogen (e.g., a bacterial or viral pathogen associated with the immunogen encoded by the adenoviral vector). For example, a composition containing an adenoviral vector described herein encoding one or more coronavirus immunogens (and / or a nucleic acid molecule capable of encoding an adenoviral vector described herein encoding one or more coronavirus immunogens) can be administered intranasally to a mammal (e.g., a human) (e.g., administered intranasally as a heterologous vaccination boost) along with one or more (e.g., 1, 2, 3, 4, 5, or more) additional agents / therapies used to increase the immune response to the coronavirus (e.g., to increase IgG and IgA antibody responses and / or to increase Th1 T cells).When one or more adenoviral vectors provided herein (e.g., adenoviral vectors encoding one or more immunogens) and / or nucleic acid molecules capable of encoding an adenoviral vector provided herein (e.g., a composition such as a vaccine composition comprising an adenoviral vector provided herein and / or a nucleic acid molecule capable of encoding an adenoviral vector provided herein) are used in conjunction with one or more additional agents / therapies used to increase an immune response (e.g., to increase IgG and IgA antibody responses and / or to increase Th1 T cells), the one or more adenoviral vectors provided herein (and / or nucleic acids capable of encoding an adenoviral vector provided herein) and the one or more additional agents / therapies can be administered simultaneously (e.g., in a single composition) or separately. For example, one or more adenoviral vectors provided herein (and / or nucleic acid molecules capable of encoding the adenoviral vectors provided herein) can be administered first and one or more additional agents / therapies can be administered second, or vice versa.

[0060] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0061] Example Example 1: Replicating Single-Cycle Adenovirus Vaccine Against COVID-19 SC-Ad6-1 (COVID-19 vaccine; Figures 8 and 9) was safe and well tolerated in early escalating dose cohorts. Furthermore, when SC-Ad6-1 was administered as a heterologous booster to humans previously vaccinated with an mRNA COVID-19 vaccine, strong mucosal and systemic neutralizing responses against the SARS-CoV-2 spike protein were observed in the majority of humans.

[0062] Ten healthy volunteers who had been fully vaccinated against COVID-19 for at least 3 months prior to day 1 of receiving SC-Ad6-1 as a heterologous vaccination booster were enrolled (Figure 2). One of the 10 volunteers was infected with SARS-CoV-2 on day 2 of the trial and was therefore excluded from the analysis. Humans were administered 8.0e8 infectious units (IU) of SC-Ad6-1 via intranasal administration, and humoral and cellular immune responses were assessed. Immunogenicity endpoints were humoral and cellular immune responses to SARS-CoV-2 as determined by (a) the presence of serum anti-SARS-CoV-2 neutralizing antibodies by live virus assay, (b) the presence of serum IgG antibodies specific for the SARS-CoV-2 spike protein by ELISA, (c) the presence of mucosal IgA antibodies specific for the SARS-CoV-2 spike protein by ELISA, and (d) the presence of cytokine-producing Th1 T cells specific for the SARS-CoV-2 spike protein by interferon-γ secretion from stimulated PBMCs.

[0063] For safety reasons, the first two human participants (sentinels) were dosed prior to the remaining eight human participants and at least one hour apart (Figure 1). The adverse event profile in the sentinels was examined 48 hours after intranasal dosing (study day 3). No safety issues were identified in the sentinels, and the remainder of the human participants were dosed with the same dose at least 48 hours after dosing of the sentinel participants (Figure 1).

[0064] Each human participant visited the clinical site on the day of dosing (Day 1) and for follow-up evaluations of safety, immunogenicity, and pharmacodynamics on Study Days 3, 8, 22, 29, and 43. A follow-up phone call to assess adverse events was conducted on Study Day 15. The end-of-study (EoS) visit was Study Day 106 (Figure 1).

[0065] Intranasal administration of SC-Ad6-1 as a nasal spray to humans as a heterologous vaccination booster resulted in increased serum IgG antibodies specific to the SARS-CoV-2 spike protein as determined by ELISA (Figure 3), increased mucosal IgA antibodies specific to the SARS-CoV-2 spike protein as determined by ELISA (Figure 4), increased serum anti-SARS-CoV-2 neutralizing antibodies as determined by live virus assay (Figure 5), and T cell responses to the SARS-CoV-2 spike that favored Th1 responses (important for pathogen immunity) over Th2 responses as determined by IL-5 secretion (Figure 7) versus interferon-γ secretion (Figure 6) from stimulated PBMCs, respectively.

