Vaccines formed by virus and antigen conjugation

Abstract

To provide methods of forming compounds and exemplary stable compounds in the nature of a conjugated compound at refrigerated or room temperature.SOLUTION: The method comprises mixing an antigen and virus particles in a conjugation reaction to form a conjugate mixture, such that the conditions and steps of forming these products allow for use of the conjugate mixture as a vaccine, including but not limited to use as a vaccine against various pathogens including for treatment of diseases caused by novel coronaviruses (including SARS-COV2).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This international patent application claims the benefit of and priority to nonprovisional U.S. patent application Ser. No. 17 / 186,941, filed February 26, 2021, which is a continuation-in-part of, and claims the benefit of and priority to, nonprovisional U.S. patent application Ser. No. 16 / 919,943, filed July 2, 2020, which also claims the benefit of and priority to nonprovisional U.S. patent application Ser. No. 16 / 709,063, filed December 10, 2019, which also claims the benefit of and priority to nonprovisional U.S. patent application Ser. No. 16 / 709,063, filed June 11, 2019. This international patent application claims the benefit of and priority to non-provisional U.S. patent application Ser. No. 16 / 437,734, which in turn claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 683,865, filed June 12, 2018, and this international patent application also claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 047629, filed July 2, 2020, and U.S. Provisional Patent Application No. 63 / 013284, filed April 21, 2020, the teachings and entire disclosures of all of which are incorporated herein by reference.

[0002] (Reference to sequence listing) This application contains a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference. The text file of the Sequence Listing submitted upon filing includes a computer readable format file "SequenceListings, PCT_ST25", which was created on March 5, 2021, and is 10,826 bytes in size.

[0003] (Technical field) Embodiments described herein include the use of the multi-set process to produce highly purified recombinant viruses as antigen carriers, and still further embodiments relate to the production of vaccines using purified viruses and purified antigens, including vaccines aimed at preventing novel coronavirus, e.g., SARS-CoV2 (referred to herein as Covid-19 or SARS-2) disease. [Background technology]

[0004] Viruses carry nucleic acid molecules within a protein coat and replicate only inside the living cells of other organisms. Often considered harmful, a wide range of viruses are capable of infecting all kinds of life forms (e.g., humans, livestock, and plants). However, they are not without positive aspects, and there is growing interest in using viruses for a range of therapeutic purposes, including, but not limited to, the creation of vaccines, gene therapy, and cancer treatment, to name a few. However, to study viruses, understand their structure, and adapt them for use as molecular tools, disease treatment vectors, and carriers, the viruses must first be purified to remove any cellular debris, polymeric fibers, organelles, lipids, and other impurities that may interfere with the intended function of the virus.

[0005] Once purified, the virus is suitable for several uses. Of importance in this disclosure is the conventional idea of ​​using viruses (considered pathogens in this context) for research and development of genetic strategies against viruses. However, the use of purified viruses as antigen carriers to prepare vaccines is also discussed at some length in this disclosure. An antigen is a molecule that, when properly delivered to an organism, can generate an immune response in that organism by stimulating antibody production through binding with antibodies in the organism that match the antigen's molecular structure. To name a few, recombinant antigens are produced from recombinant DNA that is cloned into vectors by known techniques and then introduced into specific host cells (e.g. bacteria, mammalian cells, yeast cells and plant cells). The recombinant antigen is then expressed using the translational machinery in the host cell. After expression, the recombinant antigen is retrieved and attached to the virus via a covalent bond by a process known as conjugation. After conjugation of the antigen to the virus, the virus can function as a carrier to deliver the antigen to the organism and activate the immune system's response. In this way, the virus-antigen conjugate can provide therapeutic use. Proper virus-antigen conjugation is necessary for the antigen to activate an antibody-producing immune response in the host cells of the source organism. Purification of the virus and antigen is fundamental to this proper conjugation.

[0006] Current methods of purifying viruses are generally limited to small biochemical quantities, e.g., on the order of nanograms to milligrams, and have not been demonstrated in industrial quantities on the order of grams to kilograms. For example, previous methods known as "crude infected cell lysates" utilize crude cell lysates or cell culture media derived from virus-infected cells. Infected mammalian cells are lysed by freeze-thawing or other known methods, debris is removed by low-speed centrifugation, and the supernatant is then used for experiments. Infected raw organisms are physically disrupted or ground, and the resulting extract is clarified using centrifugation or filtration to obtain a crude virus preparation. However, this method suffers from a high degree of contamination with many non-viral factors that impact the ability to perform experiments and manipulate the virus.

[0007] A second example of a traditional purification step is high speed ultracentrifugation, whereby the virus is pelleted or further purified through pelleting through a low density sucrose solution or suspended between sucrose solutions of various densities. Limitations of this method include low yields of purified virus due to limited size and scalability of high speed separations, and reduced virus purity due to the presence of other host proteins that are often co-purified with the virus sample.

[0008] A third method used to enhance conventional virus purity is density gradient ultracentrifugation. This method uses gradients of cesium chloride, sucrose, iodixanol or other solutions to separate assembled virus particles or to remove gene-free particles. The limitations of this method include the time required for virus purification (often 2-3 days), the limited number of samples, the amount of samples that can be analyzed at one time (typically 6 per rotor), and the small amount of virus that can be purified (typically micrograms to milligrams of final product).

[0009] Organic extraction and polyethylene glycol precipitation have also been used to purify viruses, such as plant-derived viruses, for example by removing lipids and chloroplasts. However, these known methods also suffer from low purity, as the product typically still remains attached to host proteins, nucleic acids, lipids and sugars, resulting in significant aggregation of the resulting viral product. These limitations impair the usefulness of the final product for compliance with Current Good Manufacturing Practice (cGMP) regulations enforced by the U.S. Food and Drug Administration (FDA).

[0010] Current cGMP regulations promulgated by the FDA include minimum requirements for the methods, facilities and regulations used in the manufacture, processing and packing of drug products. These regulations aim at the safety of the product and ensure that it has the ingredients and benefits it claims to have. Thus, for viruses used in vaccine production, gene therapy, cancer therapy and other clinical settings, the final virus product must comply with cGMP regulations. If the final virus product does not comply with cGMP regulations, its usefulness for use in clinical settings, such as products obtained from polyethylene glycol precipitation, will be nonexistent or greatly reduced.

[0011] Scalability refers to a process that consistently and reproducibly produces the same product as the amount of product increases, for example, from laboratory scale (<0.1 square meters) to a system of at least >20 square meters. As listed above, all previously used methods are problematic due to inconsistencies, low scalability (i.e., making products only in biochemical quantities) and non-compliance with cGMP regulations.

[0012] Although plant-based production has been of interest for large-scale production, there are also significant limitations in their use. Plant-based production systems can obtain industrial-scale yields at much less cost than animal cell production systems (e.g., Chinese Hamster Ovary (CHO)). However, certain conventional purification methods (suitable at some scales for non-plant viruses) do not work for plant-produced viruses and antigens. These limitations arise because purifying plant viruses is very different as opposed to purifying viruses from animal cell cultures. While animal cells produce primarily protein and nucleic acid impurities, plants are also a source of important and additional impurities not found in animal cells. Some of these include the lipid composition of chloroplast membranes and vacuolar membranes, simple and complex carbohydrate impurities, and nanoparticulate cell organelle impurities. In fact, crude plant extracts often foul the equipment used in the processing and purification of virus and antigenic material obtained from plants (e.g., by accumulation of impurities on the separation membrane or medium bed leading of the equipment). Such fouling inevitably results in pressure flow failure, poor filtration, and ultimately low yields of product. Another problem is that these impurities have a tendency to aggregate and can be co-purified with any protein, virus or other desired "product" from the plant. Thus, current methods for purifying viruses do not adequately remove all or even sufficient amounts of impurities, including but not limited to those present in plant extracts, nor have they been shown to produce adequately purified viruses.

[0013] Little progress has been made with regard to virus and antigen purification platforms capable of consistently producing highly purified viruses at commercial scale, i.e., grams to kilograms or more, and in compliance with cGMP regulations. Such improvements would enable clinical development for tools used in vaccine generation, gene therapy, and for cancer treatment. The platform described herein, according to multiple embodiments and alternatives, together with other features and advantages outlined herein, meets this and other needs.

[0014] To date, seven coronaviruses (CoVs) have been identified that can cause human infections, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and a newly identified CoV (SARS-CoV2, 2019-nCoV or Covid-19). In human populations, these three viruses pose significant public health risks and have high mortality rates: SARS-CoV: 10%, MERS-CoV: 34.4% and SARS-CoV2: 6.1%. According to the Johns Hopkins Coronavirus Resource Center, Covid-19 has now spread to more than 188 countries, infected more than 10,000,000 people and caused more than 500,000 deaths worldwide. These numbers are growing exponentially, and its spread has put global health and economics at risk. The emergence of Covid-19 and its impact on human health and the economy calls for an urgent response, especially a vaccine to prevent Covid-19 disease. Despite global efforts to find a vaccine to control or slow infections, there are currently no approved antiviral treatments that target human coronaviruses, with supportive and palliative care remaining the primary treatment.

[0015] The development of effective SARS-CoV and MERS-CoV vaccines has also been somewhat successful. Many conventional vaccine strategies have been utilized, including inactivated viruses expressed from DNA plasmids or RNA delivery systems, recombinant attenuated viruses, other live viral vectors, subunit vaccines or individual viral proteins. Conventional SARS vaccines have focused primarily on the spike (S) protein due to its functions in human receptor binding, membrane fusion and viral entry. Furthermore, the S protein is the primary antigen of coronaviruses and the binding site for protective neutralizing antibodies that block viral receptor binding and initiation of infection. Although inactivated SARS-CoV preparations obtained using the full-length S protein have elicited neutralizing antibodies in immunized animals, these conventional vaccines have proven to be ineffective in humans and raise important safety concerns (e.g., by actually enhancing viral infection in many systems). Similarly, many different SARS and MERS vaccines have been developed and tested, while none have shown protection from infection through virus neutralization without the immunopathology associated with full-length or trimerized S protein vaccines.

[0016] It is understood that various viral vectors are known as carriers for a wide range of antigens and provide effective therapeutic delivery to host organisms, such as mammals, such as humans. However, such viral vectors tend to vary with respect to the specific antigen delivered and the immune response elicited by another vector. For example, some viral vectors, such as ERVEBO® Ebola Zaire Vaccine, are live viruses that stimulate a response by the host's immune system. However, the response from the viral vector itself to the antigen delivered by the viral vector often blunts the intended immune response, for example by showing immune dominance of one antigen, which prevents the response upon subsequent administration of the same or similar vaccine. Advantageously, it has been found that the antigen of the present disclosure can be conjugated to the TMV NtK vector without stimulating subsequent host immune responses, without showing immune dominance of one antigen, and without affecting subsequent doses of subsequent vaccines using the same viral vector. Moreover, avoiding such immune responses generated by the viral vector itself provides an additional advantage to bivalent, trivalent and tetravalent vaccines since the response for each antigen can be assessed without considering the effect of the viral vector on current and future vaccine administrations. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Pat. No. 7,939,318 Summary of the Invention [Problem to be solved by the invention]

[0018] Thus, there is a significant, urgent and global need for an effective and scalable vaccine strategy for the prevention of Covid-19 disease that elicits high levels of neutralizing antibodies and induces persistent and strong neutralizing antibody titers over a long period of time following immunization. The platform described herein according to multiple embodiments and alternatives, together with other features and advantages outlined herein, fulfills this and other needs. In this regard, the vaccine according to the present embodiments has shown strong immune responses in preclinical trials and has a key advantage over conventional monovalent vaccines, being capable of conjugating to multiple CoV antigens at once (i.e., multivalent vaccines), eliciting high levels of neutralizing antibodies and potentially inducing persistent and strong neutralizing antibody titers over a long period of time following immunization. [Means for solving the problem]

[0019] In some embodiments according to the present disclosure, the virus purification method is directed to a multi-set process including recovering virus material from a source organism containing at least one virus, removing cellular debris from the at least one virus to thereby reveal the structure of the at least one virus, performing concentration of the separated and revealed virus in a filtration device with a membrane having a pore size not exceeding a predetermined limit selected by a user in some embodiments, and processing the concentrated virus by subjecting it to a series of separation steps and recovering the virus after each separation step, where at least one separation step includes ion exchange chromatography to separate host cell contaminants from the virus, and at least one separation step includes multi-mode chromatography to separate remaining impurities from the virus based on at least the size difference between the virus and the impurities and the chemical interaction occurring between the impurities and one or more chromatography ligands. In some embodiments, plants are the source organisms for recombinant expression of viruses, as non-limiting examples Nicotiana benthamiana and Lemna minor. When the source organism is a plant, recovery may include seed formation and plant germination to induce transient gene expression for expression of the desired protein, as described below. Alternatively, the source organism for recombinant expression of the virus may be a non-plant host, such as, but not limited to, a bacterium, algae, yeast, insect, or mammalian organism.

[0020] Additionally, various aspects of the embodiments described herein relate to producing and / or purifying antigens that can be conjugated to viral particles. In this embodiment and its alternatives, the viral particles include one, some, or all of the viruses and / or fragments thereof, such as, but not limited to, rod-shaped viruses, icosahedral viruses, enveloped viruses, and one or more fragments thereof. In some embodiments, the source organism for recombinant expression of the antigen is a plant, or the source organism for recombinant expression of the antigen is a non-plant host, such as, but not limited to, bacteria, algae, yeast, insects, or mammals.

[0021] Advantageously, the multi-set process carried out according to various embodiments described herein produces highly purified viruses or recombinant antigens or both on a commercial scale. Various steps are used to improve upstream purification processes, such as enriching plant viruses. In some embodiments, size exclusion chromatography and other features are used to produce purified recombinant viruses and recombinant antigens. Thus, various embodiments described herein provide one or more viruses and one or more antigens suitable for preparing one or more vaccines of conjugated viruses and antigens.

[0022] With respect to viruses, implementation of some embodiments of the inventive virus purification platform described herein has enabled purification of rod-shaped plant viruses (e.g., Tobacco Mosaic Virus (i.e., "TMV") and icosahedral plant viruses (e.g., Red Clover Mosaic Virus). Several embodiments herein have enabled purification of TMV and Red Clover Mosaic Virus, which represent two structurally distinct viruses with respect to size and structure. For example, small icosahedral viruses such as Red Clover Mosaic Virus have T=3 bilateral symmetry, a size of approximately 31-34 nm, and approximately 180 capsid proteins. In contrast, TMV contains 2160 capsid proteins, with the TMV virion being a rigid rod-shaped particle that is approximately 18 nm in diameter and 300 nm in length, and is composed of approximately 2,131 copies of a 17.5 kDa coat protein that encapsidates genomic RNA in a helical manner at a ratio of 3 nt per coat protein. The TMV NtK genome is predicted to be 6,407 nucleotides and encapsidated by 2,135 coat proteins. The following description is not intended to limit the viral carriers used with the present embodiments to any particular one, but further description and characterization of suitable TMV-NtK intermediates and coat proteins of such viruses can be found in references such as, but not limited to, U.S. Pat. No. 7,939,318 (McCormick et al., "Flexible vaccine assembly and vaccine delivery platform") and Smith et al., "Modified Tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications", Virology 2006;348(2):475-88. Taking into account the variability associated with different types of viruses, the process of the present invention is based on two structurally distinct viruses that allow for the passage of virus by filtration while retaining unwanted cellular debris.In use, controlled operating parameters allow the tangential flow (TFF) system to continue to operate efficiently without excessive or untimely contamination, while retaining chlorophyll / cell debris, while transferring all types of viruses to the filtrate. A further TFF step is designed to retain viruses while transferring small proteins to the filtrate, and a dual chromatography step is controlled to capture host cell proteins, host cell DNA, endotoxins and plant polyphenols, while eliminating both large and small viruses.

[0023] Based on the successful purification of Red Clover Mosaic Virus and TMV, it is believed that the virus purification platform according to embodiments and alternatives thereof can efficiently purify a wide array of virus particles, including viruses with various genetic material (e.g., double-stranded and single-stranded DNA and RNA viruses), geometries (e.g., rod-shaped, curved rod-shaped, and icosahedral), and families (Caulimoviridae, Geminiviridae, Bromoviridae, Closteroviridae, Comoviridae, Potyviridae, Sequiviridae, Tombusviridae).

