Patent Application
20250197885
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 17, 2023, is named 10050_011788-WOO_SL.xml and is 33.5 bytes in size.
The present application relates to virus-like particles (VLPs), compositions comprising virus-like particles (VLPs), and methods of producing or delivering such VLPs. More specifically, the present application relates to VLPs of viruses of the Adenovirus family, also identified as adenovirus virus-like particles (AdVLPs).
Adenoviruses (AdVs) are commonly known as versatile vectors, used for gene therapy, oncolytic virotherapy, and vaccine delivery applications1,2. However, AdVs can also cause infection in humans, ranging in severity from mild to severe, and in very rare cases can even be fatal3. AdVs can infect people of all ages, though the majority of cases occur in younger populations in close contact settings, such as daycares, schools, college dormitories, and military barracks4-8. To date, more than 100 types of Human AdVs have been identified, which are classified into 7 species, termed A through G9. Tissue tropism of any one specific AdV type is largely linked to its species classification, as most types within a given species have shared tropisms3. AdVs can infect several different tissues, resulting in an array of clinical manifestations including conjunctivitis, myocarditis, gastroenteritis, hepatitis, and, most commonly, respiratory tract illnesses3.
The adenovirus family is comprised of a large group of icosahedral non-enveloped double stranded linear DNA containing viruses that are able to infect a wide range of mammalian, avian and reptilian species. These viruses are classified within the adenoviridae family, which includes the following five genera: Mastadenovirus, Aviadenovirus, Atadenoviurs, Siadenovirus and Ichtadenovirus. Each of these genera includes adenoviruses that infect different species and within each species there are distinct types. For example, the genus Mastadenovirus infects only mammals and includes viruses affecting humans, bovine, ovine, canine species as well as rodents (mice).
All human adenoviruses are within the genus Mastadenovirus and are further classified into seven groups or species, A through G based on genome sequence, agglutination properties, immune cross-reactivity and genome organization. More than 70 types (a term proposed to superseded serotype) have been recognized amongst these groups; e.g., human adenovirus -A (HAdV-A) includes type: 12, 18, 31; HAdV-B includes type: 3, 7, 11, 14, 16, 21, 34, 35, 50, 55; HAdV-C include type: 1, 2, 5, 6; HAdV-D include type: 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51, 53, 54; HAdV-E includes type: 4; HAdV-F includes type: 40, 41, and HAdV-G includes type: 52. Other genera such as the Aviadenovirus genus include viruses infecting avian species and causing conditions of different levels of significance. In humans, adenovirus primarily cause respiratory infections; however, depending on the infecting serotype, they may also cause various other conditions such as gastroenteritis, conjunctivitis, cystitis, etc.
Adenoviruses are often recognized as the cause of acute respiratory disease (ARD) in military trainees and children. The clinical manifestation of adenovirus respiratory infections may range from common cold symptoms, pharyngitis, bronchitis and pneumonitis to severe illness and death.
New recombinant adenoviruses may arise from the simultaneous infection of humans with two or more types, an event which commonly occurs in military training centers or nursing homes. These events may result in the formation of new recombinant viruses possessing new biological and immunological characteristics that may overcome pre-existing immunity and rapidly disseminate within the human population. Prophylactic interventions for the control of adenovirus infections are limited. For example, human adenovirus (HAdV) species E type 4 (AdV-4) and species B type 7 (AdV-7) are the serotypes responsible for most outbreaks of acute respiratory disease (ARD) among military recruits of whom almost 80% are infected and 20% require hospitalization according to studies evaluating these trainees.
Of particular interest are AdV-4 and AdV-7, which are the most commonly circulating types in the United States10. Both AdV-4 and AdV-7 are known to cause acute respiratory disease (ARD), and in rare instances can even cause pneumonia10,11. Infections with AdV-7 are typically associated with more severe outcomes than AdV-412-15. These infections can be debilitating for several days, and in some cases require hospitalization16,17. Treatment options are limited to supportive care, as there are no approved antivirals for treating infections with AdVs12,18. Historically, AdVs have been shown to infect up to 80% of military recruits in the United States, 20% of whom required hospitalization19.
The high incidence of ARD as a result of AdV infection in the U.S. military prompted the development of live-virus oral vaccines against AdV-4 and AdV-7, administered simultaneously. These vaccines were approved by the FDA in the 1970s for use in military personnel and proved largely successful3,20,21, though they have not been made available to the general public. Protection against infection is provided within one week of immunization, and has been shown to last for at least six years22. Adverse effects associated with vaccination are minimal20,23,24. Following the introduction of these vaccines, incidence of AdV infection in military recruits decreased dramatically24. In tandem, rates of hospitalizations resulting from AdV-induced ARD were reduced by more than 90%21.
Although these vaccines against AdV-4 and AdV-7 are generally well-tolerated and effective, the use of live AdV-4 and AdV-7 as immunogens has major downsides. Vaccination with live AdV-4 and AdV-7 by oral administration causes an asymptomatic infection of the gastrointestinal tract. Shedding of vaccine-associated AdVs can be detected in stool samples, persisting for up to 28 days after vaccination23,25. If proper hygiene is not practiced, shed virus can be transmitted to people in close contact with vaccine recipients. Additionally, AdVs have the potential to undergo recombination when multiple types infect the same cell, which can generate novel subtypes26,27. Despite the risks associated with the live virus vaccines, continued vaccination is necessary. Between 1999 and 2004, vaccination of military recruits was paused due to supply issues24. During this period, incidence of AdV-4 and AdV-7 infections and consequent ARD promptly returned to pre-vaccine levels28. Thus, alternative vaccine platforms that can provide protection against AdV infection without the risk of vaccine-associated viral shedding need to be explored.
Furthermore, the emergence of new adenovirus serotypes, e.g., HAdV3, HAdV14, HAdV21 and HAdV55 among others, which are potentially able to cause epidemics and deaths, underscores the importance of these pathogens in public and military health.
Therefore, there is a need for the creation, development and production of new vaccines to protect against illnesses caused by adenoviruses in the military and in the general public.
In a first aspect, an adenovirus virus-like particle (AdVLP) is provided. The AdVLP comprises a recombinant capsid comprising: a) major capsid adenovirus (AdV) proteins including a hexon protein, a penton protein, and a fiber protein; b) minor capsid/cement AdV proteins, including a IIIa protein, a VI protein, a VIII protein, and a IX protein, wherein the minor capsid/cement AdV proteins structurally support the major capsid AdV proteins; c) a chaperone AdV protein L4-100k; and d) an accessory scaffold AdV protein L1-52/55k.
In another aspect, the AdVLP is an adenovirus type 4 virus-like particle (AdVLP4), adenovirus type 7 virus-like particle (AdVLP7), adenovirus type 14 virus-like particle (AdVLP14), or adenovirus type 55 virus-like particle (AdVLP55).
In another aspect, the major capsid AdV hexon protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, the major capsid AdV penton protein comprises SEQ ID NO: 3 or SEQ ID NO: 4, and the major capsid AdV fiber protein comprises SEQ ID NO: 5 or SEQ ID NO: 6.
In another aspect, the minor capsid/cement Adv protein IIIA protein comprises SEQ ID NO: 11 or SEQ ID NO: 12, the minor capsid/cement Adv protein VI protein comprises protein comprises SEQ ID NO: 13 or SEQ ID NO: 14, the minor capsid/cement Adv protein VIII protein comprises protein comprises SEQ ID NO: 15 or SEQ ID NO: 16, and the minor capsid/cement Adv protein IX protein comprises protein comprises SEQ ID NO: 17 or SEQ ID NO: 18.
In another aspect the chaperone AdV protein L4-100k comprises SEQ ID NO: 7 or SEQ ID NO: 8.
In another aspect, the accessory scaffold AdV L1-52/55k protein comprises SEQ ID NO: 9 or SEQ ID NO: 10.
In another aspect, the AdVLP further comprises an adjuvant. In a further aspect, the adjuvant is aluminum hydroxide or a squalene-based oil-in-water nano-emulsion.
In another aspect, the AdVLP comprises hexon proteins from two different serotypes such that the AdVLP is chimeric.
In another aspect, the hexon protein comprises one or more antigens of a different infectious agent.
In a further aspect, the different infectious agent is SARS-CoV-2.
In a second aspect, an expression plasmid comprising genes encoding adenovirus proteins is provided. The expression plasmid is suitable for the assembly of adenovirus virus-like particles (AdVLPs). The expression plasmid comprises codon-optimized genes encoding: a) major capsid adenovirus (AdV) proteins including a hexon protein, a penton protein, and a fiber protein; b) minor capsid/cement AdV proteins, including a IIIa protein, a VI protein, a VIII protein, and a IX protein; c) a chaperone AdV protein L4-100k; and d) an accessory scaffold AdV protein L1-52/55k.