[0066] These results demonstrate that SC-Ads engineered to encode SARS-CoV-2 immunogens can be administered intranasally as a heterologous vaccination boost to humans previously vaccinated with COVID-19 vaccines to induce IgG and IgA antibodies against SARS-CoV-2 and Th1 T cell immune responses (e.g., Th1-dominated T cell immune responses) against SARS-CoV-2.

[0067] Example 2: SC-Ad6-1 DNA sequence SEQ ID NO: 1 [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0068] Other embodiments While the present invention will be described in conjunction with its detailed description, it should be understood that the description is intended to be illustrative of the invention and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method of providing a heterologous vaccination boost to a human who has previously been vaccinated with a coronavirus vaccine, the method comprising intranasally administering to the human a single-cycle adenovirus (SC-Ad), the SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide, the SC-Ad comprising a nucleic acid sequence encoding a coronavirus immunogen, and the prior coronavirus vaccine did not comprise the SC-Ad.

2. 2. The method of claim 1, wherein the adenoviral polypeptide is selected from the group consisting of a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, and a pIIIa polypeptide.

3. 3. The method of claim 1 or 2, wherein the immunogen comprises a coronavirus spike polypeptide or an immunogenic fragment thereof.

4. 4. The method of claim 3, wherein the immunogen consists of, or consists essentially of, an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 of WO 2022 / 040204.

5. The method of any one of claims 1 to 4, wherein the SC-Ad further comprises a nucleic acid sequence encoding an adjuvant polypeptide.

6. 6. The method of claim 5, wherein the adjuvant polypeptide is selected from the group consisting of a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide, an interleukin-4 (IL-4) polypeptide, an interleukin-21 (IL-21) polypeptide, a CD40 ligand (CD40L) polypeptide, a 4-1BB ligand (4-1BBL) polypeptide, a transforming growth factor beta (TGF-β) polypeptide, a Clostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide, and biologically active fragments thereof.

7. 7. The method of claim 5 or 6, wherein the coronavirus spike polypeptide is fused to the adjuvant polypeptide.

8. 8. The method of claim 7, wherein the coronavirus spike polypeptide fused to the adjuvant polypeptide consists essentially of, or consists of, the amino acid sequence set forth in SEQ ID NO:5 of WO 2022 / 040204.

9. The method of any one of claims 1 to 4, wherein the SC-Ad further comprises a nucleic acid sequence encoding a chaff polypeptide.

10. 10. The method of claim 9, wherein the chaff polypeptide is a fragment of an ACE2 polypeptide.

11. The method of claim 10, wherein the fragment of the ACE2 polypeptide comprises the extracellular region of the ACE2 polypeptide and lacks the transmembrane domain.

12. 12. The method of claim 11, wherein the chaff polypeptide consists essentially of, or consists of, the amino acid sequence set forth in SEQ ID NO: 8 of WO 2022 / 040204 or SEQ ID NO: 9 of WO 2022 / 040204.

13. 13. The method of any one of claims 9 to 12, wherein the coronavirus spike polypeptide is fused to the chaff polypeptide.

14. The method of any one of claims 1 to 13, wherein the coronavirus is a β-coronavirus.

15. The method of claim 14, wherein the beta coronavirus is SARS-CoV-2.

16. 16. The method of any one of claims 1 to 15, wherein the human has received the prior coronavirus vaccine at least 90 days prior to the intranasal administration.

17. 16. The method of any one of claims 1 to 15, wherein the human has received the prior coronavirus vaccine at least 120 days prior to the intranasal administration.

18. 16. The method of any one of claims 1 to 15, wherein the human has received the prior coronavirus vaccine at least 150 days prior to the intranasal administration.

19. 16. The method of any one of claims 1 to 15, wherein the human has received the prior coronavirus vaccine at least 180 days prior to the intranasal administration.

20. 20. The method of any one of claims 1 to 19, wherein the prior coronavirus vaccine was an mRNA-based vaccine.

21. 21. The method of any one of claims 1 to 20, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of serum IgG antibodies that bind to the immunogen.

22. 22. The method of any one of claims 1 to 21, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of mucosal IgA antibodies that bind to the immunogen.

23. 23. The method of any one of claims 1 to 22, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of neutralizing antibodies in the human that bind to the immunogen.

24. 24. The method of any one of claims 1 to 23, wherein the SC-Ad affects cells of the human, and expression of the immunogen in the cells causes an increase in the number of Th1 T cells in the human that target the immunogen.

25. 1. A method of providing a heterologous vaccination boost against a pathogen to a mammal that has received a prior vaccine targeting the pathogen, the method comprising intranasally administering to the mammal a single-cycle adenovirus (SC-Ad), the SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide, the SC-Ad comprising a nucleic acid sequence encoding an immunogen for the pathogen, and the prior vaccine did not comprise the SC-Ad.