[0024] Non-limiting viruses with which the embodiments described herein may be successful include those of the genus Badnavirus (e.g., Commelinus yellow mottle virus), Caulimovirus (e.g., Cauliflower mosaic virus), SbCMV-like virus (e.g., Soybean yellows mottle virus), CsVMV-like virus (e.g., Cassava vein mosaic virus), RTBV-like virus (e.g., Rice tungro bacillus virus), Petunia vein clearing-like virus (e.g., Petunia vein clearing virus), Mastrevirus (subgroup I geminiviruses) (e.g., Corn streak virus), and the genera Curtovirus (subgroup II geminiviruses) (e.g., Beet curly top virus) and Begomovirus (subgroup III geminiviruses) (e.g., Bean golden mosaic virus), Alfamovirus (e.g., Alfalfa mosaic virus), Airau virus, and the like. Ilarvirus (e.g. tobacco streak virus), Bromovirus (e.g. brome mosaic virus), Cucumovirus (e.g. cucumber mosaic virus), Closterovirus (e.g. beet yellows virus), Crinivirus (e.g. lettuce infectious yellows virus), Comovirus (e.g. cowpea mosaic virus), Fabavirus (e.g. Broad bean wilt virus 1), Nepovirus (e.g. tobacco ringspot virus), Potyvirus (e.g. potato virus Y), Rymovirus (e.g. ryegrass mosaic virus), Bymovirus (e.g. barley yellow mosaic virus), Sequivirus (e.g. parsnip yellow spot virus), Waikavirus (e.g. rice tungro spherical virus),Carmovirus (e.g. carnation mottle virus), Dianthovirus (e.g. carnation ringspot virus), Machlomovirus (e.g. maize chlorotic mottle virus), Necrovirus (e.g. tobacco necrosis virus), Tombusvirus (e.g. tomato bushy stunt virus), Capillovirus (e.g. apple system grooving virus), Carlavirus (e.g. carnation latent virus), Enamovirus (e.g. pea fluted mosaic virus), Furovirus (e.g. soil-borne wheat dwarf virus), Hordeivirus (e.g. wheat stripe mosaic virus), Idaeovirus Idaeovirus (e.g. Raspberry yellows virus), Luteovirus (e.g. Barley yellows dwarf virus), Marafivirus (e.g. Maize rayado finovirus), Potexvirus (e.g. Potato virus X and Clover mosaic virus), Sobemovirus (e.g. Southern bean mosaic virus), Tenuivirus (e.g. Rice stripe virus), Tobamovirus (e.g. Tobacco mosaic virus), Tobravirus (e.g. Tobacco rattle virus), Trichovirus (e.g. Apple chlorotic leaf spot virus), Tymovirus (e.g. Turnip yellow mosaic virus) and Umbravirus (e.g. Carrot mottle virus).

[0025] Viruses have been successfully purified on a commercial scale and in a manner that complies with cGMP regulations. In some embodiments, the source organism is a plant, and although some variations of this embodiment include plant-based virus production, the embodiments described herein are not limited to plant virus production or purification. In some embodiments, the virus purification platform begins with growing plants in a controlled growth chamber, infecting the plants with a replication-competent virus, and recovering the virus by disrupting the cells with a crusher and removing plant fibers from the liquid via a screw press.

[0026] In some embodiments, the purification step involving plant-based and non-plant-based viruses includes concentrating the clarified extract using a tangential flow system, where the cassette pore size, transmembrane pressure, and clarified extract loading per square meter of membrane surface area are controlled. Transmembrane pressure (TMP) is the pressure difference between the upstream and downstream sides of the separation membrane, and is calculated based on the following formula: ((feed pressure + retentate pressure) / 2)-filtrate pressure. In some embodiments, the feed pressure, retentate pressure, and filtrate pressure are each controlled to obtain an appropriate TMP to ensure ceramic migration of the virus and produce a clarified extract. The clarified extract is further concentrated by ion exchange column volume and washed with ion exchange chromatography equilibration buffer. In some embodiments, the Capto Q ion exchange column is equilibrated, the feed is loaded, and collected in the flow-through fraction. The column is then washed to baseline, and host cell contaminants are removed from the column using a high salt concentration.

[0027] In some embodiments related to plant-based viruses, an extraction buffer is added prior to removal of chlorophyll and other larger cellular debris, such as polymeric fibers, organelles, lipids, etc., using tangential flow ceramic filtration. In some embodiments, ceramic filtration promotes retention of chlorophyll from the plant host, cellular debris, and other impurities, while optimizing passage of viruses. For plant-based or non-plant-based viruses, this approach promotes scalability of the process by allowing the desired objects (viruses or antigens) to pass by filtration while the impurities are retained as retentate water. Additionally, parameters such as transmembrane pressure, ceramic pore size, and biomass loaded per square meter are all controlled to ensure ceramic transfer of viruses to create a clarified extract. The ceramic TFF system is highly scalable, and parameters such as TMP, cross flow rate, pore size, and surface area can be easily scaled to accommodate larger amounts of biomass. Additional ceramic modules can be easily added into the system. The feed, retentate and filtrate pressures can also be controlled to maintain an efficient cross flow velocity with little or no fouling of the system. In some embodiments, the cross velocity and pressure differential are set and controlled to obtain a TMP of about 10-20 psi, which effectively passes viruses at smaller and larger scales. The ceramic TFF system allows for the use of highly efficient cleaning chemistries such as nitric acid, bleach and sodium hydroxide, which are cleaning tested to meet GMP and / or cGMP requirements.

[0028] Regardless of whether the virus is plant-based or non-plant-based, the purification methods according to the embodiments and alternative methods, as well as other methods for developing scalable, high-throughput methods for purifying viruses, utilize at least one separation step using multi-mode chromatography to separate remaining impurities from the virus based on the size differences between the virus and the impurities and chemical interactions that occur between the impurities and one or more chromatography ligands. For example, it is within the scope of the embodiments to perform at least one separation step using Capto® Core 700 chromatography resin (GE Healthcare Bio-Sciences). Capto® Core 700 "beads" contain octylamine ligands designed to have both hydrophobic and positively charged properties to trap molecules of a specific size (e.g., 700 kilodaltons (kDA)). Because certain viruses are quite large (e.g., greater than 700 kDa) and the exterior of the beads is inert, Capto® Core 700 allows purification of viruses by size exclusion, where the desired material (virus or antigen) passes through as the filtrate and impurities are retained as the retentate.

[0029] In some embodiments, the plant-based and non-plant-based viruses alike are equilibrated with 5 column volumes of equilibration buffer prior to the multimode chromatography column. In some embodiments, the flow-through and wash fractions from the Capto Q ion exchange chromatography are combined and loaded onto the multimode chromatography column, with the viruses being collected in the void volume of the column. The column is washed to baseline and then stripped with high conductivity sodium hydroxide. Aspects of some embodiments control the loading ratio, column bed height, residence time and chromatography buffer during this step.

[0030] The purified virus is filter sterilized, for example by diafiltration, and stored.

[0031] With respect to antigens, implementation of several embodiments of the antigen purification platform of the invention described herein has enabled the purification of H5 recombinant influenza hemagglutinin (rHA), H7 rHA, domain III of West Nile virus (WNV rDIII), Lassa virus recombinant protein 1 / 2 (LFV rGP1 / 2), H1N1 (Influenza A / Michigan), H1N1 (Influenza A / Brisbane), H3N2 (Influenza A / Singapore), H3N2 (Influenza A / Kansas), B / Colorado, B / Phuket, RBD-Fc121 (human IgG1 Recombinant antigens such as the receptor binding domain (RBD, S1 domain) of the SAR-2 spike glycoprotein fused to an Fc domain (hereinafter "RBD-Fc121" refers to the amino acid sequence of the SAR-2 spike protein as described in detail below) and RBD-Fc139 (hereinafter "RBD-Fc139" refers to a different amino acid sequence of the SAR-2 spike protein) have been produced and purified. Antigens for the various embodiments herein may be derived from many sources and can be produced using traditional recombinant protein production strategies such as bacterial, yeast, insect, mammalian, or plant-based expression methods.

[0032] In some embodiments, the antigen production platform begins with growing plants in a controlled growing room, infecting the plants for recombinant antigen replication, using a grinder, followed by removing fibers from the aqueous liquid via a screw press and recovering the antigen. An extraction buffer is added to aid in the removal of chlorophyll (in the plant context) and large cellular debris by filtration. The feed pressure, filtration pore size, clarification agent and biomass loaded per square meter of membrane surface are controlled to facilitate the movement of the antigen through the filter, whether it is a plant-based or non-plant-based antigen. A description of various (but non-limiting) in-process controls suitable for large-scale virus and antigen purification is described in more detail in the Examples section.

[0033] In some embodiments, the clarified extract, as well as the plant-based and non-plant-based antigens, are then concentrated by a tangential flow system. During this optional step, factors such as cassette pore size, transmembrane pressure, and load of revealed extract per square meter of membrane surface are controlled. In some embodiments, this optional step is skipped entirely. After this, the clarified extract is then concentrated and washed with an ion exchange chromatography equilibration buffer. One way this step is performed is by loading the feed onto an equilibrated Capto Q ion exchange column, followed by washing with equilibration buffer and elution / salt desorption. The antigen fraction is then collected in the elution and prepared for cobalt immobilized metal affinity chromatography (IMAC). The IMAC is equilibrated, the feed is loaded, then washed with equilibration buffer, and eluted. The elution fraction is diluted, the pH is checked, and then loaded onto a multi-mode ceramic hydroxyapatite (CHT) chromatography column. The CHT resin is equilibrated with equilibration buffer, and the antigen is eluted. Loading ratio, column bed height, residence time and chromatography buffer are among the factors that are controlled. Finally, the antigen is concentrated and diafiltered with saline buffer. The recombinant antigen is filter sterilized and then stored.

[0034] Still further, the following monovalent formulations were successfully conjugated according to various embodiments disclosed herein: H7 rHA to TMV, H1N1 to TMV (Influenza A / Michigan), H3N2 to TMV (Influenza A / Singapore), B / Colorado to TMV, B / Phuket to TMV, RBD-Fc121 to TMV (SARS-2), and RBD-Fc139 to TMV (SARS-2). Bivalent formulations of TMV to two influenza B viruses (B / Colorado and B / Phuket) were also successfully conjugated according to various embodiments disclosed herein, as well as tetravalent conjugations of TMV to H1N1 (Influenza A / Michigan), H3N2 (Influenza A / Singapore), B / Phuket, and B / Colorado. "Quadravalent" influenza vaccines are designed to protect against four different influenza viruses, two influenza A viruses and two influenza B viruses. For many years, trivalent vaccines were commonly used, but quadrivalent vaccines are the most common because they could beneficially provide broader protection against circulating influenza viruses by adding other B viruses. As used herein, the term "multivalent" vaccine refers to one or more antigens conjugated to a virus. In some embodiments, the protein consists of any type of therapeutic agent that can be conjugated to a virus to produce a vaccine, which is then delivered to a source organism to generate an immune response, according to several embodiments and alternatives. Thus, the disclosure herein provides compositions that include an array of virus-protein conjugates, including virus-antigen conjugates. In some embodiments, the selected virus is TMV or any of several viruses identified and / or indicated by the teachings herein.Furthermore, in some embodiments, the antigen may be a protein such as, but not limited to, influenza hemagglutinin antigen (HA), including, but not limited to, those listed in this paragraph, such as the proteins present on the surface of influenza viruses that mediate viral infection as soluble forms of HA. In some embodiments, the HA exhibits at least about 50% trimeric structure. HA is clinically important because it tends to be recognized by specific antibodies produced by the organism, and provides the main thrust for the prevention of various influenza infections. Since the antigenicity of HA, i.e., the immunogenicity of HA, is related to the conformation, it is known that trimerization of HA is more beneficial in terms of eliciting an immune response than the monomeric form.

[0035] In some embodiments, conjugation begins with concentrating and diafiltration of purified antigen and virus in a weakly acidic buffer. Antigen and virus are combined based on molar concentration and then mixed. Freshly prepared water-soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (also known as EDC), is added to the mixture and mixed based on molar concentration. A chemical reagent for converting carboxyl groups to amine-reactive N-hydroxysulfosuccinimide esters, such as ThermoFisher's Sulfo-NHS, is then added based on molar concentration. The reaction is allowed to continue until a pre-determined stop time. The reaction is then quenched, for example, by addition of amine groups (e.g., liquids containing free amines), and any chemical linkers used in facilitating the reaction (e.g., EDC, Sulfo-NHS) are removed in a multi-mode chromatography step or diafiltration with the mixture then diluted to the target concentration. In some embodiments, the conjugated and purified virus particles modified with proteins and antigens can be used as vaccines and / or diagnostic tools. These particles can be used as diagnostic tools due to their ability to track antigens of the host organism.

[0036] In some embodiments, the purified virus-antigen fusion may result from genetic fusion, in addition to the various embodiments disclosed herein. The antigen and the viral structural protein (located in the coat) form a single continuous open reading frame. In some embodiments, the antigen-coat protein is produced in a plant from a reading frame such that the coat protein self-assembles into a viral particle. The plant material is then harvested and the viral particles are purified according to the embodiments disclosed herein. The viral particles modified with the fusion-coat protein can then be used as a vaccine and / or a diagnostic tool according to the various embodiments disclosed herein.

[0037] Some viruses (e.g., icosahedral viruses, as a non-limiting example) swell at certain pH conditions, and in some embodiments, this "swelling" can be used for conjugation. According to several embodiments and alternatives, the purified virus can be conjugated to a therapeutic agent by subjecting the viral structure to acidic pH conditions to "swell" the virus. By treating the viral structure with neutral pH conditions, the viral structure relaxes and forms pores between the pentamers or other structural subunits of the virus. A therapeutic agent (e.g., a chemotherapeutic agent) is then added to the buffer and allowed to diffuse into the relaxed viral particles. By changing the pH again, the viral particles tighten, eliminating the porous structure that packages the pentamers or structural subunits together, preventing chemical diffusion of the viral particles. The plant material is then harvested, the viral particles are purified, and the viral particles containing the therapeutic agent are used for drug delivery according to the embodiments disclosed herein.

[0038] Thus, embodiments and alternatives include the production of one or more highly purified viruses. Further embodiments and alternatives include the production or purification or both of recombinant antigens. Further embodiments and alternatives include the conjugation of purified antigens and viruses for use as vaccines. Indeed, in preclinical trials, TMV-platform vaccines produced according to the embodiments described herein stimulated effective immune responses against several pathogens, including viruses and antibacterial systems. Furthermore, vaccines produced on the TMV-platform of the present invention have demonstrated the ability to readily conjugate (i.e., as multivalent combination vaccines) against multiple coronavirus (CoV) antigens and stimulate effective immune responses. This provides a significant advantage over conventional monovalent vaccines, such as in the administration of these vaccines against Covid-19 disease and influenza (as non-limiting examples). Purification of the virus may be performed alone according to this embodiment. Similarly, production or purification of recombinant antigens may be performed alone according to this embodiment. Optionally, different aspects of these multiple embodiments can be combined, whereby combining embodiments includes, among other methods of implementing these embodiments, starting with one or more source organisms, producing one or more viruses and one or more antigens, then purifying such viruses and antigens, and then forming a vaccine comprising a conjugate between at least one antigen and at least one virus.

[0039] The international application relating to this disclosure contains one or more drawings in color execution, some of which would be practically meaningless or impossible to publish in black and white execution, including, but not limited to, the protein structures and RBD-Fc fusions in Figures 51(A) and 51(B), the microscopy image in Figure 61, and the numerous line graphs in Figures 71(A)-(C), 79(A)-(B), and 81(A)-(B). Upon publication, applicant will provide the drawings in black and white, where necessary, to represent color images, but which do not add to the subject matter.