In another aspect of the expression plasmid, the AdVLP is an adenovirus type 4 virus-like particle (AdVLP4), adenovirus type 7 virus-like particle (AdVLP7), adenovirus type 14 virus-like particle (AdVLP14), or adenovirus type 55 virus-like particle (AdVLP55).
In another aspect of the expression plasmid, the major capsid AdV hexon protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, the major capsid AdV penton protein comprises SEQ ID NO: 3 or SEQ ID NO: 4, and the major capsid AdV fiber protein comprises SEQ ID NO: 5 or SEQ ID NO: 6. The minor capsid/cement Adv protein IIIA protein comprises SEQ ID NO: 11 or SEQ ID NO: 12, the minor capsid/cement Adv protein VI protein comprises protein comprises SEQ ID NO: 13 or SEQ ID NO: 14, the minor capsid/cement Adv protein VIII protein comprises protein comprises SEQ ID NO: 15 or SEQ ID NO: 16, and the minor capsid/cement Adv protein IX protein comprises protein comprises SEQ ID NO: 17 or SEQ ID NO: 18. The chaperone AdV protein L4-100k comprises SEQ ID NO: 7 or SEQ ID NO: 8, the accessory scaffold AdV protein L1-52/55k comprises SEQ ID NO: 9 or SEQ ID NO: 10.
In a third aspect, an immunogenic composition is provided that comprises at least one AdVLP of the present application.
In another aspect of the immunogenic composition, the at least one AdVLP is adenovirus type 4 virus-like particle (AdVLP4), adenovirus type 7 virus-like particle (AdVLP7), adeonovirus type 14 virus-like particle (AdVLP14), or adenovirus type 55 virus-like particle (AdVLP55).
In another aspect of the immunogenic composition, the recombinant capsid of the at least one AdVLP comprises: a) the major capsid AdV hexon protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, the major capsid AdV penton protein comprises SEQ ID NO: 3 or SEQ ID NO: 4, and the major capsid AdV fiber protein comprises SEQ ID NO: 5 or SEQ ID NO: 6; b) the minor capsid/cement Adv protein IIIA protein comprises SEQ ID NO: 11 or SEQ ID NO: 12, the minor capsid/cement Adv protein VI protein comprises protein comprises SEQ ID NO: 13 or SEQ ID NO: 14, the minor capsid/cement Adv protein VIII protein comprises protein comprises SEQ ID NO: 15 or SEQ ID NO: 16, and the minor capsid/cement Adv protein IX protein comprises protein comprises SEQ ID NO: 17 or SEQ ID NO: 18; c) the chaperone AdV protein L4-100k comprises SEQ ID NO: 7 or SEQ ID NO: 8; and d) the accessory scaffold AdV protein L1-52/55k comprises SEQ ID NO: 9 or SEQ ID NO: 10.
In another aspect of the immunogenic composition, the composition is a trivalent composition comprising AdVLP4, AdVLP7, and AdVLP14.
In another aspect of the immunogenic composition, the composition is a quadrivalent composition comprising AdVLP4, AdVLP7, AdVLP14, and AdVLP55.
In another aspect of the immunogenic composition, the composition is suitable for oral, nasal, mucosal, or parenteral administration.
In a fourth aspect, a method of generating an immune response to one or more adenoviruses in a subject is provided. In the method, an effective amount of the immunogenic composition of the present application is administered to the subject.
In another aspect of the method, the one or more adenoviruses are selected from the group consisting of HAdV-E type 4, HAdV-B type 7, HAdV-B type 14, and HAdV-B type 55.
In another aspect of the method, the immune response vaccinates the subject against the one or more adenoviruses. In a further aspect, the subject is a human.
In a fifth aspect, a method of producing an adenovirus virus-like particle (AdVLP) is provided. In the method, at least one expression plasmid is introduced into a host cell under conditions such that the host cell produces the AdVLP, wherein the at least one expression plasmid comprises codon-optimized genes encoding: a) major capsid adenovirus (AdV) proteins including a hexon protein, a penton protein, and a fiber protein; b) minor capsid/cement AdV proteins, including a IIIa protein, a VI protein, a VIII protein, and a IX protein; c) a chaperone AdV protein L4-100k; and d) an accessory scaffold AdV protein L1-52/55k.
In another aspect of the method, a) the major capsid AdV hexon protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, the major capsid AdV penton protein comprises SEQ ID NO: 3 or SEQ ID NO: 4, and the major capsid AdV fiber protein comprises SEQ ID NO: 5 or SEQ ID NO: 6; b) the minor capsid/cement Adv protein IIIA protein comprises SEQ ID NO: 11 or SEQ ID NO: 12, the minor capsid/cement Adv protein VI protein comprises protein comprises SEQ ID NO: 13 or SEQ ID NO: 14, the minor capsid/cement Adv protein VIII protein comprises protein comprises SEQ ID NO: 15 or SEQ ID NO: 16, and the minor capsid/cement Adv protein IX protein comprises protein comprises SEQ ID NO: 17 or SEQ ID NO: 18; c) the chaperone AdV protein L4-100k comprises SEQ ID NO: 7 or SEQ ID NO: 8; and d) the accessory scaffold AdV protein L1-52/55k comprises SEQ ID NO: 9 or SEQ ID NO: 10.
In another aspect of the method, in the host cell is a eukaryotic cell. In a further aspect, the eukaryotic cell is a mammalian cell.
In another aspect of the method, the method further comprises the step of purifying the AdVLP. In a further aspect, the AdVLP is purified from cell lysates and culture supernatants.
The present application relates to recombinant adenovirus capsids or adenovirus-like particles (AdVLPs), compositions comprising AdVLPs, and methods of making and using these AdVLPs, including the creation and production of homologous and heterologous AdVLP based vaccines as well as their use for drug (small molecule) and macromolecule delivery including, but not limited to, nucleic acids and genome editing systems, cancer therapy and immunotherapy and diagnostic and therapeutic applications.
In particular, the present disclosure includes strategies and methods used for the development of novel monovalent, or multivalent vaccines that are able to protect humans or other species against infection with one or more adenovirus types or serotypes included, displayed, or produced by the vaccine. Also described herein are production methods that produce AdVLPs that display certain antigenic configurations. In one or more embodiments, these VLPs feature hybrid (chimeric), modified (mutated, truncated) or reengineered antigens relevant for the generation of broad, robust and durable immune responses including high levels of neutralizing antibodies able to protect against more than one adenovirus type or serotype. Single particle monovalent, bivalent or multivalent as well as reengineered or modified AdVLPs are assembled or combined and used to formulate vaccine compositions, which allow for the immunization and subsequent protection against one or more types, serotypes or antigenically distinct adenovirus types (e.g., Mastadenvirus genus, human adenovirus species B types 7, 11, 14, 21, 55 and species C type 4; Aviadenovirus genus, fowl adenovirus 1, 4, 9, or similar combinations of members of other genera).
AdVLP vaccines can be produced in suspension culture of eukaryotic cells from where they are obtained. After purification, concentration and formulation the vaccine can be administered by any suitable route, for example via either mucosal or parenteral routes, and induce an immune response able to protect against any or all of the adenovirus types, serotypes or distinct antigenic variants for which the vaccine was designed and formulated to protect.
In one or more embodiments, AdVLPs can be comprised of combinations of adenoviruses and other antigens (e.g., other human respiratory viruses such as influenza, coronaviruses or viruses infecting other species such avian, bovine, ovine and porcine pathogens that can be combined in vaccine formulations with species specific AdVLPs). In one or more embodiments of the present application, methods of providing immune response to additional viruses are also described.
Because the assembled structures and its derivatives can interact with specific cell surface receptors and exert distinct effects including cellular uptake, they may allow for use in various areas such as therapy, gene therapy, immunotherapy, genome editing, and cancer treatment, for example.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a VLP” (or an “AdVLP”) can include a mixture of two or more such VLPs (or AdVLPs).
As used herein the term “adjuvant” refers to a compound that, when used in combination with a specific immunogen (e.g., an AdVLP) in a formulation, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
An “antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune-system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a cytotoxic T lymphocyte (CTL) epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term includes polypeptides which include modifications, such as deletions, additions and substitutions (generally conservative in nature) as compared to a native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
As use herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g., a mammal, will induce an immune response.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.