26. 26. The method of claim 25, wherein the mammal is a human.

27. 27. The method of claim 25 or 26, wherein the adenoviral polypeptide is selected from the group consisting of a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, and a pIIIa polypeptide.

28. The method of any one of claims 25 to 27, wherein the pathogen is a coronavirus.

29. 29. The method of claim 28, wherein the coronavirus is a beta coronavirus.

30. 30. The method of claim 29, wherein the beta coronavirus is SARS-CoV-2.

31. 31. The method of any one of claims 25 to 30, wherein the immunogen comprises a coronavirus spike polypeptide or an immunogenic fragment thereof.

32. 32. The method of any one of claims 25 to 31, wherein the mammal is a mammal that received the prior vaccination at least 90 days prior to the intranasal administration.

33. 36. The method of any one of claims 25 to 35, wherein the mammal is a mammal that received the prior vaccination at least 120 days prior to the intranasal administration.

34. 32. The method of any one of claims 25 to 31, wherein the mammal is a mammal that received the prior vaccination at least 150 days prior to the intranasal administration.

35. 32. The method of any one of claims 25 to 31, wherein the mammal is a mammal that received the prior vaccination at least 180 days prior to the intranasal administration.

36. 36. The method of any one of claims 256-35, wherein the prior vaccine is an mRNA-based vaccine.

37. 37. The method of any one of claims 25 to 36, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of serum IgG antibodies that bind to the immunogen.

38. 38. The method of any one of claims 25 to 37, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of mucosal IgA antibodies that bind to the immunogen.

39. 39. The method of any one of claims 25 to 38, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of neutralizing antibodies in the human that bind to the immunogen.

40. 40. The method of any one of claims 25 to 39, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the number of Th1 T cells in the mammal that target the immunogen.

41. 4. The method of claim 3, wherein the immunogen consists of, or consists essentially of, an amino acid sequence set forth in any one of SEQ ID NOs: 12, 42, 43, 44, 47, and 48 of WO 2022 / 040204.

42. 1. A method of providing a vaccination boost to a human who has been previously vaccinated against a virus that causes respiratory disease, said method comprising intranasally administering to said human a single-cycle adenovirus (SC-Ad), said SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, said SC-Ad comprising said adenoviral polypeptide, and said SC-Ad comprising a nucleic acid sequence encoding an immunogen of the virus that causes respiratory disease.

43. 43. The method of claim 42, wherein the virus causing the respiratory disease is selected from the group consisting of coronavirus; influenza virus; respiratory syncytial virus (RSV); metapneumovirus (MPV); rhinovirus; bocavirus; and parainfluenza virus (PIV).

44. The method of claim 42 or 43, wherein the immunogen comprises a coronavirus spike polypeptide or an immunogenic fragment thereof; an influenza hemagglutinin polypeptide; or an immunogenic fragment thereof; an RSV fusion (F) protein polypeptide or an immunogenic fragment thereof; an MPV fusion (F) protein polypeptide or an immunogenic fragment thereof; a rhinovirus VP1 protein polypeptide or an immunogenic fragment thereof; and a PIV fusion (F) protein polypeptide or an immunogenic fragment thereof.

45. 45. The method of claim 44, wherein the immunogen comprises a coronavirus spike polypeptide or an immunogenic fragment thereof.

46. 46. ​​The method of claim 45, wherein the immunogen consists of, or consists essentially of, an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 of WO 2022 / 040204.

47. 46. ​​The method of claim 45, wherein the immunogen consists of, or consists essentially of, an amino acid sequence set forth in any one of SEQ ID NOs: 12, 42, 43, 44, 47, and 48 of WO 2022 / 040204.

48. The method of any one of claims 42 to 47, wherein the SC-Ad further comprises a nucleic acid sequence encoding an adjuvant polypeptide.

49. 49. The method of claim 48, wherein the adjuvant polypeptide is selected from the group consisting of a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide, an interleukin-4 (IL-4) polypeptide, an interleukin-21 (IL-21) polypeptide, a CD40 ligand (CD40L) polypeptide, a 4-1BB ligand (4-1BBL) polypeptide, a transforming growth factor beta (TGF-β) polypeptide, a Clostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide, and biologically active fragments thereof.

50. 50. The method of claim 48 or 49, wherein the immunogen is fused to the adjuvant polypeptide.