[0040] The drawings and embodiments described herein are illustrative of the alternative structures, aspects, and features of the embodiments and alternatives of the invention disclosed herein, and should not be understood as limiting the scope of any of these embodiments and alternatives. It is further understood that the drawings described and provided herein are not necessarily to scale, and are not limited to the precise arrangements, depictions, and means in which the embodiments are shown. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 is a flow diagram illustrating steps in a particular virus purification platform within the present disclosure, according to multiple embodiments and alternatives. [Diagram 2] FIG. 2 is a purified icosahedral Red Clover Mosaic Virus according to multiple embodiments and alternatives. [Diagram 3] FIG. 3 is a Western blot analysis of purification of icosahedral Red Clover Mosaic Virus according to several embodiments and alternatives. [Figure 4] FIG. 4 is a purified icosahedral Red Clover Mosaic Virus according to multiple embodiments and alternatives. [Diagram 5] FIG. 5 is a Western blot analysis of purification of icosahedral Red Clover Mosaic Virus according to several embodiments and alternatives. [Figure 6] FIG. 6 is a purified rod-shaped tobacco mosaic virus according to embodiments and alternatives. [Figure 7] FIG. 7 is a Western blot analysis of purified rod-shaped tobacco mosaic virus according to several embodiments and alternatives. [Figure 8] FIG. 8 is a flow diagram illustrating steps of an antigen production platform, according to several embodiments and alternatives. [Figure 9]FIG. 9 is a Western blot analysis of several steps of the antigen production platform, according to multiple embodiments and alternatives. [Figure 10] FIG. 10 is a Western blot analysis of several steps of the antigen production platform, according to multiple embodiments and alternatives. [Figure 11] FIG. 11 is a Western blot analysis of several steps of the antigen production platform, according to multiple embodiments and alternatives. [Figure 12] FIG. 12 is a Western blot analysis of the purification of various antigens by the antigen production platform, according to multiple embodiments and alternatives. [Figure 13] FIG. 13 is an illustration of the conjugation of recombinant antigens to viruses according to several embodiments and alternatives. [Figure 14] FIG. 14 is an SDS-PAGE analysis of conjugation of antigen to virus according to several embodiments and alternatives. [Figure 15] FIG. 15 is an SDS-PAGE analysis of conjugation of antigen to virus according to several embodiments and alternatives. [Figure 16] FIG. 16 is an SDS-PAGE analysis of conjugation of antigen to virus according to several embodiments and alternatives. [Figure 17] FIG. 17 is a size exclusion-high performance liquid chromatography (SEC-HPLC) report of the free TMV product, according to several embodiments and alternatives. [Figure 18] FIG. 18 is a SEC-HPLC report of conjugation between virus and antigen for 15 minutes according to several embodiments and alternatives. [Figure 19] FIG. 19 is a SEC-HPLC report of 2 hour conjugation between virus and antigen according to several embodiments and alternatives. [Figure 20]FIG. 20 is a Western blot analysis of conjugation between virus and antigen according to several embodiments and alternatives. [Figure 21] FIG. 21 is a graph showing the infectivity of viruses treated with various levels of UV radiation, according to several embodiments and alternatives. [Figure 22] FIG. 22 is an illustration of several steps of a platform for conjugation of recombinant antigens to viruses, according to multiple embodiments and alternatives. [Diagram 23] FIG. 23 is an SDS-PAGE analysis of conjugation of antigen to virus according to several embodiments and alternatives. [Figure 24] FIG. 24 is a negative stain transmission electron microscope (TEM) image of a recombinant antigen according to several embodiments and alternatives. [Diagram 25] FIG. 25 is a negative stain TEM image of a virus, according to embodiments and alternatives. [Figure 26] FIG. 26 is a negative stain TEM image of recombinant antigens conjugated to other recombinant antigens with added virus, according to several embodiments and alternatives. [Figure 27] FIG. 27 is a negative stain TEM image of recombinant antigen conjugated to virus in a 1:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Figure 28] FIG. 28 is a negative stain TEM image of recombinant antigen conjugated to virus in a 1:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Figure 29] FIG. 29 is a negative stain TEM image of recombinant antigen conjugated to virus at a 4:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Diagram 30] FIG. 30 is a negative stain TEM image of recombinant antigen conjugated to virus at a virus to recombinant antigen ratio of 16:1, according to multiple embodiments and alternatives. [Diagram 31]FIG. 31 is a normalized sedimentation coefficient distribution of antigens according to several embodiments and alternatives. [Diagram 32] FIG. 32 is a normalized sedimentation coefficient distribution of viruses according to embodiments and alternatives. [Diagram 33] FIG. 33 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a 1:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Diagram 34] FIG. 34 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a 1:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Diagram 35] FIG. 35 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a 1:1 virus to recombinant antigen ratio, according to multiple embodiments and alternatives. [Diagram 36] FIG. 36 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a virus to recombinant antigen ratio of 4:1, according to multiple embodiments and alternatives. [Figure 37] FIG. 37 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a virus to recombinant antigen ratio of 16:1, according to multiple embodiments and alternatives. [Figure 38] FIG. 38 is a scatter plot of antigen-associated titers in a source organism following administration of viral antigen products with various virus to recombinant ratios, according to multiple embodiments and alternatives. [Figure 39] FIG. 39 is a geometric mean study showing antigen-associated titers in source organisms following administration of viral antigen products with various virus to recombinant ratios, according to multiple embodiments and alternatives. [Diagram 40] FIG. 40 is an SDS-PAGE analysis of purified recombinant antigens according to several embodiments and alternatives. [Figure 41(A)] FIG. 41(A) shows a graph of stability data for the QIV conjugate at two different temperature ranges. [Figure 41(B)] FIG. 41(B) shows a graph of the stability data for the QIV conjugate at two different temperature ranges. [Diagram 42] FIG. 42 is a graph of the immunogenicity of tetravalent vaccines in mice, according to multiple embodiments and alternatives. [Diagram 43] FIG. 43 is an illustration of immunogenicity and challenge testing of a tetravalent vaccine in ferrets, according to multiple embodiments and alternatives. [Diagram 44] FIG. 44 is an illustration of viral titers in nasal washes following viral challenge in ferrets, according to multiple embodiments and alternatives. [Diagram 45] FIG. 45 is an illustration of viral titers in nasal washes following viral challenge in ferrets, according to multiple embodiments and alternatives. [Diagram 46] FIG. 46 is an illustration of the immunogenicity of monovalent vaccines produced in mice with various virus-to-recombinant antigen ratios, according to multiple embodiments and alternatives. [Figure 47] FIG. 47 is an illustration of TMV vRNA in tissues over time following injection of a tetravalent vaccine, according to multiple embodiments and alternatives. [Figure 48] FIG. 48 is an illustration of TMV vRNA in tissues 8 days after injection of a tetravalent vaccine, according to multiple embodiments and alternatives. [Figure 49] FIG. 49 is an illustration of total anti-influenza titers based on ELISA analysis from rabbit serum samples following injection of a tetravalent vaccine, according to multiple embodiments and alternatives. [Figure 50] FIG. 50 is an illustration of neutralization titers measured in rabbits following injection of a tetravalent vaccine, according to several embodiments and alternatives. [Fig. 51(A)(B)] FIG. 51(A) is an illustration of the protein structure of the Covid-19 spike trimer in a space-solid model with the receptor binding domains circled in horizontal and vertical views. [Figure 52]FIG. 52 is an illustration of an expression plasmid containing a recombinant Covid-19 antigen construct, according to multiple embodiments and alternatives. [Figure 53] FIG. 53 is an SDS-PAGE analysis of purified recombinant Covid-19 antigens according to embodiments and alternatives. [Figure 54] FIG. 54 is a SEC-HPLC report of purified recombinant Covid-19 antigens according to embodiments and alternatives. [Figure 55] Figure 55 is an SDS-PAGE analysis of purified recombinant Covid-19 antigens according to embodiments and alternatives. [Figure 56] FIG. 56 is an illustration of some of the steps of the platform for conjugation of recombinant Covid-19 antigens to the virus and drug substance loading, according to multiple embodiments and alternatives. [Figure 57] Figure 57 is a normalized sedimentation coefficient distribution of recombinant Covid-19 antigens conjugated to virus according to embodiments and alternatives. [Figure 58] Figure 58 is a normalized sedimentation coefficient distribution of recombinant Covid-19 antigens conjugated to virus according to embodiments and alternatives. [Figure 59] Figure 59 is an SDS-PAGE analysis of recombinant Covid-19 antigens conjugated to viruses according to multiple embodiments and alternatives. [Figure 60(A)] Figure 60(A) is an ELISA standard curve showing binding of recombinant Covid-19 antigens to Covid-19 human neutralizing monoclonal antibodies. [Fig. 60(B)] Figure 60(B) is a graph showing binding of various vaccine options (sometimes referred to herein as vaccine candidates) according to embodiments and alternatives described herein to Covid-19 human neutralizing monoclonal antibodies according to embodiments and alternatives. [Figure 61]FIG. 61 consists of confocal microscopy images showing co-localization of recombinant Covid-19 antigens to ACE-2 specific antibodies according to several embodiments and alternatives. [Figure 62(A)] FIG. 62(A) is a graph showing co-localization of natural agonists and recombinant Covid-19 antigens to an ACE-2 specific antibody according to several embodiments and alternatives. [Fig. 62(B)] Figure 62(B) is a graph showing co-localization of natural agonists and recombinant Covid-19 antigens to an ACE-2 specific antibody in the presence of a co-localization control according to several embodiments and alternatives. [Fig. 63(A)(B)] FIG. 63(A) is a graph showing the results of a plaque neutralization assay of pooled serum samples pre-immunization and at days 2, 28, and 42 (booster on day 14) following the first dose with increasing dilution levels of an exemplary vaccine and varying concentrations of RBD-Fc antigen (with or without adjuvant). [Fig. 63(C)(D)] Figure 63(C) is a graph showing the results of a plaque neutralization assay of pooled serum samples pre-immunization and at days 2, 28, and 42 (booster on day 14) after the first dose with increasing dilution levels of an exemplary vaccine and varying concentrations of RBD-Fc antigen (with or without adjuvant). [Figure 64(A)] Figure 64(A) provides a graph further examining the neutralizing titers produced in the 15 and 45 g groups (but without adjuvant) on days 28 and 42 following the same dilutions. [Fig.64(B)] Figure 64(B) provides a graph further examining the neutralizing titers produced in the 15 and 45 g groups (but without adjuvant) on days 28 and 42 following the same dilutions. [Fig. 65(A)] Figure 65(A) is a graph showing the immune response of animals immunized with recombinant Covid-19 antigens and controls. [Fig. 65(B)]Figure 65(B) is a graph showing the immune response of animals immunized with recombinant Covid-19 antigens conjugated to the virus (without adjuvant).

Fig.65(C)

Fig.66(C)

Fig. 70(C)

Fig. 71(C)

Fig. 75(C)

[0042] The multi-set process according to embodiments and alternatives herein improves the upstream purification process while further enriching the plant virus, facilitating vaccine formation by conjugating the virus and antigen. Steps for producing and purifying the virus according to embodiments and alternatives are listed and discussed in relation to Table 1 and FIG. 1. Similarly, steps for producing and purifying the antigen are listed and discussed in relation to Table 2. Although the various platforms have specific embodiments described below, the scope of embodiments included in the present invention is not limited to any single specific embodiment.

[0043] Virus production and purification Table 1 and FIG. 1 illustrate steps of a virus purification platform according to several embodiments and alternatives.

[0044] [Table 1] TIFF2025076429000003.tif150166

[0045] This purification platform is designed for commercial scalability and compliance with cGMP regulations, utilizing one buffer throughout the entire purification process. According to several embodiments and alternatives, the steps of the virus purification platform are performed in the context of plant expression. However, for non-plant viruses (unless the context clearly relates to plants, e.g., references to removal of plant fiber), the subsequent steps of harvesting aerial tissues and disrupting cells are also applicable, as described below.

[0046] According to several embodiments and alternatives described herein, viral expression is achieved by methods appropriate for the particular host. In some embodiments, viral-based gene delivery to the plant host is achieved by modified TMV expression vectors that recombinantly form viruses in tobacco plants. One such option available is the GENEWARE® platform described in U.S. Pat. No. 7,939,318 ("Flexible vaccine assembly and vaccine delivery platform"). This plant-based transient expression platform described in this patent uses the plant virus TMV to exploit the protein production machinery in plants to express various viruses with a short recovery period (e.g., less than 21 days) after inoculation. Tobacco plants inoculated with viral genes express the specific virus in infected cells, which are extracted upon harvest. In the methods described herein, inoculation is performed by manual inoculation of the leaf surface, mechanical inoculation of the plant bed, high pressure spraying of the leaves, or vacuum infiltration, as examples selected by the user.

[0047] In addition to Nicotiana benthamiana, other plant and non-plant hosts are contemplated by the present disclosure, including those described in the Summary of the Invention. In addition to the GENEWARE® platform, other strategies can be used for gene delivery to plants (Lemna gibba or Lemna minor, as non-limiting examples) and non-plant organisms (algae, as non-limiting examples). These other strategies include agro-infiltration, which introduces viral genes via Agrobacterium bacterial vectors to many cells throughout the transfected plant. Another is electroporation into open pores in the host cell membrane to introduce viruses and genes that recombinantly produce antigens, as described, but not limited to, in Examples 1 and 3 below. The other is the TMV RNA-based overexpression (TRBO) vector, which utilizes a TMV replicon driven by the 35S promoter, lacking the TMV coat protein gene sequence, as described in John Lindbo, "TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector," Plant Physiol. Vol. 145, 2007.

[0048] In some embodiments, the growth of wild-type Nicotiana benthamiana plants is carried out in a controlled growth chamber. Plant cultivation is controlled by irrigation, light and fertilization cycles. Plants are grown in a soil-less medium and temperature is also controlled throughout the process.

[0049] At an appropriate day post sowing (DPS), e.g., 23-25 ​​DPS, plants are infected with the virus replicating. After infection, plants are irrigated only and controlled via light cycle and temperature for a set number of days post infection (DPI) depending on the type of virus.

[0050] Plants are closely examined for height and signs of infection, and aerial tissue is sampled.

[0051] Virus recovery / cell disruption is accomplished by grinding in a grinder configured with optimized blade / sieve sizes, followed by removal of residual cellulosic plant fibers from the aqueous liquid (e.g., by a screw press, in one example).

[0052] An appropriate extraction buffer (e.g., 200 mM sodium acetate, pH 5.0, see step 201 in FIG. 1 for a non-limiting example) is added to the resulting extract in a ratio of buffer:tissue=1:1. For pilot-scale chlorophyll and large cellular debris removal, tangential flow (TFF) ceramic filtration (1.4 micron / 5.0 micron) is used. In some embodiments, an additional 0.1 micron ceramic tangential flow filtration step is used. Transmembrane pressure, ceramic pore size, and biomass loaded per square meter of surface membrane are all controlled to ensure passage of viruses through the ceramic. In some embodiments, feed pressure, retentate pressure, and filtrate pressure are set and controlled to produce a transmembrane pressure in the range of about 1.5-2 bar TMP. Optionally, ceramic tangential flow filtration is followed by dead-end filtration, e.g., 1.2 micron glass fiber filtration.

[0053] The ceramic filtrate is further clarified through the use of fiberglass depth filtration (as a non-limiting example, step 203 of FIG. 1).

[0054] The clarified extract is concentrated by a TFF system (Sartorius AG) by controlling the cassette pore size (100-300 kDa), the appropriate TMP as described herein, and the loading amount of clarified extract per square meter of membrane surface.

[0055] The clarified extract is concentrated and washed 7 times with NMT 2X ion exchange column volumes with ion exchange chromatography equilibration buffer (200 mM sodium acetate, pH 5.0, shown as a non-limiting example in FIG. 1, step 204). A Capto Q ion exchange column is equilibrated with 5 column bed volumes with 200 mM sodium acetate, pH 5.0 (shown as a non-limiting example in FIG. 1, step 205), the feed is loaded, and the flow-through fraction is collected. The column is washed to baseline and host cell contaminants are removed from the column with high salt concentration.

[0056] The flow-through and wash fractions are collected, combined, and prepared for multimode Capto® Core 700 chromatography. The multimode chromatography column is equilibrated with 5 column bed volumes of equilibration buffer (200 mM sodium acetate, pH 5.0, step 206 in FIG. 1 shows a non-limiting example).