As used herein an “effective dose” generally refers to that amount of VLPs (e.g., AdVLPs) of the present application sufficient to induce immunity, to prevent and/or ameliorate an infection or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of a VLP. An effective dose may refer to the amount of VLPs sufficient to delay or minimize the onset of an infection. An effective dose may also refer to the amount of VLPs that provides a therapeutic benefit in the treatment or management of an infection. Further, an effective dose is the amount with respect to VLPs of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection. An effective dose may also be the amount sufficient to enhance a subject's (e.g., a human's) own immune response against a subsequent exposure to an infectious agent. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and/or reduces the severity of symptoms.
As used herein, the term “effective amount” refers to an amount of VLPs (e.g., AdVLPs) necessary or sufficient to realize a desired biologic effect. An effective amount of the composition would be the amount that achieves a selected result, and such an amount can be determined as a matter of routine experimentation by a person skilled in the art. For example, an effective amount for preventing, treating and/or ameliorating an infection can be the amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to VLPs of the invention. The term is also synonymous with “sufficient amount.”
An “immunogenic composition” is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest.
An “immunological response” or “immune response” to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytotoxic T lymphocytes (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or γΔ T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
As used herein, the term “multivalent” refers to VLPs (e.g., AdVLPs) which have multiple antigenic proteins against multiple types or strains of infectious agents or alternative conformations of the same antigen/protein (metastable), which naturally transition from one conformation to the next, but in the context of a vaccine formulation may contain stabilized (fixed) form of one conformation or both.
A “nucleic acid” molecule can include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any unacceptable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein the term “protective immunity”, “protective immune response” or “protective response” refers to an immune response mediated by antibodies against an infectious agent, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. VLPs of the present application can stimulate the production of antibodies that, for example, neutralize infectious agents, blocks infectious agents from entering cells, blocks replication of said infectious agents, and/or protect host cells from infection and destruction. The term can also refer to an immune response that is mediated by T-lymphocytes and/or other white blood cells against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates adenovirus infection or reduces at least one symptom thereof.
“Purified” or “Substantially purified” general refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
As used herein, the term “spike receptor binding domain” (RBD) refers to a part of a virus located on its ‘spike’ domain that allows it to dock to body receptors to gain entry into cells.
By “subject” is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The system described above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.
As used herein, “treatment” refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
As used herein, the term “vaccine” refers to a formulation which contains VLPs (e.g., AdVLPs) of the present application, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of VLPs. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved. In this form, the composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
A “vector” is capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of one or more sequences of interest in a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The vector is usually a plasmid designed for gene expression in cells. The term is used interchangeably with the terms “nucleic acid expression vector”, “expression plasmid”, and “expression cassette”.
As used herein, the terms “virus-like particle”, “VLP”, “recombinant virus-like particle” or “recombinant VLP” (e.g., AdVLP) refer to a nonreplicating, viral shell. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can also be described as “enveloped” if they contain a cell derived lipid membrane or non-enveloped if assembly with protein without a lipid membrane. The terms nanoparticles or nanospheres have also been used to described virus particles which do not defer in composition from the virus like particles delineated in this disclosure. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art and discussed more fully below. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical and immunological characterizations, and the like. See, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994) 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions. Additional methods of VLP purification include, but are not limited to, chromatographic techniques such as affinity, ion exchange, size exclusion, and reverse phase procedures.
As used herein, the term “about” or “approximately” for a numerical value means ±3% of the numerical value.
VLPs29 are structurally similar to native viruses, but are completely devoid of genetic material, rendering them non-infectious. This combination allows antigens to be presented to the immune system in their native conformation without the risk of vaccine-associated viral shedding or recombination. VLPs have been safely and successfully applied as vaccine platforms against human papillomavirus, hepatitis B virus, and hepatitis E virus30-34. Self-assembly of VLPs is typically driven by recombinant expression of viral structural proteins in mammalian, bacterial, yeast, or plant-based expression systems35. In AdVs, the bulk of the capsid is composed of major capsid proteins (hexon, penton, and fiber), which are structurally supported by the minor capsid/cement proteins (IIIa, VI, VIII, and IX)36,37. The major capsid proteins, specifically hexon, are the primary target of neutralizing antibodies38-41, which are associated with protection against disease42,43. The VLP platform of the present application is an effective alternative to the existing live virus Adenovirus vaccines for administration to both military recruits and to the general public, for example.
Furthermore, VLPs can also be used for the diagnosis of infection or for therapeutic indications. VLP vaccines can be produced via transient transfection of suspension culture of eukaryotic cells or suspension culture of stably transfected cells that constitutively produce the VLPs, which are released into the culture medium. After purification, concentration, and formulation, the vaccine can be administered by any suitable route, for example, via either mucosal or parenteral routes, and induce an immune response able to protect against any or all coronaviruses, antigenic variants, etc. VLPs comprising therapeutics, immunomodulatory functions and diagnostic application are also provided.
Virion Structure: Adenoviruses (AdVs) are non-enveloped icosahedral particles of approximately 90 nm in diameter, which do not contain either membranes or lipids. This icosahedral capsid is composed predominantly of proteins and encases a linear double-stranded DNA (dsDNA) genome-containing core. The AdV capsid is constructed by the assembly of 14 proteins including the hexon (protein II of 109 kDa in size), the penton base (protein III of 63.3 kDa in size) and the fiber (protein IV of 38-61.1 kDa in size), which are referred to as the major capsid proteins. Twelve copies of these hexon trimers form each one the 20 facets of the icosahedron that represents the most abundant capsid protein component. Each of the 12 vertices of the icosahedron is occupied by a pentameric penton base, which forms a complex with a trimer of the protruding fiber. The fiber trimers assemble into a shaft that extends outward from the center of the pentons and terminates forming a knob structure, which serves as the adenoviruses' attachment protein to cell surface receptors during infection. The minor capsid components include protein IIIa (63.5 kDa), VIII (24.0 kDa) and IX (14.4 kDa) that function as cement proteins linking the major structural building blocks with each other and the viral core. Protein VI (27 kDa) is also a cement protein that plays additional roles in virion maturation, entry, trafficking and early gene expression. The viral core is composed of the dsDNA genome (˜36Kbp that varies based on virus type and genus) that is associated with four proteins: V (41.6 kDa), VII (19.4 kDa), μ (4 kDa) and terminal protein (TP-55 kDa), in addition to the viral protease (23 kDa). Furthermore, other viral-encoded proteins present in infected cells seem also to participate in capsid assembly and/or genome packaging such as proteins IVa2, L4 33K, L4 22K, E2 72K and L1-52/55K.
AdV morphogenesis appears to follow a sequential pathway that involves numerous consecutive steps. The capsid formation begins with the assembly of hexon and penton capsomers followed by the assembly of empty capsids and also uses the minor capsid cement proteins and involvement of non-structural proteins. Assembly of the hexon trimers depends upon a virus-encoded protein, L4 100K, that serves as a chaperone-like element guiding hexon trimerization. Subsequent to capsid assembly, packaging proteins recognize the packaging domain in the viral genome and initiate its insertion into the empty capsid, likely through a single opening located in one vertex of the structure. Completion of the process entails cleavage of some of the capsid components by the viral protease and release of scaffolding and some packaging proteins allowing for the maturation of viral particles.
Adenoviruses enter target cells via a receptor-mediated endocytosis mechanism. During the initial step the fiber protein, which protrudes from each vertex of the icosahedron and terminates as a globular head (the knob), attaches with high affinity to cell surface receptors. Adenoviruses from species A, C through G attach to the coxsackie and adenovirus receptor (CAR) whereas members of the B species use CD46 and desmoglein-2 (DSG-2) as their primary receptor. Following attachment, virus uptake is mediated by the binding of the penton base protein with the αvβ3/αvβ5 integrins, which enable cell uptake and initiation of infection. Some other cell surface molecules have also been described as co-receptors for adenovirus such as MHC class I, sialic acid, and coagulation factor X. After infection, the adenovirus completes its replication cycle inside the cells.
Human adenoviruses are known to cause respiratory infections, conjunctivitis and gastroenteritis in normal individuals. In neonates and immunosuppressed individuals, however, adenovirus can cause serious illness and fulminant fatal pneumonia, hepatitis or encephalitis. Human adenovirus infections account for a small portion of acute respiratory illnesses in the general population and for about 5% to 10% of respiratory ailments in children, however, they can cause serious respiratory epidemic outbreaks amongst military recruits and in nursing homes. A live adenovirus vaccine formulated with human adenovirus types 4 and 7, which are the most prevalent serotypes causing disease in military recruits, is currently in use in military centers. These live viruses are enclosed in an enteric-coated pill and administered via the oral route. It is likely that due to only serotype-specific immunity offered by current vaccines and in consideration of the fact that many human adenoviruses are able to cause significant respiratory illness, it is distinctly possible that many are at risk of serious illness from multiple strains of the disease. Furthermore, virus recombination events may result in new antigenic serotypes, which may further increase the rate of respiratory epidemic outbreaks.