51. 51. The method of claim 50, wherein the immunogen fused to the adjuvant polypeptide consists essentially of, or consists of, the amino acid sequence set forth in SEQ ID NO: 5 of WO 2022 / 040204.

52. The method of any one of claims 42 to 47, wherein the SC-Ad further comprises a nucleic acid sequence encoding a chaff polypeptide.

53. 53. The method of claim 52, wherein the chaff polypeptide is a fragment of an ACE2 polypeptide.

54. 54. The method of claim 53, wherein the fragment of an ACE2 polypeptide comprises the extracellular region of an ACE2 polypeptide and lacks the transmembrane domain.

55. 55. The method of claim 54, wherein the chaff polypeptide consists essentially of, or consists of, the amino acid sequence set forth in SEQ ID NO: 8 of WO 2022 / 040204 or SEQ ID NO: 9 of WO 2022 / 040204.

56. 56. The method of any one of claims 52 to 55, wherein the immunogen is fused to the chaff polypeptide.

57. 57. The method of any one of claims 42 to 56, wherein the human has received the prior coronavirus vaccine at least 90 days prior to the intranasal administration.

58. 57. The method of any one of claims 42 to 56, wherein the human has received the prior coronavirus vaccine at least 120 days prior to the intranasal administration.

59. 57. The method of any one of claims 42 to 56, wherein the human has received the prior coronavirus vaccine at least 150 days prior to the intranasal administration.

60. 57. The method of any one of claims 42 to 56, wherein the human has received the prior coronavirus vaccine at least 180 days prior to the intranasal administration.

61. 61. The method of any one of claims 42 to 60, wherein the prior vaccine is an mRNA-based vaccine.

62. 62. The method of any one of claims 42-61, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of serum IgG antibodies that bind to the immunogen.

63. 63. The method of any one of claims 42 to 62, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of mucosal IgA antibodies that bind to the immunogen.

64. 64. The method of any one of claims 42-63, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the amount of neutralizing antibodies in the human that bind to the immunogen.

65. 65. The method of any one of claims 42-64, wherein the SC-Ad affects cells of the human and expression of the immunogen in the cells causes an increase in the number of Th1 T cells in the human that target the immunogen.

66. 1. A method for boosting a vaccination against a pathogen in a mammal that has been previously vaccinated against the pathogen, the method comprising intranasally administering to the mammal a single-cycle adenovirus (SC-Ad), the SC-Ad comprising a genome lacking at least a portion of a nucleic acid sequence encoding an adenoviral polypeptide, the SC-Ad comprising the adenoviral polypeptide, and the SC-Ad comprising a nucleic acid sequence encoding an immunogen for the pathogen.

67. 67. The method of claim 66, wherein the mammal is a human.

68. 68. The method of claim 66 or 67, wherein the pathogen causes a respiratory disease.

69. 69. The method of claim 68, wherein the virus causing the respiratory disease is selected from the group consisting of coronavirus; influenza virus; respiratory syncytial virus (RSV); metapneumovirus (MPV); rhinovirus; bocavirus; and parainfluenza virus (PIV).

70. 70. The method of any one of claims 66 to 69, wherein the adenoviral polypeptide is selected from the group consisting of a fiber polypeptide, a V polypeptide, a hexon polypeptide, a penton base polypeptide, and a pIIIa polypeptide.

71. 71. The method of any one of claims 66 to 70, wherein the mammal is a mammal that has been vaccinated with the prior coronavirus vaccine at least 90 days prior to the intranasal administration.

72. 72. The method of any one of claims 66 to 71, wherein the mammal is a mammal that has been vaccinated with the prior coronavirus vaccine at least 120 days prior to the intranasal administration.

73. 72. The method of any one of claims 66 to 71, wherein the mammal is a mammal that has been vaccinated with the prior coronavirus vaccine at least 150 days prior to the intranasal administration.

74. 72. The method of any one of claims 66 to 71, wherein the mammal is a mammal that has been vaccinated with the prior coronavirus vaccine at least 180 days prior to the intranasal administration.

75. 75. The method of any one of claims 66 to 74, wherein the prior vaccine is an RNA-based vaccine.

76. 76. The method of any one of claims 66 to 75, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of serum IgG antibodies that bind to the immunogen.

77. 77. The method of any one of claims 66 to 76, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of mucosal IgA antibodies that bind to the immunogen.

78. 78. The method of any one of claims 66 to 77, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the amount of neutralizing antibodies in the human that bind to the immunogen.

79. 79. The method of any one of claims 66 to 78, wherein the SC-Ad affects cells of the mammal and expression of the immunogen in the cells causes an increase in the number of Th1 T cells in the mammal that target the immunogen.

Images