[0057] The flow-through and wash fractions from the Capto Q ion exchange chromatography are combined and loaded onto the column, with the virus being collected in the void volume of the column. The column is washed to baseline and stripped with high conductivity sodium hydroxide. The loading ratio, column bed height, residence time and chromatography buffer are all controlled. The virus (step 208 in FIG. 2) is formulated and concentrated, in some embodiments, by a TFF system (e.g., a Sartorius AG system). The pore size (30-300 kDa), appropriate TMP as described herein, load per square meter of membrane surface area and pore material are all controlled. The virus is concentrated to an appropriate concentration, e.g., 10 mg / ml, and in some embodiments, diafiltered with an appropriate buffer, e.g., sodium phosphate. The formulated virus is sterilized and appropriately stored. In some embodiments, sterilization is performed via a PES filter. EXAMPLES

[0058] All examples provided herein are provided as illustrations of virus production, virus purification, antigen production, antigen purification, and virus-antigen conjugation according to some or all of the various aspects of the embodiments and alternatives. These examples are non-limiting and merely illustrative of the features of the alternative embodiments herein.

[0059] Example 1 - Purification of icosahedral red clover mosaic virus Western blotting (shown in FIG. 3 as a known technique for detecting various proteins in a mixture) shows successful purification of the icosahedral Red Clover Mosaic Virus shown in FIG. 2. Similarly, Western blotting in FIG. 5 shows successful purification of the icosahedral Red Clover Mosaic Virus shown in FIG. 4. Both viruses were purified according to the embodiments described herein. Target proteins were extracted from tissues according to known detection techniques. The proteins of the samples were then separated using gel electrophoresis based on their isoelectric point, molecular weight, charge, or various combinations of these factors. The samples were then loaded into various lanes of the gel, with a lane set up as a "ladder" containing a mixture of known proteins with known molecular weights. For example, lane 12 in FIG. 3 serves as a ladder. A voltage was then applied, causing the various proteins to migrate through the gel at different speeds based on the factors mentioned above. Separation of the different proteins into visible bands occurred within each lane, as provided in FIGS. 3 and 5, respectively. Pure products were characterized by clearly visible bands by Western blotting and are marked in these figures.

[0060] Figures 3 and 5 illustrate a virus purification platform that efficiently purifies icosahedral red clover mosaic virus. Each lane of the Western blot shows the purity of the virus after completion of a different step in the virus purification platform. In Figure 3, the lanes are as follows: lane 1 - untreated, lane 2 - TFF ceramic clarified retentate, lane 3 - TFF ceramic clarified filtrate, lane 4 - TFF cassette retentate, lane 5 - TFF cassette filtrate, lane 6 - ion exchange, lane 7 - ion exchange, lane 8 - multimode, lane 9 - multimode, lane 10 - 30K TFF filtrate, lane 11 - 30K retentate, lane 12 - marker. In Figure 5. Western blot lanes are as follows: lane 1 - untreated, lane 3 - TFF ceramic clarified retentate, lane 5 - TFF ceramic clarified filtrate, lane 7 - TFF cassette retentate, lane 9 - TFF cassette filtrate, lane 11 - ion exchange, lane 13 - multimode and lane 14 - marker.

[0061] After the final step is performed on the virus purification platform, the resulting virus product is highly purified, as shown by the visible bands in lane 11 of FIG. 3 and lane 13 of FIG.

[0062] Example 2 - Purification of rod-shaped TMV FIG. 6 shows purified rod-shaped TMV, and FIG. 7 illustrates the viral purification platform used to obtain this purified TMV within the scope of the embodiments and alternatives disclosed herein. Similar to FIGS. 3 and 5, FIG. 7 illustrates the purity of the viral product after completion of various steps of the viral purification platform of the present invention. After the final purification step, the resulting product is a highly purified viral product, consistent with the clearly visible band in lane 13 of FIG. 7. Exemplary, prior to such conjugation, the identity of the intermediate TMV-NtK can be confirmed by mass spectrometry, e.g., MALDI-TOF mass spectrometry. In some embodiments, the physiochemical properties of the intermediate TMV-NtK are a pH of about 7.0-7.4, an osmolality of about 15-45 mOsm / kg·H2O, a bioburden of 100 CFU (colony forming units) / mL or less, and less than 100 ng / mg of residual host cell-derived protein.

[0063] Thus, the virus purification platform of the present invention efficiently purifies any virus, including icosahedral and rod-shaped viruses, to which the inventors have applied these methods, and it is believed that the platform will reproducibly and consistently purify any (if not all) types of viruses on a commercial scale.

[0064] Recombinant antigen production and purification Table 2 and FIG. 8 illustrate steps of an antigen purification platform according to several embodiments and alternatives.

[0065] [Table 2] TIFF2025076429000005.tif185166TIFF2025076429000006.tif71165

[0066] This purification platform is designed for commercial scalability and compliance with cGMP regulations, utilizing one buffer throughout the entire purification process. According to multiple embodiments and alternatives, the steps of the antigen purification platform are as follows:

[0067] Growth of wild type plants of Nicotiana benthamiana in a controlled growth chamber. Plant growth is controlled by irrigation, light and fertilization cycles. Plants are grown in soil-free medium and temperature is controlled throughout the process. After an appropriate number of DPS, e.g. 23-25 ​​days, plants are infected for protein replication of the selected antigen. Once attached, the protein is sufficient for retention in the ER of the transgenic plant cells. After infection, plants are only irrigated and controlled by light cycle and temperature for an appropriate number of days post-infection, e.g. 7-14 days depending on the type of antigen. Plants are examined for height, infection symptoms and aerial tissues are harvested.

[0068] Recovery of antigens produced by the plant is performed by a grinder configured with optimized blade / sieve sizes followed by removal of residual cellulosic plant fibers from the aqueous liquid (e.g., by a screw press, in one example).

[0069] An appropriate extraction buffer is added to the resulting extract in an appropriate ratio, for example, 1:1 buffer:tissue ratio, or 2:1 buffer:tissue ratio. In some embodiments, the extraction buffer may be 50-100 mM sodium phosphate + 2 mM EDTA + 250 mM NaCl + 0.1% Tween 80, pH 8.5. Removal of chlorophyll and large cell debris involves the use of filtration. Celpure 300 is added at a ratio of 33 g / L and mixed for 15 minutes. Feed pressure (<30 PSI), filter pore size (0.3 microns), clarifier (Celpure 300) and biomass loaded per square meter of membrane surface are all controlled to ensure passage of antigens.

[0070] The clarified extract is concentrated by a TFF system (e.g., a Sartorius AG system). In some embodiments, the cassette pore size (e.g., 30 kDa), appropriate TMP as described herein, and the loading amount of clarified extract per square meter of membrane surface area are controlled.

[0071] The clarified extract is concentrated and washed seven times with an appropriate ion exchange chromatography equilibration buffer (e.g., 50 mM sodium phosphate + 75 mM NaCl, pH 6.5). A Capto Q ion exchange column is equilibrated with 5 column volumes of 50 mM sodium phosphate + 75 mM NaCl, pH 6.5, the feed is loaded, washed with equilibration buffer, and the column is eluted / desorbed with high salt.

[0072] The antigen fraction is collected in the elution and prepared for cobalt IMAC chromatography. The IMAC is equilibrated with 50 mM sodium phosphate + 500 mM sodium chloride, pH 8.0 for 5 column volumes, the feed is loaded, washed with equilibration buffer and eluted using imidazole.

[0073] The eluted fractions are diluted, checked for conductivity and pH, and loaded onto a multi-mode ceramic hydroxyapatite (CHT) chromatography column. The CHT resin is equilibrated with 5 column volumes of equilibration buffer (5 mM sodium phosphate, pH 6.5). The antigen is eluted using a gradient of phosphate and NaCl. The loading ratio, column bed height, residence time, and chromatography buffer are all controlled. Antigen formulation and concentration are performed by a TFF system (e.g., Sartorius AG system). Pore size (kDa), TMP, loading amount per square meter of membrane surface area, and pore material are all controlled as detailed further.

[0074] The antigen is then concentrated to an appropriate concentration, for example 3 mg / ml, and diafiltered with a suitable buffer (e.g., phosphate buffered saline, pH 7.4). The formulated antigen is sterilized and appropriately stored. In some embodiments, sterilization is performed through a PES filter.

[0075] Figures 9, 10 and 11 illustrate various steps of an antigen purification platform according to multiple embodiments and alternatives: Figure 9 shows the purity of the antigen product after the Capto Q chromatography step, Figure 10 shows the purity of the antigen product after the affinity chromatography step, and Figure 11 shows the purity after the CHT chromatography column.

[0076] Examples 3, 4, 5 and 6 - H5 rHA, H7 rhA, WNV rDIII and LFV rGP1 / 2 As shown in Figure 12, the antigen purification platform according to multiple embodiments and alternatives efficiently purified H5 rHA, H7 rhA, WNV rDIII and LFV rGP1 / 2. Figure 12 shows two images of the results of the antigen purification platform: The image on the left contains an SDS Page gel showing the purity of the viral vector TMV NtK (NtK is an abbreviation for N-terminal lysine) and influenza antigens, and the image on the right contains a Western blot showing immunoreactivity to West Nile and Lassa antigens. As shown by the clearly visible bands in FIG. 12, each antigen product is highly pure. Thus, the antigen purification platform according to several embodiments and alternatives has been used in a manner that is also cGMP compliant to consistently purify each type of antigen on a commercial scale. Similarly, it is believed that the platform will purify virtually any type (if not all) of antigens with high reproducibility.

[0077] Production of recombinant antigen-virus conjugates FIG. 3 illustrates the steps of recombinant antigen conjugation according to several embodiments and alternatives.

[0078] [Table 3] TIFF2025076429000008.tif167150

[0079] In an embodiment, the steps of the conjugation platform are as follows: The purified antigen and virus are separately concentrated and diafiltered into a weakly acidic buffer, such as 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing NaCl.

[0080] A water-soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (known as EDC), is formulated in purified water at a molar concentration of 0.5M.

[0081] A chemical reagent for converting carboxyl groups to amine-reactive N-hydroxysulfosuccinimide esters, such as Sulfo-NHS from ThermoFisher, is formulated in purified water to a molar concentration of 0.1 M.

[0082] Antigen and virus are combined on a weight or molar basis (eg, 1:1 mg:mg addition) and mixed homogeneously.

[0083] Freshly prepared water-soluble carbodiimide (eg, EDC) is added to the mixture and mixed on a molar basis.

[0084] A chemical reagent (e.g., Sulfo-NHS) for converting carboxyl groups to amine-reactive esters is added based on molar concentration within 1 minute of EDC addition. The conjugation reaction is initiated and allowed to continue for a predefined mixing dwell time, e.g., 4 hours, at controlled room temperature.

[0085] The reaction is quenched by adding free amines and the chemical linkers (e.g., EDC and Sulfo-NHS) are removed by a multimodal chromatography step, such as Capto® Core 700 or diafiltration into phosphate buffered saline. According to several embodiments and alternatives, remaining impurities are removed from the result of the conjugation reaction (sometimes referred to herein as the conjugate mixture) based on the size difference between the impurities as the retentate and the conjugate mixture as the filtrate.

[0086] The conjugate mixture is diluted to the target concentration. At this point, the virus-antigen conjugate is prepared for use as a purified vaccine / drug substance. Suitable delivery mechanisms for the vaccine include liquid vials or lyophilized materials that are reconstituted with physiological buffers for project injection. Injection may be intramuscular or subcutaneous. Other delivery methods are contemplated, including but not limited to intranasal administration.

[0087] Example 7 - Conjugation of H7 rHA to TMV FIG. 13 provides an example of conjugation of recombinant antigens (meaning "vaccine antigens") to viruses, where light and dark shaded ovals represent the conjugation area of ​​the vaccine antigens represented in the examples. The light shade represents free virus, while the dark shade represents antigens conjugated to viral coat proteins. Also, as shown in FIG. 13, some viruses contain proteins that are positioned around the RNA genome. For example, the viral vector TMV NtK contains an N-terminal lysine that serves as a connector point to the coat protein. In some embodiments, the portion of the virus that associates with the N-terminal lysine residue is modified to enhance presentation for binding of recombinant antigens that provide targeted conjugation (e.g., antigen to virus) to the amines of the protein. In relation to the discussion of radius measurements herein, the viral radius is greatly increased after conjugation of recombinant antigens to viral coat proteins. In some embodiments, modifications are made when attempting to modify to enhance presentation of those residues by enveloped viruses.

[0088] As shown in Figures 14-20, the recombinant antigen-to-virus conjugation platform efficiently conjugated H7 rHA to TMV. Figures 14-16 show the analysis of the conjugation between TMV and H7 rHA based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis ("SDS-PAGE") at pH 5.50. As illustrated in these figures, almost all of the H7 rHA was conjugated to TMV within 2 hours. The simultaneous observation of disappearance of the rHA protein band and staining of the complex above the 200 KDa marker indicates the formation of the complex. The reactivity of the band with an HA-specific antibody further supports this conclusion.

[0089] The SEC-HPLC report also showed successful conjugation of H7 rHA to TMV according to an embodiment of the conjugation platform of the present invention. Figure 17 shows the SEC-HPLC report of the free TMV product. The SEC-HPLC report of the free TMV product in Figure 17 gave the signal data detailed in Table 4 below.

[0090] [Table 4]

[0091] Figure 18 shows the SEC-HPLC report after H7 rHA was conjugated to TMV for 15 minutes according to an embodiment of the conjugation platform of the present invention. From the SEC-HPLC report in Figure 18 after H7 rHA was conjugated to TMV for 15 minutes, the signal data detailed in Table 5 was obtained.

[0092] [Table 5]

[0093] Figure 19 shows the SEC-HPLC report after H7 rHA was conjugated to TMV for 2 hours according to an embodiment of the conjugation platform of the present invention. From the SEC-HPLC report in Figure 19 after H7 rHA was conjugated to TMV for 2 hours according to an embodiment of the conjugation platform of the present invention, the signal data detailed in Table 6 below was obtained.

[0094] [Table 6]

[0095] As illustrated in Figures 19 and 20, the SEC-HPLC reports show that all TMV rods were covered with some H7 rhA after 15 minutes of conjugation, and more H7 rhA was added to the rods up to 2 hours. After 2 hours, no further conjugation was detected. According to various embodiments and alternatives, the SEC-HPLC reports show that the conjugation reaction achieved at least about 50% reduction of unconjugated, native molecular weight, viral coat protein, with about 3% free TMV remaining after 4 hours of conjugation had occurred.

[0096] As illustrated in Figure 20, Western blot analysis of the conjugation product showed successful conjugation of H7 rhA to TMV via covalent attachment. Figure 20 shows Western blot analysis of various steps of the conjugation platform according to an embodiment of the present invention, where all samples were loaded at 10 μL. Each lane illustrates a different conjugation reaction time between antigen and virus. Lanes 14 and 13 show that all TMV rods were covered with antigen after 15 minutes. Lanes 6-9 after 2 hours show that no further conjugation occurred.

[0097] Example 8 - UV inactivation of TMV NtK To avoid viral contamination of biopharmaceutical products, it is often necessary to inactivate (or sterilize) the virus to ensure that it is no longer infectious. In addition, many regulatory agencies have enacted regulations (e.g., cGMP regulations) that mandate at least one effective inactivation step in the purification process of viral products. While UV-C radiation has been used in water treatment systems for many years, its use for biopharmaceutical products has remained unexplored and research on its ability to effectively inactivate viruses has been limited.

[0098] Therefore, various UV-C conditions (i.e., energy density and wavelength) and various TMV concentrations were evaluated to effectively inactivate and sterilize TMV NtK after virus production and purification and prior to conjugation with recombinant antigens. Although many energy densities were tested, only high levels of energy density efficiently inactivated TMV NtK. In addition, it was found that the success of virus inactivation was concentration-dependent, as UV-C irradiation did not effectively sterilize all viruses in the samples when the TMV solution was not diluted to the appropriate concentration. Therefore, the TMV solution must be appropriately diluted to allow UVC irradiation to interact with and effectively inactivate each virus.

[0099] As shown in Figure 21, various amounts of UVC irradiation (300 J / m 2 ~2400J / m 2 The energy density of 2400 J / m was tested on Nicotiana tabacum to evaluate infectivity. As shown in Figure 21, lesions were formed at 2400 J / m 2 After a UVC energy dose of 4800 J / m, the dose was reduced to zero, thus indicating good inactivation of the virus. In addition, many higher energy doses were also tested, up to 4800 J / m. 2 ~5142J / m 2 It was revealed that even at an energy density of 1000 nm, good inactivation of TMV NtK occurred.