To address the limitations of the existing live adenovirus vaccine, the present application, in accordance with one or more embodiments, provides a new generation of adenovirus vaccines that can not only protect against multiple adenovirus types, but also, in certain embodiments, can be upgraded to include emerging serotypes. Safe non-replicating vaccines such as the ones described in this application can be used to prevent outbreaks in crowded settings such as nursing home, military facilities, etc. as well as to prevent disease in the general populations.
Protection provided by the AdVLP vaccines of the present application against adenovirus infection is primarily mediated by neutralizing antibodies targeting the major capsid proteins, particularly the most abundant hexon polypeptide that is both a type-specific and species-specific antigen. In addition, neutralizing antibodies that result from immunization with the proposed vaccine target the fiber protein and contribute to virus neutralization by synergizing with antibodies directed to the penton base, the latter of which mediate binding with the secondary HAdV receptor.
The external surface of the hexon protein exhibits loops with hypervariable sequences that are significant for adenovirus type-specific immunogenicity and elicitation of protective neutralizing antibodies. The ability of these domains to tolerate a significant amount of variability likely aids in evasion of the immune response. These hexon loops, however, are suitable for modifications or grafting of antigenic domains from related or unrelated sources.
In accordance with one or more embodiments, provided herein are adenovirus virus-like particles (AdVLPs). AdVLPs are recombinant capsids or shells that comprises capsid proteins of one or more adenoviruses. In particular, in one or more embodiments, the AdVLP is comprised of a recombinant capsid that includes major capsid adenovirus (AdV) proteins, specifically a hexon protein, a penton protein, and a fiber protein. The recombinant capsid of the AdVLP further includes minor capsid/cement AdV proteins, including a IIIa protein, a VI protein, a VIII protein, and a IX protein. The minor capsid/cement AdV proteins structurally support the major capsid AdV proteins in the recombinant capsid. The recombinant capsid further includes a chaperone AdV protein L4-100k, and an accessory scaffold AdV protein L1-52/55k. The major and minor capsid proteins, along with the chaperone and accessory scaffold proteins assemble into icosahedral (recombinant) capsids to form the AdVLPs, which are analogous to the empty capsids present in adenovirus-infected cells. In one or more embodiments, these structures appear to be primarily composed of 240 capsomeres of hexon trimers (12 per each triangular facet of the icosahedron), 12 pentameric penton capsomeres each occupying a vertex of the 12 vertices of the icosahedron and 12 trimeric fibers each projecting outward from the pentons. Exemplary amino acid and nucleotide sequences of various major and minor capsid proteins, and chaperone and accessory scaffold proteins of exemplary AdVLPs of the present application are provided following the examples section.
In one or more embodiments, the AdVLPs of the present application are formed from major and minor capsid proteins, chaperone proteins, and accessory scaffold proteins of one or more types of adenoviruses, including but not limited to: human adenovirus-A (HAdV-A) type: 12, 18, or 31; HAdV-B type: 3, 7,11,14,16, 21, 34, 35, 50, or 55; HAdV-C type: 1, 2, 5, or 6; HAdV-D type: 8-10, 13,15,17,19, 20, 22-30, 32, 33, 36-39, 42-49, 51, 53, or 54; HAdV-E type: 4; HAdV-F type: 40 or 41, and HAdV-G type: 52. In at least one embodiment, proteins from genera other than human adenovirus, such as the Aviadenovirus genus that include viruses infecting avian species, can be utilized to form the AdVLPs. In one or more embodiments, the AdVLPs of the present application are formed from major and minor capsid proteins, chaperone proteins, and accessory scaffold proteins of one or more of HAdV-E type 4 (AdVLP4), HAdV-B type 7 (AdVLP7), HAdV-B type 14 (AdVLP14), and HAdV-B type 55 (AdVLP55).
In one or more embodiments, the AdVLP can comprises hexon proteins from different serotypes (e.g., two different adenovirus serotypes) such that the AdVLP is chimeric.
In one or more embodiments, the AdVLP can further include one or more adjuvants. For example, in one or more embodiments, the adjuvant can be aluminum hydroxide (Alum) or a squalene-based oil-in-water nano-emulsion (e.g., ADDAVAX (InvivoGen, San Diego, CA, USA)).
The AdVLPs and compositions of the present application can be administered to a subject by any mode of delivery, including, for example, by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal (e.g., see International Publication No. WO99/27961) or transcutaneous (e.g., see International Publication Nos. WO02/074244 and WO02/064162), intranasal (e.g., see International Publication No. WO03/028760), ocular, aural, pulmonary or other mucosal administration and/or inhalation of powder compositions. Multiple doses can be administered by the same or different routes.
The AdVLPs (and AdVLP-containing compositions) can be administered prior to, concurrent with, or subsequent to delivery of other vaccines, for example. Additionally, the site of AdVLP administration may be the same or different as other vaccine compositions that are being administered.
Dosage treatment with the AdVLP composition may be a single dose schedule or a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response, for example at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the potency of the modality, the vaccine delivery employed, the need of the subject and be dependent on the judgment of the practitioner.
In one or more embodiments, an expression plasmid is provided comprising genes encoding adenovirus proteins, such that the expression plasmid is suitable for the assembly of the adenovirus virus-like particles (AdVLPs) of the present application. The expression plasmid can comprise codon-optimized genes that encode for major capsid adenovirus (AdV) proteins (hexon, penton, and fiber), the minor capsid/cement AdV proteins (IIIa, VI, VIII, and IX protein), a chaperone AdV protein L4-100k, and an accessory scaffold AdV protein L1-52/55k. In one or more embodiments, the codon-optimized genes encode for proteins of one or more types of adenoviruses such as HAdV-A type, B type, C type, D type, E type, F type, and G type adenoviruses as described above, or other types of adenoviruses. For instance, in at least one embodiment, the genes encode for proteins of one or more of AdVLP4, AdVLP7, AdVLP14, and AdVLP55.
In accordance with one or more embodiments, an immunogenic composition (e.g., vaccine) is provided that comprises at least one AdVLP of the present application (e.g., AdVLP4, AdVLP7, AdVLP14, AdVLP55). In one or more embodiments, the immunogenic composition can be monovalent, bivalent, trivalent, quadrivalent, polyvalent or multivalent, such that the immunogenic composition can be attached to one type of antigen (monovalent), two types of antigens (bivalent), three types of antigens (trivalent), etc., wherein the different antigens can be different types of adenoviruses, as described above. Thus, in one or more embodiments, the immunogenic composition can immunize a subject (e.g., human) against one or more types of adenoviruses.
In at least one embodiment, the immunogenic composition is a trivalent composition comprising AdVLP4, AdVLP7, and AdVLP14 (such that it immunizes a subject against HAdV-E type 4, HAdV-B type 7, and HAdV-B type 14). In at least one embodiment, the immunogenic composition is a quadrivalent composition comprising AdVLP4, AdVLP7, AdVLP14, and AdVLP55 (such that it immunizes a subject against HAdV-E type 4, HAdV-B type 7, HAdV-B type 14, and HAdV-B type 55).
In one or more embodiments, a carrier is optionally present in the compositions (e.g., immunogenic compositions, AdVLPs) described herein. Typically, a carrier is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993. Such carriers are well known to those of ordinary skill in the art.
Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Exemplary adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu), (d) ADDAVAX™ vaccine adjuvant, a squalene-based oil-in-water nano-emulsion (InvivoGen, San Diego, CA, USA); (3) saponin adjuvants, such as Stimulon™. (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (IL-I, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes, etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63), LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (see, e.g., International Publication Nos. WO 93/13202 and WO 92/19265); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
In at least one embodiment, a method of generating an immune response to one or more adenoviruses in a subject is provided. The method comprises administering an effective amount of the immunogenic composition to the subject. In one or more embodiments, the immunogenic composition can be administered by oral, nasal, mucosal, or parenteral administration, or other mode of administration, as described above. In one or more embodiments of the method, the one or more adenovirsues are selected from the group consisting of HAdV-E type 4, HAdV-B type 7, HAdV-B type 14, and HAdV-B type 55, and in one or more embodiments, the immune response vaccinates the subject (e.g., human) against the one or more adenoviruses.
As such, the immunogenic compositions of the present application (e.g., AdVLP-based vaccines) can trigger, upon human or other species administration, a strong and balanced immune response characterized by the induction of high levels of neutralizing antibodies against one or more adenovirus species, types, serotypes or antigenic variants.