[0100] According to multiple embodiments and alternatives, the viral inactivation steps (after purification, before conjugation) are as follows:

[0101] Dilute the TMV NtK solution to a concentration of less than 50 μg / ml as measured by A260 (a common method of quantifying nucleic acid by exposing a sample to UV light at a wavelength of 260 nm and measuring the amount of light that passes through the sample).

[0102] Filter the TMV solution at 0.45 microns to remove bacteria and any other large species that may interfere with UV light measurement, another purification step according to this embodiment, immediately prior to UV light inactivation.

[0103] Approximately 2400J / m 2 ~Approx. 5142J / m 2 Inactivating TMV NtK by exposing the virus to UV spectrum light having an energy density of about 4800 J / m. In some embodiments, the energy density of the UV light is about 4800 J / m. 2 ~Approx. 5142J / m 2 According to some embodiments and alternatives, the wavelength of the ultraviolet light is 254 nm.

[0104] The inactivated TMV NtK is then prepared for conjugation to a recombinant antigen.

[0105] These viral inactivation steps are designed for commercial scalability and compliance with cGMP regulations.

[0106] Example 9 - pH Dependence of Conjugation To evaluate whether incubating the virus at acidic pH would result in high quality conjugation, experiments were performed using the same batch of virus, antigen, buffer, and ester, varying only the virus formulation. In reaction 1, according to several embodiments and alternatives, TMV was formulated in 1×MES conjugation buffer at pH 5.50 at a concentration of 3.1 mg / ml. In reaction 2, TMV was concentrated to 11.0 mg / ml in phosphate buffer and added directly as 15% of the conjugation reaction volume. Following these steps, the conjugation process was monitored by SEC, where an orderly decrease in free TMV from 0 min (denoted by T=0) indicates good conjugation.

[0107] As shown in Tables 7 and 8, reaction 1 showed successful conjugation (orderly decrease in free TMV from 0 min) while reaction 2 was unsuccessful as indicated by the percent of TMV remaining free.

[0108] [Table 7]

[0109] [Table 8]

[0110] Thus, incubation of the virus at acidic pH results in greater than 90% conjugation, as shown in Table 7. Without the acidic pH incubation step, % conjugation remains below 50% (shown in Table 8).

[0111] Based on this experiment, a model of conjugation (shown in FIG. 22) was developed. According to embodiments and alternatives, conjugation between purified virus and purified antigen (shown as "rHA" in FIG. 22) is greatly enhanced by improving the chemical readiness of the virus (referred to herein as "activating", "activating" or "activating") to associate with the antigen by exposing the virus to a conjugation environment. In some embodiments, virus activation is achieved by formulating the virus at an acidic pH prior to the conjugation reaction to concentrate positive charges on the virus surface. In some embodiments, the activation step includes exposing the virus to a pH of about 5.5 or less for a time sufficient for activation. In some embodiments, such exposure time to the conjugation environment is about 18-72 hours. According to embodiments and alternatives, treating the purified virus at an acidic pH activates the virus by charging the lysines of the coat protein. This activation step results in amine groups and positive charge concentrations on the viral surface (as shown in Figure 22) via viral clustering in a conjugation environment, ready for conjugation to the carboxyl terminus of recombinant antigens.

[0112] The virus activation step according to several embodiments and alternatives contrasts with conventional approaches, where the pH of the virus during storage is generally maintained at or near neutral pH. As shown in Figure 22, conventional approaches do not concentrate positive charges on the virus surface, resulting in % conjugation remaining below 50% (see Table 8). Additionally, conventional approaches utilize phosphate buffers that promote solubility at the expense of having a favorable surface charge.

[0113] In investigating successful conjugation with TMV, it was observed that successful conjugation generally occurs when the radius of the virus, as measured by dynamic light scattering (DLS), increases by at least 2.75-fold during the activation step (see Table 9A, compared to Table 9B). Generally, as shown in these tables, successful TMV conjugation (e.g., as discussed in Table 9C) was characterized by an increase in DLS radius of about 70 nm to about 195 nm or more.

[0114] Based on the successful conjugation using viral activation, a platform for conjugating purified antigens to purified viruses was developed. According to several embodiments and alternatives, the steps for preparing purified antigens for conjugation are as follows: To ensure pH control of the conjugation reaction, the purified antigen is formulated into the reaction buffer immediately prior to initiation of the reaction.

[0115] Prior to conjugation, the purified antigen is stored in phosphate buffered saline at slightly neutral to basic pH.

[0116] Depending on the nature of the molecule, the target pH for an antigen is typically pH 5.50-6.50.

[0117] To facilitate conjugation to the virus, the storage buffer is exchanged into a MES / NaCl buffer by ultrafiltration using an acidic pH and the protein concentration is increased to >3 mg / mL.

[0118] The conjugation reaction is then initiated within 4 hours of completing the antigen preparation to prevent destabilization of the protein structure.

[0119] According to multiple embodiments and alternatives, the steps for preparing purified virus for conjugation are as follows: After storage at neutral pH, the virus is activated at acidic pH before conjugation. For good reaction, the virus is formulated from a phosphate buffer at pH 7.4 into an acetate buffer at pH 5.50 for a minimum of about 18 hours and a maximum of about 72 hours before the start of the conjugation reaction. In some embodiments, the virus is formulated from a phosphate buffer at pH 7.4 into an acetate buffer at pH 4.50 for a minimum of about 18 hours and a maximum of 72 hours before the start of the conjugation reaction. It has been observed that storing the virus at acidic pH for more than 72 hours leads to self-association between the viruses, which makes the virus less soluble and reduces the conjugation efficiency.

[0120] Tables 9A and 9B further demonstrate that the activation step proceeds by increasing the radius of the virus (in this case TMV) as measured by DLS. Specifically, Table 9A shows data in which the DLS radius of TMV was increased after activation and before successful conjugation with the antigens listed in the right column. The "fold increase in radius" is the TMV radius after activation divided by the typical TMV radius of about 70 nm at neutral pH. Conversely, Table 9B shows data in which the DLS radius of TMV was increased after the activation step was started and prior to unsuccessful conjugation attempts with the antigens listed in the right column. In Tables 9A and 9B, the left column represents the standard radius of TMV rods at neutral pH and typical storage conditions, i.e., before activation.

[0121] [Table 9] TIFF2025076429000015.tif66166

[0122] [Table 10]

[0123] After these preparation steps, the antigen and virus reactants were mixed to form a conjugate mixture, and the progress of conjugation was monitored using DLS and SDS-PAGE methods. Table 9C shows the average molecular radius over the time course of the conjugation reaction after the virus was activated using acidic pH using DLS. As shown in Table 9C, the molecular radius is one indicator of successful coating of the virus rods with antigen molecules.

[0124] [Table 11]

[0125] Next, Figure 23 shows an SDS-PAGE based analysis of conjugation between activated TMV NtK and purified antigen, according to multiple embodiments and alternatives. As shown in Figure 23, the orderly decrease of both free TMV NtK and free antigen, corresponding to the appearance of a >200 kDa protein band, over time indicates successful conjugation.

[0126] Example 10 - TEM imaging of conjugations with different ratios of purified virus to purified antigen The desired conjugation reaction between purified virus and purified antigen is represented by the following formula: Virus + antigen → virus·antigen (Equation 1)

[0127] However, antigens are known to be prone to self-conjugation, which may result in less than desirable activity, as shown in the following formula: Virus + antigen → virus·antigen + antigen·antigen (Equation 2)

[0128] Self-conjugation of purified antigens is a challenge to successful vaccine development because antigen-antigen conjugates are not removed during the size chromatography step, resulting in a minimized or reduced immune response.

[0129] To address this self-conjugation challenge, various experiments were performed to determine how to consume unreacted antigens and antigen conjugates. First, the antigens were capped by exposing them to a self-conjugation suppression reagent. Although this conventional approach was expected to be successful, it failed because the reaction occurred too rapidly.

[0130] The virus-to-antigen ratio was then adjusted to determine the appropriate conjugation ratio. Seven different samples were analyzed by performing negative staining transmission electron microscopy (TEM) imaging, as shown in Tables 10 and 11 and Figures 24-30. Samples 1-3 served as controls, while samples 4-7 contained different hemagglutinin (HA)-to-TMV ratios (mixing step of the conjugation platform, as shown in operational step 5 in Table 3).

[0131] [Table 12]

[0132] [Table 13]

[0133] In the various designations recited herein, "KBP-VP" refers to TMV Antigen Presentation and is provided for reference purposes only. Figure 24 is a TEM image of Sample 1 (free HA, Lot 19UL-SG-001) at 52,000x magnification and a scale bar of 200 nm. In Figure 24, this sample contained small spherical (indicated by arrow A) and elongated particles (indicated by arrow B) ranging in size from about 5 nm to about 9 nm. The appearance of these particles indicates a regular structure consistent with ordered aggregation of HA, which matches the native trimeric conformation. In addition, the particles were well dispersed by minimal clumping.

[0134] FIG. 25 is a TEM image of sample 2 (TMV NtK alone, lot 18HA-NTK-001) at 52,000× magnification and 200 nm scale bar. In FIG. 25, rod-shaped particles (arrow A) were observed with sizes varying from about 125 nm to about 700 nm in length and about 18 nm to about 20.5 nm in width. These dimensions are consistent with the size and shape of TMV particles. In addition, a central about 4 nm channel was observed in the rod (arrow B), which is a known feature of TMV. Multiple rods were often aligned parallel to their long axes, and the surfaces of the rods were generally smooth. In some cases, small about 8 nm to about 10 nm spherical particles (arrow C) were observed, associated with the surface of the rods but not with the rod-shaped particles in the background. These spherical particles (arrow C) did not resemble individual HA trimers.

[0135] FIG. 26 is a TEM image of Sample 3 (HA:HA self-conjugate, spiked with TMV NtK, Lot 19UL-SG-004) at 52,000× magnification and 200 nm scale bar. In FIG. 26, rod-shaped particles were observed that varied in length from about 25 nm to about 885 nm, width from about 18 nm to about 20.5 nm (arrow A), and a central about 4 nm inner channel (arrow B). The rods were either not decorated at all or sparsely decorated with small, proteinaceous particles of various sizes and shapes (arrow C). Some of the small, proteinaceous particles were also seen in the background and were not associated with the rods (arrow D). FIG. 26 shows large clumps of HA particles, but the TMV appeared identical to the unconjugated TMV (shown in FIG. 25) as expected.

[0136] FIG. 27 is a TEM image of Sample 4 (TMV:HA, 1:1 ratio, Lot 18 KBP-VP-SG-002) at 52,000× magnification and 200 nm scale bar. In FIG. 27, rod-shaped particles were observed that varied in length from about 50 nm to over about 1000 nm, widths from about 18 nm to about 20.5 nm (arrow A), and a central about 4 nm inner channel (arrow B). The particle rods were similar in size and shape to the conjugated TMV observed in FIG. 28, with the exception that most of the rods were heavily decorated with a density of small proteins on their surfaces (arrow C). Some of the small, proteinaceous particles were also seen in the background and were not associated with the rods (arrow D). Sample 5 shown in FIG. 27 appears superior to the other TEM images, most likely due to differences in virus treatment prior to conjugation. For this batch, the virus was prepared at pH 5.50, the pH was lowered to 4.50 over 15 minutes, and returned to pH 5.50 at the start of the conjugation reaction. For the batch shown in Figures 28-30, the virus was formulated directly at pH 4.50 and held overnight prior to conjugation.

[0137] FIG. 28 is a TEM image of sample 5 (TMV:HA, 1:1 ratio, Lot 19UL-SG-001) at 52,000× magnification and 200 nm scale bar. In FIG. 28, many rod-shaped particles were observed, varying in length from about 65 nm to about 720 nm, width from about 18 nm to about 20.5 nm (arrow A), and a central about 4 nm inner channel (arrow B). The particle rods were similar in size and shape to the free TMV NtK (sample 2) observed in FIG. 25. However, in contrast to the unconjugated virus shown in FIG. 25, the particle rods observed in FIG. 28 were decorated with a modest density of proteins (arrow C). These densities were irregular in shape and size and appeared to be randomly associated with the surface of the rods with no obvious pattern. Some small, proteinaceous particles were also seen in the background and were not associated with the rods (arrow D).

[0138] Figure 29 is a TEM image of sample 6 (TMV:HA, 1:4 ratio, lot 19UL-SG-002) at 52,000x magnification and 200 nm scale bar. In Figure 29, rod-shaped particles were observed that varied in length from about 25 nm to over about 1000 nm, with widths ranging from about 18 nm to about 20.5 nm (arrow A), and a central about 4 nm inner channel (arrow B). The particle rods observed in Figure 29 were similar in size to the previously conjugated sample, but the level of surface modification with small protein density (arrow C) varied from moderate to sparse. Some of the small, proteinaceous particles were also seen in the background and were not associated with the rods (arrow D).

[0139] FIG. 30 is a TEM image of Sample 7 (TMV:HA, 1:16 ratio, Lot 19UL-SG-003) at 52,000× magnification and 200 nm scale bar. In FIG. 30, rod-shaped particles were observed, about 30 nm to about 1000 nm or more in length, about 18 nm to about 20.5 nm in width (arrow A), with a central about 4 nm inner channel (arrow B). The particle rods observed in FIG. 30 were similar in overall morphology to the previous conjugated samples. However, the rods were only sparsely decorated with protein (arrow C) or not decorated at all. Small, proteinaceous particles were seen in the background and were not associated with the rods (arrow D).

[0140] Figures 24-30 show that the 1:1 ratio showed complete rod decoration, the 4:1 ratio showed moderate decoration, and the 16:1 ratio showed sparse decoration. In other words, the 1:1 ratio generated viral rods with heavy antigen decoration (i.e., more density) of HA antigens, while the 16:1 ratio generated viral rods with less antigen decoration (i.e., less density) of HA antigens on each rod. As a by-product of the conjugation reaction, HA-HA self-conjugates were observed mainly in the 1:1 ratio reaction. Furthermore, in TEM images as well as reaction analysis by SDS-PAGE, less free HA or HA-HA conjugates were seen in the 4:1 reaction and less in the 16:1 reaction compared to the 1:1 reaction (data not shown). In other words, the conjugation efficiency of exclusively HA to TMV rods was higher overall at the 16:1 ratio, but the density of HA per rod was less than that of the 1:1 reaction.

[0141] Example 11 - Sedimentation velocity analysis of different conjugation conditions Sedimentation velocity ("SV") measured in an analytical ultracentrifuge ("AUC") is an ideal method to obtain information about protein heterogeneity and the association state of aggregates. Specifically, aggregates or different oligomers can be detected on the basis of different sedimentation coefficients. This method also detects aggregates or other trace components at levels below 1% by weight. Moreover, SV provides high quality quantification of the relative amounts of species and provides accurate sedimentation coefficients for any aggregates.

[0142] To determine the content of self-conjugated, unreacted HA and the amount of HA occupancy on TMV NtK by different conjugation conditions, the total signal associated with free antigen, free virus and centrifugation of various TMV:HA ratios was measured using SV-AUC. The following samples and descriptions are shown in Table 12:

[0143] [Table 14]

[0144] These stocks were shipped refrigerated (not frozen) and stored at 2-8°C until further analysis. 1X PBS from Corning was used as a blank reference for sample dilution. Sample 1 was diluted 1:1 and samples 2-7 were diluted 1:3 in 1X PBS to prepare samples for sedimentation velocity. These dilutions were made to ensure that the total absorbance of the samples was within the linear range of the absorbance detection system.

[0145] Methods: Diluted samples were loaded into cells with a two-channel Charcoal-Epon centerpiece with an optical path length of 12 mm. 1X PBS was loaded into the reference channel of each cell. The loaded cells were placed into the analytical rotor, loaded into an analytical ultracentrifuge, and cooled to 20°C. The rotor was then spun at 3000 rpm and samples were scanned (280 nm) to confirm proper cell loading. For samples 2-7, the rotor was rotated to a final speed of 9,000 rpm. Scans were recorded at this rotor speed (every 3 min) as fast as possible for approximately 11 h (250 total scans for each sample). For sample 1 (free HA), the rotor was rotated to 35,000 rpm and scans were recorded every 4 min for 5.3 h. The data were then analyzed using the c(s) method described in Schuck, P. (2000), "Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling", Biophys. J. 78, 1606-1619. Using this method, the raw scans were directly fitted to derive the distribution of sedimentation coefficients, while the effects of diffusion on the data were modeled to enhance resolution.

[0146] Results and Discussion: High-resolution sedimentation coefficient distributions for samples 1-7 are shown in Figures 31-37. In these figures, the vertical axis shows concentration and the horizontal axis shows separation based on sedimentation coefficient. To ensure that the area under each peak represents the proportion of that species, each distribution was normalized by setting the total area under the curve to 1.0 (100%). Because samples 2-7 contain material that precipitates over a wide range of sedimentation coefficients, the data analysis was pushed to cover species that precipitate as rapidly as 2000 Svedburg units (S), so that the horizontal axis is log scaled. To compensate for the fact that log scaling can distort the visible areas of the peaks, the vertical axis is multiplied by the sedimentation coefficient, so that the relative peak areas are correctly scaled. The data for sample 1 (free HA) is conventionally shown using a linear sedimentation coefficient scale.

[0147] Figure 31 shows the distribution of normalized sedimentation coefficients for sample 1 (HA alone, lot 19S-G-001). Because the free antigen is much smaller in size than the virus, this sample was analyzed at a much faster rotor speed (35,000 rpm) than samples 2-7 (9,000 rpm) to characterize the proper particle size distribution. As shown in Figure 31, sample 1 is somewhat homogeneous and shows a 73.7% main peak at 8.967 S. This was the expected result for a sample with only HA antigen. The width of the main border and this sedimentation coefficient imply that this main peak species has a molar mass of about 222 kDa, which may indicate that this main peak corresponds roughly to the expected trimer of about 70 kDa monomer of HA. It is physically illogical for this sedimentation coefficient to correspond to a monomer, instead the main peak corresponds to an oligomeric state larger than the monomer. As highlighted in the SEC HPLC data for HA3 Singapore release in Table 13 below, >90% of the HA was identified in the trimeric state, with three out of four samples analyzed having greater than 50% trimer formation.

[0148] [Table 15]

[0149] As also shown in Figure 31, seven minor peaks were detected that sedimented more rapidly than the main peak, together representing 6.2% of the total sedimentation absorbance. Presumably, those two peaks represent product aggregates rather than high molecular weight impurities. The major aggregate species of 12.4S (4.25%) sedimented 1.4 times more rapidly than the monomer, a ratio that falls within the range of 1.4-1.5 typically observed for dimers. The ratio suggests that this species is a dimer of the main peak material (probably a hexamer of the approx. 70 kDa monomer), and its sedimentation coefficient may suggest that it is a highly extended or partially open trimer of the main peak material (probably a nonamer of the approx. 70 kDa monomer).

[0150] In Figure 31, the next peak at 15.3S (0.96%) sedimented 1.7 times faster than the monomer, suggesting trimerization of the main peak material. No absorbance was detected with a sedimentation coefficient greater than 30.9S. Also, three minor peaks were detected that sedimented more slowly than the main peak: 2.8S (2.81%), 4.5S (12.44%), and 6.0S (4.94%). Of these minor peaks, the 4.5S peak most likely corresponds to the antigen monomer.

[0151] Figure 32 is the distribution of normalized sedimentation coefficients for sample 2 (free TMV NtK, lot. 18HA-NTK-001). As shown in Figure 32, no sedimenting material was detected at approximately 60S. This sample appeared very heterogeneous, with the most abundant peak being sedimentation at 229S (30.9%). The second most abundant peak was detected at 191S (28.7%). It is not clear which peak corresponds to fully assembled virus. In addition, 25.3% of the total signal was observed as sedimentation from 229S to 2,000S, the largest sedimentation coefficient seen in this Example 11. It is unclear what the partially split peak from approximately 60S to 2000S represents.

[0152] Figures 33-37 show the normalized sedimentation coefficient distributions of virus-antigen conjugates. Each of these figures shows a significant absorbance of approximately 0.15 OD that did not precipitate. This was established by increasing the rotor speed to 35,000 RPM after completion of each run to pellet all remaining material. This material was not observed in either the free antigen or free TMV NtK samples. However, this material did not precipitate and therefore did not affect the measured size distribution results.

[0153] Figure 33 is the distribution of normalized sedimentation coefficients for sample 3 (lot 19UL-SG-004 at a 1:1 ratio of TMV to HA). As shown in Figure 33, the results in sedimentation coefficient ranged from about 40S to 2000S, similar to the results observed with free virus (shown in Figure 33). Three peaks were also observed with sedimentation coefficients ranging from 1 to 40S: 9.9S (28.3%), 18.7S (7.8%), and 34.5S (1.0%). The peak observed at 9.9S may correspond to the main peak observed in the free HA sample (shown in Figure 32). The diversity of smaller peaks may reflect HA-HA self-conjugation events.

[0154] Figure 34 is the distribution of normalized sedimentation coefficients for sample 4 (lot 18 KBP-VP-SG-002 with a 1:1 ratio of TMV to HA) and Figure 35 is the distribution of normalized sedimentation coefficients for sample 5 (lot 19 UL-SG-001 with a 1:1 ratio of TMV to HA). The results shown in Figures 34 and 35 are similar to those described for sample 3 (and shown in Figure 33). However, some notable differences were observed. First, due to the low resolution at this rotor speed, it is difficult to note the differences observed for the free antigen samples (1-40S). Nevertheless, Figures 34 and 35 show that there is more total signal from 40S-2,000S (indicating virus-related material) than from sample 3.

[0155] Figure 36 is the distribution of normalized sedimentation coefficients for sample 6 (lot 18 19UL-SG-002 with a 4:1 ratio of TMV to HA) and Figure 37 is the distribution of normalized coefficients for sample 7 (lot 19UL-SG-003 with a 16:1 ratio of TMV to HA). Figure 36 shows 91.1% virus-related material (i.e., virus-antigen conjugate) and Figure 37 shows 99.4% virus-related material (i.e., virus-antigen conjugate).

[0156] The virus-antigen normalized sedimentation coefficient distribution results shown in Figures 33-37 are shown in Table 14. As mentioned above, according to various embodiments and alternatives, the fraction between 1-40S indicates the percent HA monomer / trimer, and the fraction between 40-2000S indicates the percent TMV NtK-HA conjugates.

[0157] [Table 16]

[0158] The results in Table 14 show that the 1:1 ratio has more HA self-conjugates and HA products compared to the 4:1 and 16:1 ratios. Furthermore, increasing the TMV:HA ratio results in substantially complete association of the HA products in the TMV conjugation event (approaching nearly 100% conjugation in sample 7).

[0159] According to several embodiments and alternatives, decreasing the amount of HA in the conjugation reaction by increasing the TMV NtK to HA ratio from 1:1 to 16:1 (1) reduces the aggregation of HA antigen on each TMV rod, as observed by Example 10 and Figures 24-30; (2) reduces the amount of self-conjugated and unreacted HA events to nearly zero, as shown in Figures 31-37 and Table 14; and (3) increases the association (percentage) of HA to TMV compared to self-conjugated and unreacted HA events, as shown in Figures 31-37 and Table 14.

[0160] Example 12 - Immune responses in mice To determine the immune response following administration of the virus-antigen conjugates of the present invention, mice were administered the conjugates as a vaccine via intramuscular injection. Each vaccine was a TMV:HA conjugate produced at a 1:1 (TMV:HA) ratio as described herein and was administered to most animals on days 0 and 14 of the study (control animals received buffer alone, TMV alone, or HA alone). Vaccines administered received either 15, 7.5, or 3.75 mcg (micrograms) of antigen, as shown in Table 15 below. One cohort was sampled on day 7, another on days 14 and 21, and a third on days 28, 42, and 90, and the samples were then subjected to a hemagglutination inhibition (HAI) assay.

[0161] Based on this assay, no animals had measurable responses to either vaccine on days 7 or 14. However, early responses were noted in some animals on day 21. Specifically, 10 / 27 animals responded at low levels (only one of them >80 HAI titer) to the H1N1 vaccine (Influenza A / Michigan / 45 / 2015 (H1N1pdm09)), and 22 / 27 animals responded at low levels (only two of them >80) to the H3N2 vaccine (Influenza A / Singapore / INFIMH-16-0019 / 2016). On day 28, the number of animals in this cohort measurably responding to the H1N1 vaccine was 8 / 29, with a single animal at an 80 HAI titer and all others lower. For the H3N2 vaccine, there were 14 / 29 measurable responses, with one animal having an HAI titer of 80, all others lower.

[0162] The most striking results were observed from blood samples taken on days 42 and 90, and are shown in Table 15 below. In this table, the standard error of the mean (SEM) is shown along with the mean and percentage of responding animals (Fr.Resp.). Note that in each cohort, some of the mice were vaccinated against influenza B virus (B / Colorado / 06 / 2017(V) and B / Phuket / 3073 / 2013(Y) respectively). As expected, no responses were detected in these animals on any of the days, since influenza B virus and the corresponding HA immunogens are known not to generate HAI titers in mice with the efficiency and effectiveness as type A HA immunogens.

[0163] [Table 17] TIFF2025076429000024.tif19169

[0164] Apart from the immune response studies mentioned above, to further evaluate the system of the present invention with respect to suitable virus-to-antigen ratios, humoral immune responses in mice were evaluated after vaccination with various TMV:HA conjugate ratios (i.e., 1:1, 4:1, 16:1) of both influenza A and influenza B antigens, along with controls, as described below. In this way, different conjugation ratios and their effect on immune responses were tested. Vaccinated mice were administered 15mcg of HA by injection in the dorsal subcutaneous area on days 0 and 14 of the study. Serum antibody responses to vaccination were then analyzed for HA-specific activity. Tables 15 (H3 influenza virus used as capture protein) and 16 (recombinant H3 protein used as capture protein) show the groups of mice (12 mice per group) and the drugs administered, and the right-hand column of each table shows the results of ELISA antibody (Ab) titers.

[0165] [Table 18]

[0166] Figure 38 is a scatter plot related to Table 16, providing a graphical analysis of H3:HA antibody titers after administration of vaccines at ratios of 0, 1:1, 4:1, and 16:1 (TMV:HA). Figure 39 also illustrates the results of a geometric mean antigen-associated Ab titer study using recombinant H3 antigen (Table 17) as a coating or captured H3 virus (Table 17) as a capture protein that binds to anti-influenza A H3 antigen antibodies. In terms of density (surface area of ​​TMV occupied by HA), the trend of the three ratios was in the order 1:1 (highest charge density)>4:1>16:1 (lowest charge density), as demonstrated by TEM and AUC analysis. In these figures, which represent ELISA results obtained with the H3 antigen, the highest immune response was observed when the lowest density conjugate was used. That is, the trend of immune response was 16:1>4:1>1:1, which was the opposite of the trend of density. Thus, it was surprisingly found that at these ratios of TMV:HA, low conjugation density tends to provide a better immune response. Explanations for this surprising finding that antigenicity does not correlate with maximum HA conjugation events include: (1) at a relatively low density, more uniformity of antigens, as well as less unreacted or self-conjugated proteins, (2) more efficient processing of conjugated antigens and more conserved / uniform conformation of antigens, and (3) (for example) TMV rods may stimulate more antigen-presenting cells to migrate to the injection site and stimulate processing of attached antigens or some combination of these factors. However, it should be noted that the presence of TMV particles alone does not replace the need for conjugation (see, for example, Tables 14 and 15).

[0167] In addition to the Influenza A H3 antigen, an Influenza B antigen (B-Phuket HA) was also tested using recombinant Influenza B Phuket antigen and its corresponding antibody binding trends. Table 17 below shows the results of this part of the study which were not found to show 16:1>4:1>1:1 based on the mean ELISA Ab titer results.

[0168] [Table 19]

[0169] Nevertheless, the 16:1 ratio showed the highest mean antibody titers. It is therefore reasonable to expect the same relationship between density and immune response to apply to the testing of influenza B antigen (B-Phuket HA). That is, similar to the results for the H3 antigen, the immune response would be higher for the formation of lower density conjugates. There is also reason to believe that the conjugation reaction at the 4:1 ratio did not proceed as well as the reactions at the other ratios, possibly due to anomalies during conjugation, and no electron microscopy or ultracentrifugation analysis was performed on this sample. In any case, the data here show immune responses at all three ratios. The fact that immune responses were seen at multiple ratios highlights the robustness of the system in not being bound to any one particular ratio. This flexibility seen with certain TMV conjugate vaccines perhaps provides further indication that the system will also work well when other antigens are conjugated to TMV in addition to the H3 and H1 antigens included in these tests, as well as when other viral carriers are used as carriers in addition to TMV.

[0170] With regard to clinical utility, products conjugated according to any of the embodiments and alternatives described herein may be utilized as vaccines by delivering purified antigens via purified viruses, such as, but not limited to, the viral antigen conjugates described in Examples 7, 9, 10, 11, and 12. Still further, embodiments of the present disclosure include any vaccine product produced from any of the virus-protein conjugate compositions and conjugates thereof provided herein, packaged in any number of forms (e.g., vials) with appropriate buffers and additives. In this regard, embodiments include those in which such vaccine products are suitable for delivery in the form of a unit dose provided to a subject, such as, but not limited to, administration by syringe or spray via routes such as subcutaneous, intramuscular, intradermal, and nasal, and, to the extent clinically appropriate, oral administration by mouth and / or topical administration. As a non-limiting example, and without prejudice to the breadth and scope of the embodiments herein, the size of TMV (typically 18 nm x 300 nm) and its rod-like shape facilitate antigen uptake by antigen-presenting cells (APCs), thus enhancing immunity driven by T cells (such as Th1 and Th2) including cellular responses, and providing adjuvant activity to surface-conjugated subunit proteins. This activity is also stimulated by viral RNA / TLR7 interactions. As a result, the combined effect of vaccine uptake directly stimulates APC activation. Humoral immunity is typically maintained in balance between IgG1 and IgG2 subclasses via subcutaneous and intranasal delivery. Responses upon mucosal vaccine delivery also include substantial systemic and mucosal IgA. Cellular immunity is also very robust, inducing antigen-specific secretion similar to live virus infection responses. Whole antigen fusion allows epitope processing by natural cytotoxic T lymphocytes (CTLs) without regard for human leukocyte antigen (HLA) variance.

[0171] The broad (humoral and cellular) and enhanced (amplified and effective) immune responses associated with the multi-set purification platform according to the present embodiment are in sharp contrast to the subunit proteins tested without TMV conjugation, which induce little or no cellular or humoral immunity. The impact of these immune responses is that the vaccines produced via the multi-set platform according to the present embodiment promote highly protective responses as single-dose vaccines, offering speed and safety not offered by other conventional vaccine platforms. Indeed, the conjugation platform has been shown to be combined in a wide range of ratios, administered well at various doses, and work with a wide range of viruses and proteins (including antigens), again demonstrating the robustness of the system. Further advantages of the multi-set platform for producing vaccines in the present embodiment include a proactive antigenic stimulation approach for systemic immune defense against pathogen challenge, which is highly adaptable to produce antigen domains from disease pathogens (including viral glycoproteins or non-secreted pathogen antigens), and which serves as an effective vaccine platform against both viral and bacterial pathogens.

[0172] In addition to the advantages for vaccination, plant viral particles produced via the multi-set platform according to embodiments of the present invention can be formulated for various drug delivery purposes, which may include: 1) immunotherapy: by conjugating therapeutic antibodies to the surface of the viral particles and delivering them to enhance the cytotoxic effect, 2) gene therapy: by loading specific nucleic acids for introduction into specific cell types for genetic modification, and 3) drug delivery: by loading chemotherapeutic agents onto viral particles for delivery to target tumors.

[0173] To give a brief example of the many advantages of the method discussed herein, in the multi-set platform according to several embodiments, purified virus can be utilized as a drug delivery tool by first exposing it to a pH shift as discussed above to cause it to swell.The virus in this state is then incubated with a solution of a concentrated chemotherapeutic agent, such as doxorubicin, and then the pH is returned to neutral, thereby returning the virus to its pre-swelling state, thereby capturing the chemotherapeutic agent molecules.The virus particles can then be delivered to an organism by a delivery mechanism, such as, but not necessarily limited to, injection for targeted treatment of tumors.