In one or more embodiments, the immunogenic composition may induce a humoral immune response in the subject administered the immunogenic composition. In some embodiments, the induced humoral immune response may be specific for one or more adenoviruses. The humoral immune response may be induced in the subject administered the immunogenic composition by about 1.5-fold to about 100-fold, about 2-fold to about 90-fold, or about 3-fold to about 80-fold. The humoral immune response can be induced in the subject administered the immunogenic composition by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more. The humoral immune response induced by the immunogenic composition may include an increased level of neutralizing antibodies associated with the subject administered the immunogenic composition as compared to a subject that is not administered the immunogenic composition. The neutralizing antibodies may be specific for one or more adenoviruses. The neutralizing antibodies can provide protection against and/or treatment of infections from one or more adenoviruses and their associated pathologies in the subject administered the immunogenic composition.
In one or more embodiments, the humoral immune response induced by the immunogenic composition may include an increased level of IgG antibodies associated with the subject administered the immunogenic composition as compared to a subject not administered the immunogenic composition.
In at least one embodiment, the humoral response may be cross-reactive against two or more strains or types of adenovirus. The level of IgG antibody associated with the subject administered the immunogenic composition may be increased by about 1.5-fold to about 100-fold, about 2-fold to about 50-fold, or about 3-fold to about 25-fold as compared to the subject not administered the immunogenic composition. The level of IgG antibody associated with the subject administered the immunogenic composition can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more.
An appropriate effective amount of the immunogenic composition for administration to the subject can be determined by one of skill in the art. Such an amount will fall in a relatively broad range that can be determined through routine trials and will generally be an amount on the order of about 0.1 μg to about 10 (or more) mg, more preferably about 1 μg to about 300 μg, of VLP/antigen. In another aspect, the immune response vaccinates the subject against one or more coronaviruses. For instance, in at least one embodiment, the immune response vaccinates the subject against one or more adenoviruses.
In accordance with one or more embodiments, a method of producing an AdVLP is provided. The method of producing the AdVLP includes introducing into a host cell at least one expression plasmid under conditions such that the host cell produces the AdVLP. The at least one expression plasmid introduced into the host cell comprises codon-optimized genes encoding: a) major capsid adenovirus (AdV) proteins (hexon, penton, fiber); b) minor capsid/cement AdV proteins (IIIa, VI, VIII, and IX); c) a chaperone AdV protein L4-100k; and d) an accessory scaffold AdV protein L1-52/55k. In one or more embodiments, the host cell is a eukaryotic cell, for example, a mammalian cell.
In at least one embodiment, the method of producing the AdVLP can further include a step of purifying the AdVLP. The AdVLP can be purified from cell lysates and culture supernatants, as exemplified in the example section below. It should be understood, however, that in other embodiments, the AdVLP can be purified using other approaches as is known and understood in the art.
The strategies for adenovirus vaccine development of the present application are based on the recombinant expression of adenovirus-like particles (AdVLPs) composed of alternative combinations of the structural elements involved in the morphogenesis of complete icosahedral virion particles. AdVLPs lack a viral genome and therefore are unable to replicate or cause infection thereby serving as suitable candidates for vaccine development. Lack of infectivity of AdVLP precludes the need for chemical inactivation, better preserving the immunological attributes of a vaccine based on this approach. Furthermore, stimulation of immunity by AdVLP is independent of viral replication avoiding viral interference caused by the replication dominance of one virus over the others in the composition. This may lead to an imbalance of the immune response when multiple replicating viral strains are combined in a live vaccine.
In accordance with one or more embodiments, the AdVLPs of the present application can provide utility as vaccines, delivery vehicles, therapeutics, drug enhancers, or diagnostic tools. For instance, in one or more embodiments, the AdVLPs of the present application can be used for a diverse range of applications because as biocompatible biological nanoparticles (e.g., AdVLP size ˜90 nm), they may serve as carriers of peptides, nucleic acids, glycans, and small molecules, etc. Furthermore, they can be modified, via genetic or chemical methods, to display homologous or heterologous proteins, antigens or peptide sequences and through bioconjugation, be able to display diverse molecular entities such as glycans, proteins, lipids, drugs, etc.
These various AdVLP designs can allow for their use in numerous fields such as for vaccines, imaging, targeted and non-targeted therapeutics, gene therapies, cancer vaccines and therapies, immunomodulation agents, nanodevices, among others. For instance, the hypervariable region (HVR) of the hexon protein, the Arg-Gly-Asp (RGD) domain loop of the penton base protein, the carboxyl terminal and HI loop of the fiber knob and the carboxyl terminal of the protein IX in addition to other possible sites may be suitable for genetic modifications or chemical conjugation of different molecular entities. For example,
In one or more embodiments, the AdVLPs of the present application can feature hybrid (chimeric) antigens. For instance, in at least one embodiment, the hexon protein of the AdVLP can comprise one or more antigens of a different infectious agent (an infectious agent other than an adenovirus). For example,
In addition to the functionalization of the exterior or interior surfaces of the particles, encapsulation or incorporation of new material within the interior cavity of these particles can further expand their range of use. Different techniques can be deployed for the incorporation, packaging or entrapment of small molecules, proteins, nucleic acids, or other molecular entities. Furthermore, understanding of the adenovirus assembly pathways may guide the discovery of new antiviral drugs and further advance nanotechnological applications. Thus, the assembly of native or modified AdVLPs broaden their utility beyond the field of adenovirus vaccine use as described above.
Exemplary embodiments and applications of the AdVLPs of the present application are described in further detail in the Examples section below.
An exemplary methodology for the self-assembly of recombinant adenovirus capsids or adenovirus virus-like particles (AdVLPs) is described herein. These are structures which exist as empty capsids that lack the viral genome and found in adenovirus infected cells. To assemble and produce these structures, multi-gene expression plasmids are utilized which, following their transfection into mammalian cell suspension cultures, direct the production of the proteins needed for the self-assembly of recombinant adenovirus capsids or adenovirus-like particles (AdVLPs). In one instance, two quintuple expression plasmids are utilized, one carrying the adenovirus major capsid proteins, hexon (two copies), penton and fiber together with the chaperone 100K protein and a second plasmid carrying the genes encoding minor adenovirus capsid proteins or the cement proteins, e.g., IX, IIIa, VI and VIII, together with a gene encoding for the scaffold protein 52/55, in order to construct the unique AdVLP. An example of expression plasmids utilized for the production of the AdVLP55 (adenovirus type 55) are shown in
Alternative methods for the production of AdVLPs include, but are not limited to, the use of differing numbers of plasmids, engineered cells for the stable production of some or all the needed proteins, modified cells that retain and amplify the expression plasmids for the prolong production of AdVLPs and other developed technology. Examples of additional or alternative methods use for the production and/or stabilization of the AdVLP particles may include other adenovirus genes such as IVa2, L4 22K, L4 33K, E2 72K and the viral protease (AVP-23 kDa) as well as the internal proteins, V (41.6 kDa), VII (19.9 kDa), and mu (4 kDa).
These AdVLPs can be purified from cell lysates and culture supernatants utilizing either cesium chloride (CsCl) gradient ultra-centrifugation methods, (e.g., discontinuous and isopycnic density gradients) or alternatively via tangential flow filtration and ionic-exchange chromatography.
The protein content of the AdVLPs are analyzed via Western blot and Coomassie blue staining techniques. Examples of the results of a Western blot from a CsCl purified AdVLP55 and a Coomassie blue staining of an AdVLP55 purified via chromatography are shown in
Moreover, the utilization of a fewer number of structural proteins such as the pair of penton base and fiber alone may result in the assembly of dodecahedron particles, which represent much smaller and less complex structures than the complete empty capsids or mature capsids containing the viral genome. The recombinant capsids or shells described here named adenovirus-like particles (AdVLP) are composed of the major capsid proteins hexon, penton base and fiber which together with the minor capsid proteins or cement protein, IIIa, VI, VIII and IX and other accessory elements assemble into icosahedral capsids or AdVLPs that are analogous to the empty capsids present in adenovirus infected cells. These structures appear to be primarily composed of 240 capsomeres of hexon trimers (12 per each triangular facet of the icosahedron), 12 pentameric penton capsomeres each occupying a vertex of the 12 vertices of the icosahedron and 12 trimeric fibers each projecting outward from the pentons. During adenovirus infection, these structures seem to be preformed and then utilized for threading the genomic DNA into place within the capsid. In the present application, recombinant empty capsids (or AdVLPs as described), have been produced and their immunogenicity have been evaluated in animal models. Results from these studies showed that these structures elicited robust immune responses that can protect against adenovirus infection.