[0174] Thus, the above description provides multiple embodiments and several alternative approaches for (i) plant-based production and purification of viruses, (ii) plant-based production and purification of antigens, and (iii) formation of virus-antigen conjugates outside plants that are therapeutically useful as vaccines and antigen carriers, and (iv) delivery of therapeutic vaccines comprising purified viruses and purified antigens.

[0175] Example 13 - Vaccine stability under refrigerated and room temperature conditions Vaccines have dramatically improved human and animal health. For example, in the 20th century alone, vaccines have eradicated smallpox, eliminated polio in the Americas, and controlled a variety of diseases around the world. However, vaccines are highly unstable and highly sensitive to temperature changes. As discussed in F. Coenen et al., Stability of influenza sub-unit vaccine. Does a couple of days outside the refrigerator matter? Vaccine 24 (2006), 525-531, influenza vaccines generally cannot be stored at room temperature (i.e., about 25°C) for 5 weeks or they will become inactivated. Of all the influenza vaccines discussed in F. Coenen's paper, only one vaccine showed stability at room temperature for 12 weeks. In many situations, this is also a significant issue for other types of vaccines, as well as for individual specific components of the vaccine (e.g., intermediates). Thus, vaccines generally must be kept refrigerated throughout the entire supply chain, from the moment of production by the manufacturer to administration, often referred to as the "cold chain."

[0176] While in a refrigerated environment, the majority of vaccines remain stable for the typical target period of 78 weeks of stability. However, the absolute requirement for a cold chain is often difficult to ensure in developing countries, resulting in widespread vaccine loss, and is therefore a global issue limiting vaccine availability worldwide. Many efforts have been made to create vaccines with room temperature stability, but as discussed in the literature, these efforts have not been successful. Furthermore, the cold chain is very costly to maintain for manufacturers, as well as for physicians and organizations that receive, store, and administer vaccines to the population. Thus, there is a critical and global need to increase vaccine stability and enhance vaccine antigen stability to reduce the dependency on the cold chain and ensure that the vaccine retains its potency until administration. With regard to the stability of the antigen itself, i.e., as an intermediate ready to be conjugated with a suitable viral carrier, there are advantages to maintaining antigen stability after production and purification and before conjugation. These advantages include, but are not limited to, the ability to manufacture the antigen in a separate manufacturing facility or at a different time than the production and purification of the viral carrier. This allows for more flexibility in the supply chain. Furthermore, improving stability can extend the shelf life of the vaccine, which facilitates stockpiling of vaccines in preparation for potential pandemics and prevents vaccine loss under inappropriate conditions. The scope of the present embodiments, together with other features and advantages outlined herein, meet these and other needs. In doing so, the purification and conjugation platform of the present invention advances the stability of protein-virus conjugates under both refrigerated and room temperature conditions.

[0177] There are several methods to measure the quality of the antigen and the stability of the vaccine, for example: (1) Protein concentration measured by BCA protein assay (proteins react with Cu in alkaline solution). 2+ Cu +1(2) preservative efficacy as measured by VaxArray® antibody array binding (utilizing a multiplex sandwich immunoassay); (3) purity as a single migration band on SDS-Page; (4) pH as a measure of physical contamination properties; and, where possible, (5) size exclusion chromatography to characterize the multimeric structure of the antigen. In some embodiments, bioburden testing is also performed to analyze the presence of bacterial or mold contamination (in the table below, "TAMC" is an abbreviation for total aerobic microbial count and "TCYM" is an abbreviation for total yeast / mold count). Furthermore, if the vaccine fails the BCA protein assay, the VaxArray® test, or the SDS-Page analysis, the vaccine is considered unacceptable for use. In other words, if the vaccine fails any one of these three tests, the vaccine is unacceptable for use and is inactive. The VaxArray® potency test is used to assay for improved susceptibility to and efficacy of vaccines in treating a variety of different virus strains, including SARS-CoV2 and pandemic strains of influenza. Some aspects of the VaxArray® assay related to influenza viruses and vaccines are described in Byrne-Nash, Rose T. et al., "VaxArray potency assay for rapid assessment of 'pandemic' influenza Vaccines", npj vaccines 3, 43 (2018). With the 2020 SARS-CoV-2 pandemic, the VaxArray® potency assay is commercially available for testing coronaviruses, vaccines and vaccine intermediates.

[0178] Thus, the five studies referred to in the above paragraphs were performed on the following influenza HA antigens produced and purified according to multiple embodiments and alternatives: H1NI (A / Michigan), H3N2 (A / Singapore), H1N1 (A / Brisbane), H3N2 (A / Kansas), B / Colorado, and B / Phuket. The following table shows the stability data and preservative efficacy measured at release and at various times after filling into vials and storing under refrigerated conditions (2-8° C.). As used herein, initial concentration or integrity refers to the concentration or integrity of a compound, conjugate mixture, drug product, vaccine, etc., at the date of its release (i.e., after constitution or dilution of the drug product, often referred to as "day 0"), as determined in accordance with 21 CFR Part 11 and ICH Q1A Stability Testing of New Drug Substances and Products, Revision 2 (November 2003), and references cited therein, the entire contents of all of the above being incorporated herein by reference for all purposes.

[0179] [Table 20]

[0180] [Table 21]

[0181] [Table 22]

[0182] [Table 23]

[0183] [Table 24]

[0184] [Table 25]

[0185] Tables 18-23 show that purified free antigens show different patterns of stability. For example, some antigens, such as H1NI (A / Michigan) and H3N2 (A / Singapore), appear to be stable after 6 months with no significant deviations in measurements (as typically observed). However, other antigens, such as B / Colorado and H1N1 (A / Brisbane), and to a lesser extent H3N2 (A / Kansas) and B / Phuket, showed degradation, loss of trimer, or loss of other important properties under these conditions. For example, Figure 40 shows an SDS-PAGE analysis of purified B / Phuket after 1 month under refrigerated conditions. In Figure 40, there is a degradant band with a lower molecular weight than the intact band at about 60 kDA, indicating that the purified B / Phuket antigen has degraded. As expected, the data in Tables 18-23 and Figure 40 show that different proteins show different stability under refrigerated conditions.

[0186] When the same purified antigen is conjugated to TMV, the stability profile and storage efficacy change according to several embodiments and alternatives. In some embodiments, the method of the present invention enhances a measure of stability of a conjugated compound comprising a protein and a viral particle, comprising activating the viral particle and then mixing the viral particle and antigen in a conjugation reaction to form a conjugate mixture, thereby enhancing the stability of the conjugated compound when placed in a non-refrigerated environment and after a period of at least 42 days after the release date. An exemplary storage temperature is at least 20°C. The enhanced stability can be measured by comparing the stability of the conjugated mixture to that of the antigen alone. Suitable measures are any one or more of antigen concentration, antigen integrity, or antigen potency. For example, if the measure of stability is antigen concentration measured by BCA or other suitable method, a difference of at least 10% between the concentration of the conjugated compound and the concentration of the antigen alone is within the scope of this embodiment. Similarly, where the measure of stability is antigen integrity as measured by SDS-PAGE, SEC-HPLC or other suitable method, a difference of at least 10% between the integrity of the conjugated compound and the integrity of the antigen alone is within the scope of this embodiment. Similarly, where the measure of stability is antigen-antibody interaction based on ELISA results, or antigen potency as measured by VaxArray®, surface plasmon resonance or other suitable methodology, a difference of at least 30% between the potency of the conjugated compound and the potency of the antigen alone is within the scope of this embodiment.

[0187] Thus, the following table provides stability data for several monovalent formulations (1:1 TMV to antigen ratio) at the time of release and at various times after filling into vials and storage under refrigerated conditions (2° C. to 8° C.):

[0188] [Table 26]

[0189] [Table 27]

[0190] [Table 28]

[0191] [Table 29]

[0192] In addition, the following table provides stability data for several monovalent formulations (TMV to antigen ratio 8:1) at the time of release and at various times after filling into vials and storage under refrigerated conditions (2° C. to 8° C.):

[0193] [Table 30]

[0194] [Table 31]

[0195] [Table 32]

[0196] [Table 33]

[0197] [Table 34]

[0198] [Table 35]

[0199] [Table 36]

[0200] [Table 37]

[0201] The following table provides stability data for several other monovalent formulations (TMV to antigen ratio 8:1) at the time of release and at various times after filling into vials and storage under refrigerated conditions (2° C. to 8° C.) or room temperature conditions (22° C. to 28° C.):

[0202] [Table 38]

[0203] [Table 39]

[0204] [Table 40] TIFF2025076429000048.tif66168

[0205] [Table 41]

[0206] [Table 42] TIFF2025076429000051.tif46168

[0207] [Table 43]

[0208] [Table 44] TIFF2025076429000054.tif45168

[0209] [Table 45]

[0210] In each of the conjugates listed in Tables 24-39, purity, pH, protein concentration, and storage potency are maintained through at least 6 months of storage under refrigerated conditions, and for the others, through at least 12 months of storage. Furthermore, polydiversity is also consistent over this time frame. Polydiversity refers to the variability in particle size in the conjugate product, and generally, the better the product, the lower the polydiversity. Similarly, the purification and conjugation platform according to multiple embodiments and alternatives successfully maintained the stability and potency of various antigens, utilizing various conjugation ratios, illustrating that diversity.

[0211] In addition to the monovalent formulations, the following tetravalent conjugates produced according to several embodiments and alternatives at a 1:1 TMV to antigen ratio exhibit high stability under both refrigerated (2° C.-8° C.) and room temperature (22° C.-28° C.) conditions:

[0212] [Table 46] TIFF2025076429000057.tif120166

[0213] [Table 47] TIFF2025076429000059.tif120166

[0214] [Table 48]

[0215] [Table 49]

[0216] In addition, the following tetravalent conjugate produced according to several embodiments and alternatives at a TMV to antigen ratio of 8:1 demonstrated high stability under both refrigerated (2° C.-8° C.) and room temperature (22° C.-28° C.) conditions: Results for this tetravalent conjugate were obtained for each of the four HAs conjugated to the modified TMV NtK carrier, including influenza strains A / Michigan, A / Singapore, B / Colorado, and B / Phuket. Further efficacy data for these and other conjugates and vaccine components is provided herein.

[0217] [Table 50]

[0218] [Table 51]

[0219] [Table 52]

[0220] [Table 53]

[0221] Additionally, the potency of tetravalent conjugates described in Tables 42A, 42B, 43A and 43B are shown in Figures 41A and 41B, respectively. Figure 41(A) is a graph showing QIV stability at refrigerated temperatures of 2-8°C (referred to as routine stability in the figures) from Tables 42A and 42B. Figure 41(B) is a graph showing QIV stability at elevated temperatures of 22-28°C (referred to as accelerated stability in the figures) from Tables 43A and 43B. As shown in Figures 41A and 41B, no statistically significant differences were observed in the potency of tetravalent conjugates produced according to several embodiments and alternatives at a TMV to antigen ratio of 8:1 under room temperature or refrigerated conditions.

[0222] Additionally, the following tetravalent conjugates produced according to several embodiments and alternatives at a TMV to antigen ratio of 8:1 demonstrated high stability under both refrigerated (2°C-8°C) and room temperature (22°C-28°C) conditions: Unlike the tetravalent conjugates discussed above in Tables 42-43, the following tetravalents utilize four different HAs conjugated to a modified TMV NtK carrier, including the following antigens: H1 (Brisbane), H3 (Kansas), B / Y (Phuket), B / V (Colorado).

[0223] [Table 54] TIFF2025076429000067.tif153169

[0224] [Table 55]

[0225] Tables 40-45 and Figures 41A and 41B show that the tetravalent conjugates maintain consistency and stability in terms of protein concentration, storage potency, pH and appearance under both refrigerated and room temperature conditions for at least 3-12 months. The data also show that the purification and conjugation platform, according to several embodiments and alternatives, successfully conjugates and increases stability across a wide range of antigens and various conjugation ratios.

[0226] Table 46 provides the percent change in preservative potency of various antigens listed in Tables 41A and 41B by comparing the initial potency with the preservative potency at a particular time point.

[0227] [Table 56]

[0228] Thus, as shown in Table 46, when the conjugates were placed in a non-refrigerated environment, the preservative efficacy after 30 days was at least 70% of the initial efficacy of the conjugate mixture within 1 day after conjugation. At the end of 90 days, the preservative efficacy of the conjugate mixture stored in a non-refrigerated environment was at least 68% of the initial efficacy, and the preservative efficacy of the conjugate mixture was at least 75% at the end of at least 180 days.

[0229] The following table illustrates the stabilizing effect of the embodiments described herein by comparing the release conditions of the same protein conjugated to TMV with purified recombinant antigen according to several embodiments and alternatives. Additionally, the stability after 6 months under refrigerated conditions (4°C-8°C) was compared between purified antigen and the same antigen conjugated to TMV by analyzing protein concentration, potency, SDS-page purity and pH as follows:

[0230] [Table 57]

[0231] [Table 58]

[0232] [Table 59]

[0233] [Table 60]

[0234] Tables 46-50 show the stability-inducing properties of purification and conjugation embodiments, most notably for the B / Colorado, B / Phuket, and H1NI (A / Michigan) antigens in terms of purity measurements. For the H3N2 (A / Singapore) and B / Colorado antigens, the stability of the conjugates is also shown in terms of antigen concentration. As shown in Tables 31-34, the purification and conjugation process, according to multiple embodiments and alternatives, stabilized the physical properties, antigen reactivity, and other quantitative stability characteristics of the antigens.

[0235] Additionally, Tables 41A-45 and Figure 41B show that tetravalent conjugates produced according to embodiments and alternatives exhibit robust stability measurements at room temperature (22°C-28°C) for at least 6 months or 24 weeks. Compared to conventional vaccines that exhibit an average stability of about 5 weeks at room temperature (as discussed in the F. Coenen paper above), vaccines according to embodiments and alternatives exhibit stability at least 5 times higher than conventional influenza vaccines and several times longer than purified antigens. Thus, the formulation and conjugation process according to embodiments and alternatives stabilizes highly unstable antigens such as B / Colorado and extends the stability of other antigens such as H3N2 (A / Singapore), H1NI (A / Michigan), and B / Phuket well beyond the stability limits of free antigens and conventional vaccines.

[0236] Example 14(a)-14(h) - Trial of Quadrivalent Influenza Vaccine To demonstrate the safety, efficacy and utility of the embodiments disclosed herein for immunogenicity and protection against seasonal viral challenge, several preclinical studies were conducted using a tetravalent seasonal vaccine candidate (referred to in this example as the "QIV vaccine"). The vaccines used in the studies were manufactured according to multiple embodiments and alternatives disclosed herein. The QIV vaccines of interest included the World Health Organization, Centers for Disease Control and Prevention recommended 2018 / 2019 North American seasonal influenza vaccine strains conjugated to inactivated TMV NtK, as well as the FDA's Vaccine and Related Biological Products Advisory Committee (VRBPAC) recommended influenza vaccines for 2018 / 2019 (A / Michigan / 45 / 2015(H1N1)pdm09, A / Singapore / INFIMH-16-0019 / 2016(H3N2), B / Phuket / 3073 / 2013 (B Yamagata lineage), and B / Colorado / 06 / 2017 (B The following influenza HA antigens from the QIV seasonal vaccine (Victoria lineage) were included: Influenza HA antigens from the QIV seasonal vaccine (Victoria lineage) from the QIV seasonal vaccine (Victoria lineage) from the QIV seasonal vaccine (Victoria lineage). In some embodiments, the four vaccine antigen conjugates are blended together in phosphate buffer solution (as a non-limiting example) with 0.01% thimerosal as a preservative to create a single injectable tetravalent vaccine formulation. As discussed in more detail below, these studies demonstrated that the embodiments disclosed herein enhance the immunogenicity of recombinant hemagglutinin protein antigens, as determined by various measurements and analyses including hemagglutination inhibition and neutralizing antibody titers. Similarly, the studies described in this example demonstrate that the QIV seasonal vaccine is immunogenic, as challenge with a virus strain homologous to the vaccine strain HA antigen demonstrated levels of protection in all mammalian disease models tested to date. Unless otherwise noted, the content of inactivated TMV NtK and HA intermediates incorporated into the conjugation reaction was 1:1 equivalent on a mg:mg (i.e., weight (wt)) basis in the studies. As a non-limiting example, the 8:1 TMV of drug substance intermediate was used to determine the immunogenicity of the QIV seasonal vaccine (Victoria lineage). Subsequent trials with QIV vaccines conjugated at NtK:HA ratios (mg:mg basis) demonstrated favorable humoral responses.