The strategy and methods described in this submission are applicable for the assembly and production of AdVLPs for any member of the Adenoviridae family as well as for viral families exhibiting capsid structures with essentially identical design to that seen with adenovirus such as bacteriophages PRD1 (family Tectiviridae), PM2 (family Corticoviridae), PBCV-1 (family Phycodnaviridae) as well as members of the Iridoviridae family.
The immune response elicited in small animal models was tested following the administration of monovalent AdVLP as vaccine compositions (e.g., containing AdVLP4, AdVLP7, AdVLP14 or AdVLP55), as well as polyvalent formulations combining different monovalent AdVLPs, (e.g., a trivalent composition of AdVLP4, AdVLP7 and AdVLP14) or a tetravalent composition including of AdVLP4, AdVLP7, AdVLP14 and AdVLP55. These studies demonstrate that the monovalent vaccine compositions stimulated the production of high titers of specific serum total IgG antibodies as measured by ELISA. Either, AdVLP vaccine alone or when formulated with an adjuvant, stimulated production of significant levels of specific IgG as compared to the pre-immune control. A representative example of an ELISA using adenovirus 4 (AdV4) as antigen to evaluate the immune response elicited by the monovalent AdVLP4 is shown in
Furthermore, the specific neutralizing activity of the serum samples obtained from animals immunized with the monovalent and polyvalent AdVLP vaccine compositions were tested utilizing recombinant reporter adenovirus (rrAdV) which, following cell infection, express the reporter proteins luciferase and green fluorescent proteins (Luc/GFP). Detection of the levels of expression of these reporter genes is used to assess the power of the serum to prevent/neutralize viral infection. Interestingly, these vaccine compositions elicited high titers of neutralizing antibodies as compared to the response elicited by homologous inactivated adenovirus vaccine controls. An example of the neutralizing activity elicited against rrAdV4/Luc virus detected in serum samples from mice immunized with the monovalent AdVLP4 alone or admixed with an adjuvant as well as the inactivated adenovirus 4 (AdV4) control is shown in
Both monovalent AdVLP4 vaccine formulations elicited strong neutralization activity with an average titer of about −750 ID50, which were greater than that elicited by the inactivated AdV4 virus immunized control. This difference, however, was not statistically significant. These results demonstrate that the AdVLP vaccine formulated either adjuvant or with buffer alone is able to elicit a potent neutralizing antibody response,
The other monovalent compositions AdVLP7, AdVLP14 and AdVLP55 as well as a quadrivalent formulation that included AdVLP4, 7, 14 and 55 were also able to stimulate strong and specific serum IgG responses as well as robust neutralizing antibody responses as compared to naïve controls or live adenovirus vaccinated controls. An example of the IgG responses elicited by monovalent vaccines and a quadrivalent formulation is shown in
In summary, the adenovirus-like particles (AdVLPs) assembled and produced using the methods described above stimulate robust and specific immune responses when administered as monovalent or polyvalent vaccine compositions. These vaccines can be delivered as a liquid formulation via the parenteral route; however, alternative formulation, (e.g., powder, gels, pills, etc.), and ways of delivery (e.g., epidermal, oral, intranasal, etc.) can also be utilized.
In the following examples, in accordance with one or more embodiments, a set of proteins are defined that are used for the self-assembly of AdV-7 VLPs (AdVLP-7) in a mammalian expression system, and it is shown that these AdVLPs are comparable to wild type AdV-7 virus particles. These examples demonstrate that recombinant AdV capsid assembly can be driven by the expression of plasmid-encoded structural proteins. Immunogenicity studies were performed in mice, revealing that AdVLP-7 induces a potent humoral response against AdV-7.
HEK-293 cells (Gibco, Waltham, MA, USA) were grown in suspension culture in EX-CELL® CD HEK293 Viral Vector medium (Sigma Aldrich, St. Louis, MO, USA, 14385C) supplemented with 5 mM L-glutamine. A549 cells (ATCC, Manassas, VA, USA, CCL-185) were grown as adherent monolayers in Ham's F-12K (Kaighn's) medium (Gibco, 21127022) supplemented with 10% heat-inactivated FBS and 1× penicillin/streptomycin (100 U penicillin and 100 μg streptomycin per mL). All cultures were incubated in a humidified incubator at 37° C. with 5% CO2. HEK-293 cells were incubated while shaking at 125 rpm.
Wild type AdV-7 was obtained from ATCC (strain Gomen, ATCC, VR-7). A replication-competent reporter AdV-7 (rAdV-7) with a deleted E3 region replaced with GFP, generated as previously described70, was used for neutralization assays. Prior to use, wild type AdV-7 was passaged in HEK-293 cells, and rAdV-7 was passaged in A549 cells.
The genes encoding the major capsid proteins (hexon, penton, and fiber), minor capsid/cement proteins (VIII, VI, IX, and IIIa), and accessory proteins L4-100k and L1-52/55k were codon optimized, chemically synthesized, and individually cloned into cloning vectors by Blue Heron Biotech (Bothell, WA, USA). Genes were subcloned from cloning vectors into the expression plasmid pcDNA3.4 by restriction enzyme digestion and ligation. Genes were consolidated into four plasmids, as follows: i. pcDNA3.4-hexon-IRES-100k (pHexon-100k), ii. pcDNA3.4-penton-IRES-fiber (pPenton-Fiber), iii. pcDNA3.4-CMV-VIII-IRES-VI-CMV-IX-IRES-IIIa (pVIII-VI-IX-IIIa), and iv. pcDNA3.4-52/55k (p52/55k). Constructs were verified by restriction enzyme digestion and sequencing.
AdVLPs were produced by transfecting HEK-293 cells with the four plasmids listed above (i. pHexon-100k, ii. pPenton-Fiber, iii. pVIII-VI-IX-IIIa, and iv. p52/55k, in a 2:1:1:1 ratio). Cells were seeded at 1.0×106 cells/mL one day prior to transfection. Transfection was conducted using PEI Max® (Polysciences, Warrington, PA, USA, 24765). PEI Max® was mixed with DNA in a 4:1 ratio (PEI:total DNA) in a volume of EX-CELL® CD HEK293 Viral Vector medium equal to 5% of the total culture volume. Mixtures were incubated for 15 minutes at room temperature to allow PEI/DNA complexation, and subsequently added to cells dropwise. Valproic acid was added to cells 24 hours after transfection at a final concentration of 3.75 mM. At 72 hours post transfection, transfected cultures containing intracellular AdVLPs were supplemented with 50 mM NaCl, 1 mM MgCl2, and 1× Halt™ protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA, 78440).
Cells containing AdVs or AdVLPs were subjected to three freeze-thaw cycles to lyse cells. Cell lysates were centrifuged at 10,000×g for 20 minutes at 4° C., and pellets of cellular debris were discarded. For AdVLPs, supernatants were concentrated 10× by tangential flow filtration using a Pellicon® XL50 with Biomax® 300 kDa membrane (Millipore Sigma, Burlington, MA, USA, PXB300C50). For both AdVs and AdVLPs, supernatants were loaded onto a two-step cesium chloride (CsCl) gradient (1.41 g/mL and 1.26 g/mL) in ultracentrifuge tubes and separated by ultracentrifugation using an SW28 rotor for two hours at 10° C. AdV samples are separated into two distinct bands, the lower (heavier) of which contains mature, infectious particles with packaged genomic DNA (referred to as WT AdV-7), while the higher (lighter) band consist of empty capsids which do not contain DNA (referred to as empty capsids). AdVLP samples contain only a single band. Each tube was punctured using an 18-gauge needle and separate fractions were collected for each band of interest. Fractions were diluted with 1.3 g/mL CsCl to a total volume of 11.5 mL. Samples were ultracentrifuged using an SW40ti rotor for 16 hours at 10° C. Bands of interest were again collected by puncturing the sidewall of the tube and fractions were stored in CsCl with 2 mM MgCl2 at 4° C. until just prior to use. Immediately before use, samples were buffer exchanged using Amicon® Ultra centrifugal filter units with 100 kDa NMWCO (Millipore Sigma, UFC9100) into VLP suspension buffer (PBS with 187 mM NaCl, 2 mM MgCl2, 6 μM Tween-80, and 0.1 mM EDTA).
Protein composition of purified AdVLP-7 and AdV-7 preparations was assessed using SDS-PAGE. Purified samples were mixed with lithium dodecyl sulfate sample buffer with β-mercaptoethanol and incubated at 85° C. for 5 minutes. Samples were separated by SDS-PAGE using 4-12% Bis-Tris gels (Invitrogen, Waltham, MA, USA). For visualization of total protein, gels were stained with Coomassie brilliant blue R-250 for 24 hours and de-stained with destaining solution (40% methanol, 10% glacial acetic acid) for 1 hour (destaining solution replaced with fresh solution every 15 minutes). For western blots, separated proteins were transferred to nitrocellulose membranes. Membranes were then blocked with 5% non-fat dry milk in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20). Membranes were incubated overnight with one of the following antibodies, as indicated: i. goat anti-adenovirus 5 antibody (1:1,000 dilution, Novus Biologicals, Centennial, CO, USA, NB600-1386), ii. anti-IIIa, iii. anti-AdV-14, iv. anti-VII, v. anti-IX, or vi. anti-L1-52/55k (ii.-vi. used at 1:200 dilution, generated in house via immunization of rabbits with the respective His-tag purified proteins). Membranes were washed 3× with TBS-T for 10 minutes. HRP-conjugated rabbit anti-goat IgG (1:6,000 dilution, Abcam, Cambridge, UK, ab97100) or goat anti-rabbit IgG (1:6,000 dilution, Invitrogen, 65-6120) was added to membranes and incubated for 1 hour. Membranes were again washed 3× with TBS-T for 10 minutes and subsequently developed using the SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific, 34580). Coomassie-stained gels and developed western blots were imaged using an Azure Biosystems C600 Imaging System. Protein quantification was performed via densitometry analysis of western blots using AzureSpot software (Azure Biosystems, Dublin, CA, USA). Serial dilutions of purified hexon protein were used as standards.
Particle size of purified samples was determined by dynamic light scattering using a Litesizer 500 (Anton Paar, Graz, Austria). Purified samples were diluted 20× in PBS and loaded into a cuvette. Measurements were performed using default settings for protein samples in PBS. The hydrodynamic diameter of each sample was measured in series of at least 5 replicates.
CF200-CU carbon film 200 mesh copper grids (Electron Microscopy Sciences, Hartfield, PA, USA) were held with forceps and washed with 10 μL of 0.01% BSA solution. After a 5 second incubation, grids were dried using filter paper to draw liquid off from the edge. Samples of AdVLP-7 (5 μL) were immediately loaded onto grids and allowed to incubate at room temperature for 5 minutes. After incubation, grids were again dried with filter paper. Grids were then immediately stained with 2% phosphotungstic acid and incubated for 1 minute at room temperature. After incubation, grids were dried a final time with filter paper, and further air-dried overnight. Grids were examined using a JOEL 2100 transmission electron microscope at 200 kV and imaged with a 2048×2048-pixel CCD (Gatan Inc, Pleasanton, CA, USA).
Immunogenicity of AdVLP-7 vaccines was tested in 6-8 weeks old BALB/c mice purchased from Charles River Laboratories (Wilmington, MA, USA). AdVLP-7 vaccines were given to groups of mice, adjuvanted with either aluminum hydroxide (Alum) or AddaVax (InvivoGen, San Diego, CA, USA), with a third group receiving AdVLP-7 formulated without adjuvant. For control, a fourth group was administered wild type AdV-7 without adjuvant. Finally, a fifth group received only a sham injection with VLP suspension buffer. Mice were immunized with primary and booster doses of vaccines or sham, administered via intramuscular injection into a hind leg on days 0 and 14, respectively. Each dose of AdVLP-7 or AdV-7 contained 4 μg of hexon protein, determined by densitometry analysis of western blots. Serum samples were collected three weeks after the booster immunization (day 35). All sera were heat-inactivated by heating at 56° C. for 30 minutes and subsequently stored at −80° C.
Antibody titers were determined for all serum samples via ELISA. Total IgG titers against each of the major capsid proteins were measured individually, in addition to titers against total virion. IgG subclass analysis was also performed against purified total virion. Purified major capsid proteins (hexon, penton, or fiber) or purified AdV-7 particles were diluted to a concentration of 0.5 μg/mL in coating buffer (0.1 M sodium bicarbonate, pH 9.6) and 100 μL was added to each well of a 96-well plate. Plates were incubated overnight at 4° C. Following overnight incubation, plates were blocked with 1% BSA in PBS for 1 hour at room temperature and subsequently washed with 0.05% Tween 20 in TBS. Serum samples (n=10 per group) were added to plates, serially diluted 3-fold in blocking buffer, and incubated for 2 hours at room temperature. Plates were then washed and incubated with one of the following HRP-conjugated secondary antibodies: for quantification of total IgG, goat anti-mouse IgG (1:10,000 dilution, IgG heavy and light chain, Invitrogen, 31430); for IgG subclass analysis, either goat anti-mouse IgG1 (1:4,000 dilution, IgG1 heavy chain, Southern Biotech, 1071-05) or goat anti-mouse IgG2a (1:4,000 dilution, IgG2a heavy chain, Southern Biotech, 1081-05). Secondary antibodies were diluted in blocking buffer and added to plates for 1 hour at room temperature. Plates were washed a final time and developed with TMB substrate (Thermo Scientific, 34029) for 15 minutes at room temperature. The reaction was stopped by adding 2 M sulfuric acid. The absorbance at 450 nm was determined for each well. For each serum sample, absorbance was plotted against the dilution factor. Binding titers, defined as the serum dilution at which absorbance readings were at 50% of their maximum value, were calculated using a sigmoidal 4-paramter logistic regression. Serum samples that showed binding titers that fell below the lower limit of detection were assigned a value equal to half of the starting serum dilution to enable calculation and statistical analyses.
Neutralizing antibody titers in sera were assessed using a recombinant reporter AdV-based microneutralization assay. Heat-inactivated sera (n=10 per group) were 2.5-fold serially diluted in 96-well plates in Ham's F-12K (Kaighn's) medium with 5% FBS and 1× penicillin/streptomycin. Serum dilutions were mixed with an equal volume of GFP-expressing rAdV-7 containing ˜500 FFU, for a total mixture volume of 100 μL. Each serum sample was run in triplicate, with a starting dilution of 1/40 (indicative of the serum dilution after mixing with rAdV-7). Serum/virus mixtures were incubated for 1 hour at 37° C., at which point 2.25×104 A549 cells in 100 μL F-12K were added to each well and mixed. The following controls were included: i. cells exposed only to the rAdV-7 (no serum); and ii. cells unexposed to either serum or rAdV-7 (background fluorescence control). Plates were incubated at 37° C. with 5% CO2 for 28 hours. Following incubation, plates were imaged using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA, USA). The number of green fluorescent cells in each well was measured and plotted against the serum dilution factor. Plots were fitted with a non-linear regression, which was used to calculate the serum dilution at which 50% of the rAdV-7 was neutralized relative to control wells that contained cells infected with rAdV-7 unexposed to serum (ID50). Neutralizing antibody titers that fell below the lower limit of detection were assigned a value of 20, equal to half the starting serum dilution factor, to allow for statistical analysis.
For comparison of results of ELISA and microneutralization assays between groups, data were analyzed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. Correlation analyses were performed using Pearson's correlation analysis. For all analyses, alpha=0.05. All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).
The minimum set of proteins needed for formation of stable AdVLP-7 particles was identified, which includes the major capsid proteins hexon, penton, and fiber, minor capsid/cement proteins IIIa, VI, VIII, and IX, the chaperone protein L4-100k, and the accessory scaffold protein L1-52/55k. To produce AdVLP-7, four expression plasmids encoding the required viral genes were introduced into HEK-293 cells by transient transfection. Particles were harvested by repeated freeze/thaw cycles of transfected cells, and purified by cesium chloride gradient ultracentrifugation. Purified particles were examined by negative staining electron microscopy, which revealed that the structure of AdVLP-7 mimics the typical icosahedral morphology of wild type AdVs (
Protein compositional analysis of AdVLP-7 was performed by SDS-PAGE with subsequent Coomassie blue staining (
To determine the immunogenicity of these recombinant capsids, BALB/c mice were immunized with purified AdVLP-7, administered alone or in combination with an adjuvant (aluminum hydroxide or AddaVax™, a squalene-based oil-in-water nano-emulsion). To examine how AdVLP-7 compares to the wild type virus, a separate group of mice were immunized with WT AdV-7. As a control, an additional group of mice received only a sham injection of buffer. Mice were given the initial dose and then boosted two weeks later. Three weeks after the final immunization, serum samples were collected and AdV-7-specific antibody titers were measured via ELISA. All vaccinated mice generated higher titers of IgG antibodies against AdV-7 as compared to sham-injected mice, which did not show any binding activity against AdV-7 (
To better understand the effects of vaccination with AdVLP-7 on the cellular immune response, the titers of both IgG1 and IgG2a were determined, as these are markers of Th2- and Th1-skewed responses, respectively. AdVLP-7-immunized mice generated high titers of both IgG1 and IgG2a against AdV-7, regardless of adjuvant formulation (
While the cellular immune response is critical to the resolution of AdV infection45,46, the hallmark of protection against infection is neutralizing antibodies (NAbs). Major capsid proteins hexon, penton, and fiber are the predominant targets of NAbs generated during AdV infection38-41. The IgG titers against each major capsid protein were determined for all animals via ELISA. Immunization with AdVLP-7, alone or adjuvanted, resulted in significant IgG titers against all major capsid proteins (
Given the high binding titers observed against the major capsid proteins, the functionality of the antibody response against AdV-7 in immunized mice was also assessed. Neutralizing activity was determined using a microneutralization assay based on a reporter AdV-7 that expresses GFP (rAdV-7). Incubation of rAdV-7 with serum from vaccinated mice led to a reduction in the number of infected cells as compared to the sham-injected group, which did not show neutralizing activity (representative images,
As exemplified by the above Examples, development of an alternative to the live virus vaccines against Adenoviruses, such as AdV-4 and AdV-7, is critical for combating adenoviral infection not only in military populations but also in the general public. The existing live virus vaccines against AdV-4 and AdV-7 have proven to be very effective with >93% seroconversion rate in vaccinated individuals, and a vaccine efficacy of 99% against infection24. However, live AdVs have the ability to undergo recombination which can produce new subtypes26,27, and therefore pose a significant safety hazard. Considering the potential for recombination and vaccine-associated viral shedding that can persist for nearly a month after vaccination, the existing vaccines cannot be safely administered to the general public. While AdV infections are typically mild, more severe infections can occur, especially in susceptible populations including children, the immunosuppressed, and during pregnancy17,47-51. The clinical manifestations of AdV infections are broad, but can include gastroenteritis, hepatitis, and pneumonia among many others, and can be fatal in both immunocompromised and immunocompetent patients3,11,52. Given the significant risks and limitations associated with the current live virus vaccines, and considerable threat posed by AdV infections, better approaches to vaccination need to be developed.
One such approach is through the use of the virus-like particle platform of the present application. AdVLPs are structural mimics of native viruses, enabling antigens to be presented to the immune system in the same conformation as the live virus vaccines. However, AdVLPs are non-replicating as they lack genomic material, and therefore present no risk of recombination or vaccine-associated shedding. While VLP-based vaccines have been shown to be safe and effective in humans, only a few have been successfully developed and brought to market30-34. The challenge in developing VLP vaccines is determining the correct conditions and composition that enable the formation of stable particles. As exemplified in the examples, the necessary components for the generation of AdVLPs are defined, and demonstrate the first construction of a recombinant capsid that mimics both the size and icosahedral structure of wild type AdVs.
For example, it was determined that formation of stable AdVLPs requires the expression of the major and minor capsid proteins, the chaperone protein L4-100k which is required for proper folding of hexon, and the accessory protein L1-52/55k. While L1-52/55k is primarily involved in genome packaging53 and is not present in mature WT AdV-7 virions44, for instance, it was found that AdVLPs that lack this protein are not stable for long durations. Indeed, AdVLPs formed without the minor/cementing proteins and the accessory protein L1-52/55k become unstable and are unsuitable.
Not included in AdVLP-7, for example, was the adenovirus protease (AVP), which is important for the final proteolytic processing of several proteins within assembled capsids, some of which are components of AdVLP-7 (IIIa, VI, VIII, and L1-52/55k)54-57. Cleavage of these proteins primarily functions in rendering the virus infectious, as it allows capsid uncoating to occur upon cell entry58,59.
Additionally, AVP requires multiple cofactors, including viral DNA60-62, for proper activation and therefore would not function correctly in VLPs which lack genomic material and are non-infectious in nature. The lack of AVP was apparent in western blot analyses, which showed that IIIa, VI, VIII, and L1-52/55k exist in their uncleaved conformations in AdVLP-7, but are processed or absent in the case of L1-52/55k in mature WT AdV-7, as expected. Differences in banding patterns between AdVLP-7 and WT AdV-7 are consistent with previous studies regarding precursor proteins and the AVP55,56,63-65. Even without AVP, AdVLP-7 is stable for more than 40 weeks when stored at refrigeration temperatures.
In addition to defining the components needed for generation of stable AdVLPs, it was also found that these recombinant capsids are highly immunogenic. As exemplified in the above examples, AdVLP-7 elicited a robust humoral response in mice, resulting in high titers of antibodies that bind to each of the major capsid proteins and potently neutralize AdV-7. Importantly, the observed response was equivalent between the males and females of each individual group. AdVLP-7 not only mimics the size and structure of WT AdV-7, the recombinant capsids also induce an equal antibody response. When administered without an adjuvant, AdVLP-7 elicited nearly identical binding and neutralizing antibody titers to those observed in mice immunized with an equivalent dose of WT AdV-7.
Additionally, the immunogenicity of AdVLP-7 was significantly increased when adjuvanted with either alum or AddaVax, though both adjuvants elicited similar levels of total IgG and NAbs. This trend between the different vaccine groups was observed for all measures of total IgG titers (against hexon, penton, fiber, and total AdV-7), as well as NAb titers. Furthermore, there were significant correlations between all datasets, most notably between the total IgG and NAb titers. In general, mice that showed higher binding titers against AdV-7 and its major capsid proteins also tended to show higher neutralizing activity. Binding titers against hexon, the primary target of neutralizing antibodies38-40, expectedly correlated most strongly with NAb titers. These results highlight the overall consistency of the AdVLP vaccine strategy and the striking similarity between recombinant and wild type AdV capsids.
Neutralizing antibody titers are used as the benchmark for protection against AdV infection after vaccination22,24, with less of an emphasis placed on the cellular immune response. However, the development of a strong T cell response is critical for controlling AdV infections66. Both clinical and in vitro experiments have highlighted the role of AdV-specific T cells in immunity against infection and prevention of severe disease67. In stem cell transplant recipients, the reconstitution of hexon-specific CD4+ and CD8+ T cells resulted in spontaneous resolution of disseminated AdV infection45. To elucidate the effects of AdVLP-7 immunization on the T cell response, IgG1 and IgG2a titers were determined, as they are markers of the Th2 and Th1 response, respectively. The results showed that immunization with AdVLP-7 adjuvanted with alum results in a Th2-skewed response, as indicated by the high levels of IgG1 production and relatively low IgG2a titers. A more balanced response was induced when AdVLP-7 was administered either alone or adjuvanted with AddaVax. The administration of AdVLP-7 with AddaVax resulted in significantly higher titers of IgG2a antibodies than other AdVLP-7 formulations, comparable to those generated in mice immunized with WT AdV-7. However, the ratio of IgG1 to IgG2a was much lower in the WT AdV-7 group, indicative of a Th1 polarization. While this IgG subclass analysis presents a preliminary assessment, additional studies are needed to further understand the cellular response elicited by AdVLP-based vaccines.
Overall, these examples highlight the ability of AdVLPs to serve as a platform for the next generation of vaccines against human AdVs. There are several directions that can be explored to continue the development of the AdVLP platform. As shown and described in the above examples and throughout the present application, in accordance with one or more embodiments herein, a template for AdVLP generation has been developed.
Exemplary amino acid and nucleotide sequences of various proteins of the AdVLPs of the present application are shown below in accordance with or more embodiments.
In accordance with one or more embodiments of the present application, exemplary AdVLPs, expression plasmids, compositions, and methods are set out in the following items:
Item 1. An adenovirus virus-like particle (AdVLP) comprising:
All publications, patents, and patent documents are incorporated by reference herein in their respective entireties, as though individually incorporated by reference. This statement of incorporation by reference is intended by applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. No limitations inconsistent with this disclosure are to be understood therefrom.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims. Such equivalents are intended to be encompassed by the following claims.
This application is a National Stage of International Application No. PCT/US2023/064176, filed Mar. 10, 2023, which claims priority from U.S. Provisional Application No. 63/318,742, filed Mar. 10, 2022. The International Application was published on Sep. 14, 2023 as International Publication No. WO 2023/173114 A2. The entire disclosures of each of the above identified applications are incorporated herein by reference in their entireties.
This invention was made with government support under contract number W81XWH-19-C-01 awarded by the U.S. Army Medical Research and Development Command/Military Infectious Diseases Research Program/Walter Reed Army Institute of Research. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/064176 | 3/10/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63318742 | Mar 2022 | US |