[0237] Table 51 provides an overview of the studies conducted on the QIV vaccine according to this example. "GLP" refers to Good Laboratory Practice, such as the CPMP Note on Guidance for Preclinical Pharmaceutical and Toxicological Testing of Vaccines (CPMP / SWP / 465 / 95) and the World Health Organization guidelines on nonclinical evaluation of vaccines (WHO Technical Report Series, No. 927), both of which are incorporated herein by reference in their entirety. For further discussion of various aspects of the studies, see the table below.

[0238] [Table 61] TIFF2025076429000075.tif209165TIFF2025076429000076.tif210166TIFF2025076429000077.tif206166TIFF2025076429000078.tif221166

[0239] As summarized in Table 52, immunogenicity studies in BALB / c mice were performed with monovalent and tetravalent preparations, respectively, to evaluate preferred formulation ratios and to monitor injection site reactions and clinical signs of toxicity.

[0240] [Table 62]

[0241] The study was conducted in the spirit of GLP regulations. BALB / c mice (N=5 / group) were immunized with each vaccine preparation on days 0 and 14. Animals were bled to prepare serum for pre-dose HAI antibody titer analysis on days 0 and 14, and a terminal bleed was performed on day 28.

[0242] Table 53 (below) shows the number of animals that developed detectable HAI titers, the titer range of positive animals, and the geometric mean titers (GMT). GMT values ​​were calculated using the following formula as set forth in Armitage and Berry, Statistical Methods in Medical Research, 2nd Edition (1987), pages 31-33, the entire contents of which are incorporated herein by reference: GM={x1x2x3.....xn} 1 / n (Formula 3)

[0243] As shown in Table 53, HAI titers for serum samples from all study groups prior to the first vaccination (day 0) were below the limit of detection (<10). On day 14 (prior to the second vaccination), antibody titers were below detectable levels in all groups except: 1 / 5 mice in group 3 (received 30 μg monovalent vaccine) had an HAI titer of 10 against A / Singapore / INFIMH-16-0019 / 2016(H3N2) and 2 / 5 mice in group 7 (received 30 μg quadrivalent vaccine) had a titer of 10 against A / Singapore / INFIMH-16-0019 / 2016(H3N2). In Table 37, HAI antibody titers were taken as the reciprocal of the highest dilution of serum that inhibited hemagglutination with 4 HA units of virus.

[0244] [Table 63]

[0245] On day 14, HAI titers were detected against H3N2 virus at the high (30 μg) dose only in the monovalent and QIV vaccine groups. On day 28, there was a dose-dependent increase in HAI titers against A / Michigan / 45 / 2015 (H1N1) in the quadrivalent vaccine and A / Singapore / INFIMH-16-0019 / 2016 (H3N2) in both the monovalent and quadrivalent vaccines. HAI titers were detectable at antigen doses as low as 1.5 μg in some animals, with the majority of animals raising antibody titers at 7.5 μg per HA antigen. No detectable titers were raised by mice vaccinated with the monovalent vaccine against the other three strains tested. Although less pronounced, the QIV vaccine also induced HAI titers in a subset of mice against B / Colorado / 06 / 2017. No detectable HAI titers were generated against the Yamagata lineage B / Phuket / 3073 / 2013 component.

[0246] In summary, based on the HAI assay data, the monovalent formulation vaccine induced detectable humoral immune responses against the H3N2 virus. The QIV formulation induced detectable humoral immune responses against three of the four antigens. The induced immune responses against influenza H1N1 and H3N2 antigens seen in mice vaccinated with the monovalent and QIV vaccine formulations were dose-dependent. As shown in Table 37, H1N1 GMTs ranged from 20 to 279 (increasing in a dose-dependent manner) and H3N2 GMTs ranged from 20 to 52. Additionally, no adverse clinical signs or injection reactions were observed with the monovalent vaccine or QIV vaccination.

[0247] An immunogenicity study in naive mice (BALB / c mice) was conducted by evaluating the immunogenicity of the tetravalent vaccine with and without TMV conjugation (1:1 ratio of TMV NtK:HA antigens) over time, as summarized in Table 54. The objective of this study was to confirm the results from previous studies, shown in Table 53, compare the immunogenicity of QIV to a vaccine in which the same antigens were not conjugated to the TMV NtK carrier, and analyze the durability of the immune response over a 90-day period. The vaccines in the following table were conjugated in a 1:1 ratio of TMV NtK:HA antigens.

[0248] [Table 64] TIFF2025076429000082.tif66166

[0249] Figure 42 shows QIV vaccine induction of H1N1 and H3N2 hemagglutination inhibition (HAI) titers in mice over time. Data includes geometric mean titers (GMT) of BALB / c mice administered 15 μg QIV vaccine per HA. GMT values ​​were calculated using the following formula: GM={x1x2x3.....xn} 1 / n (Formula 3)

[0250] Mice with a titer value ≦10 were assigned a titer value of 5 for GMT calculations according to Armitage and Berry, supra.

[0251] As shown in FIG. 42 and Tables 55 and 56 below, HAI titers were observed only in animals that received the QIV vaccine (referred to as "KBP-VP H1NI" and KBP-VP H3N2 in FIG. 42) and only against H1N1 and H3N3 antigens. No detectable immune response occurred against unconjugated antigens. Titers were below detectable levels for A / Michigan / 45 / 2015 (H1N1) and A / Singapore / INFIMH-16-0019 / 2016 (H3N2) through day 21. HAI titers remained detectable on days 28 and 42 and were highest on day 90. Furthermore, humoral responses continued to increase for at least 90 days, a feature not known from conventional influenza vaccines. This study further supports the effectiveness of conjugating antigen to the TMV NtK carrier in generating an immune response, as there was no detectable response with antigen alone.

[0252] [Table 65]

[0253] [Table 66]

[0254] Similar to HAI titers, virus neutralization (VN) titers were only observed in animals that received the QIV vaccine. As shown in Table 57, VN titers against A / Michigan / 45 / 2015 (H1N1) and A / Singapore / INFIMH-16-0019 / 2016 (H3N2) were also not observed until Day 21. VN titers remained detectable on Days 28 and 42 and were highest on Day 90.

[0255] [Table 67]

[0256] Mice were also monitored for clinical signs and injection site reactions in the studies outlined in Table 54. At necropsy, organ weights were measured and gross necropsy and histopathology of tissues were performed. No test article related gross findings were noted in animals necropsied on days 21 or 90.

[0257] For animals euthanized on Day 21, test article-related microscopic findings of minimal to mild mixed cellular infiltrate at the injection site were noted in 3 of 9 animals in Group 2 (TMV NtK control), 1 of 9 animals in Group 3 (HA alone), and 9 of 9 animals in Group 6 (QIV). Minimal degeneration / regeneration of muscle fibers at the injection site was also noted in 1 of 9 tested animals in Group 6. No other microscopic test article-related findings were noted for mice euthanized on Day 21 or Day 90.

[0258] In conclusion, the QIV vaccine elicited detectable humoral immune responses based on HAI and virus neutralization assays. The strongest responses were against A / Michigan / 45 / 2015 (H1N1) and A / Singapore / INFIMH-16-0019 / 2016 (H3N2). HAI and VN titers were first detectable on day 21 and highest on day 90. These data indicate that the QIV vaccine is safe and immunogenic in mice.

[0259] Example 14(c) - Immunogenicity and challenge studies in ferrets Immunogenicity and challenge studies with the QIV vaccine were also performed in ferrets, which are accepted as the most representative animal model of influenza infection, to assess vaccine efficacy via reduction in viral load after challenge in relation to a licensed vaccine comparator. As shown in the study design in Figure 43, blood for immunogenicity studies was collected on the following study days: -3, 7, 14, 21, 28, and 42. Animals were challenged on study day 43, and nasal washes were performed and viral titers were analyzed on study days 45 and 47.

[0260] Briefly, on study days 0 and 14, N=30 ferrets per group (15M / 15F) were immunized with one of the following: 1. Placebo buffer as a negative control (same amount as in Group 4) 2. Fluzone® tetravalent (15 μg per HA, 60 μg total HA) vaccine as licensed control 3. 15 μg QIV per HA antigen (60 μg total HA antigen; 60 μg TMV NtK carrier) 4. 45 μg QIV per HA antigen (total HA 180 μg) After each dose, injection sites and clinical signs were monitored daily for seven consecutive days. Animals were bled at regular intervals for measurement of HAI and neutralizing antibody titers, as shown in Figure 43. Animals from each dose group were subdivided into two groups (N=12, 6M / 6F) and immunized with 1x10 IgG of either A / Michigan / 45 / 2015(H1N1)pdm09 or A / Singapore / INFIMH-16-0019 / 2016(H3N2) on day 43. 6 The animals were challenged with plaque forming units (PFU) (non-limiting example). Animals were monitored and nasal washes were performed 2 and 4 days after challenge to quantitate residual influenza virus titers and assess viral clearance.

[0261] In ferrets administered either the 15 or 45 μg dose level of QIV and Fluzone®, hemagglutination inhibition (HI) was detected against all four viruses tested [A / Michigan / 45 / 2015(H1N1)pdm09, A / Singapore / INFIMH-16-0019 / 2016(H3N2), B / Colorado / 06 / 2017, and B / Phuket / 3073 / 2013]. The QIV vaccine induced the strongest HI responses against B / Phuket, B / Colorado, and A / Singapore / INFIMH-16-0019 / 2016(H3N2). The weakest HI response was against the A / Michigan / 45 / 2015(H1N1) virus. Virus neutralizing (VN) titers were detected against all viruses, with the following descending responses: A / Michigan / 45 / 2015(H1N1)pdm09, A / Singapore / INFIMH-16-0019 / 2016(H3N2), B / Phuket, and B / Colorado / 06 / 2017 showed the lowest responses.

[0262] As shown in Figure 43, after influenza challenge with A / Michigan / 45 / 2015(H1N1)pdm09, virus was detected in the nasal washes of all challenged ferrets on days 2 and 4 post-challenge. Figure 44 shows the nasal wash titers (log10 TCID 50 / mL), where the QIV vaccine is referred to as "QIV". As shown in Figure 44, there was no statistical difference in viral titers on day 2 post-challenge, but a mean temperature increase of 1.0°C was only observed in the placebo control. Compared to the placebo group on day 4 post-challenge, all three vaccine groups had a statistically reduced viral titer with the QIV vaccine, which showed a dose-dependent log reduction (2.3 and 1.9 log10 TCID50 / mL) that was statistically greater than Fluzone® (0.83 log10 TCID50 / mL). In Figure 44, the p-value for Fluzone® compared to placebo is less than 0.05, and the p-value for QIV compared to Fluzone® is less than 0.001. Additionally, the weight loss observed in Fluzone® and QIV vaccinated ferrets was comparable to the placebo control.

[0263] Figure 45 shows the nasal wash titers (log10 TCID 50 45 shows the mean nasal washes of all challenged ferrets after challenge with A / Singapore / INFIMH-16-0019 (H3N2) virus at 2 and 4 days post-challenge. The QIV vaccine 45 μg group (1.0 log10 TCID 50 The greatest log reduction in viral titer was observed on day 2 in the vaccinated groups (1.3 log TCID / mL), which was statistically lower than placebo. QIV vaccine and Fluzone® produced log reductions of 0.7 log TCID / mL and 0.5 log TCID / mL, respectively, which were not statistically reduced compared to the placebo group (p value less than 0.05). As shown in Figure 45, on day 4, all vaccinated groups, Fluzone® (1.3 log TCID 50 / mL), QIV vaccine (15 μg dose, 1.4 log10 TCID 50 / mL), and QIV vaccine (45 μg, 1.2 log10 TCID 50Comparable levels of significant log reductions in virus titers were observed for both vaccinated and non-vaccinated animals (15 μg QIV and 1.2% QIV, respectively). Furthermore, placebo and Fluzone® vaccinated groups had comparable maximum mean weight losses of 3.1% and 2.6%, respectively. Animals immunized with QIV had infection-induced maximum mean weight losses of 2.1% (15 μg QIV) and 1.2% (45 μg QIV).

[0264] With regard to testing of immunogenic responses, the QIV vaccine of this example induced detectable humoral immune responses to the four antigens tested in HI and VN assays. The strong VN responses were consistent with the reduction in nasal wash virus titers observed at day 4 post-challenge in vaccinated ferrets challenged with A / Michigan / 45 / 2015(H1N1)pdm09. The log reduction in virus titers observed at day 4 in QIV vaccinated ferrets was 1.1-1.5 logs greater compared to Fluzone®. Strong HI responses were observed in all three groups of vaccinated ferrets against A / Singapore / INFIMH-16-0019 / 2016(H3N2). In ferrets challenged with A / Singapore / INFIMH-16-0019 / 2016 (H3N2), improved protection was observed in the high dose group on days 2 and 4 post-infection, with similar log reductions in virus titers observed in QIV vaccinated ferrets and Fluzone®, both of which yielded statistically lower virus titers than placebo. These data indicate that the QIV vaccine is immunogenic and provides a level of protection in ferrets against challenge with H1N1 and H3N2 homologous viruses.

[0265] Example 14(d) - Matrix immunogenicity study in mice The objective of this study was to evaluate the immunogenicity of QIV vaccines with different conjugation ratios of TMV NtK to HA antigen. For this study, A / Singapore / INFIMH-16-0019 / 2016 (H3N2) was used to formulate a monovalent vaccine. As shown in Table 58, a fixed dose of HA + increased dose of TMV was compared to a fixed dose of TMV + decreased dose of HA (data not shown).

[0266] [Table 68]

[0267] Eight-week-old female BALB / c mice (5 per group) were immunized with A / Singapore / INFIMH-16-0019 / 2016 (H3N2) monovalent influenza vaccine by subcutaneous route of administration using the indicated vaccine compositions on days 1 and 14. Mice were bled and sera were collected on days 12, 28, 42, and a final serum was collected on day 60.

[0268] Figure 46 shows the immunogenicity of monovalent vaccines obtained with various ratios of TMV NtK:HA antigen. Relative IgG titers are expressed as ng of IgG per mL of serum. As shown in Figure 46, at day 60 (end of study), IgG titers induced by monovalent vaccines prepared with increasing TMV-NtK:HA antigen conjugation ratios continued to show significant improvement compared to the benchmark 1:1 ratio group.

[0269] Furthermore, in a HA antigen study with a fixed dose of 15 μg HA with increasing concentrations of TMV NtK, the 8:1 and 16:1 vaccine formulations induced the highest mean responses that were stable over time. This data supports the use of vaccines produced according to multiple embodiments and alternatives as an effective strategy to generate humoral immunity.

[0270] Thus, the immunogenicity test in mice and efficacy test in ferrets clearly demonstrates a significant increase in the development of humoral immunity by the QIV vaccine of this embodiment. Furthermore, immunogenicity was shown to be associated with the conjugation of the HA antigen to the inactivated TMV NtK carrier. In mice, humoral responses continued to increase over 90 days. Furthermore, QIV vaccine immunization could significantly reduce H1N1 and H3N2 viral loads after virus challenge, to a greater or equal extent than the conventional vaccine comparator Fluzone®. In other words, QIV vaccine immunization could reduce morbidity and virus levels caused by infection with homologous H1N1 and H3N2 influenza strains to vaccine strains to a greater or equal extent than the conventional vaccine comparator Fluzone®. It is expected that the vaccine produced according to this embodiment will show improved efficacy similar to that of the QIV vaccine of Example 14 across a wide range of antigens conjugated to viruses such as TMV used against many types of viruses.

[0271] Examples 14(e) and 14(f) - Pharmacokinetic studies A pharmacokinetic study was performed to evaluate the biodistribution of the QIV vaccine after a single intramuscular injection over 8 days in male New Zealand White rabbits. The study used a developed and refined RT-qPCR method to measure TMV NtK viral RNA extracted from tissues or blood, and the study compared two different QIV vaccine formulations prepared with various amounts of the TMV NtK carrier. Tissues analyzed were blood, skeletal muscle (injection site), lymph nodes, spleen, thymus, heart, liver, lung, kidney and testis.

[0272] Figure 47 shows a comparison of the distribution of TMV vRNA in tissues over time as measured by RT-qPCR following a single injection of two different QIV formulations incorporating either 45 μg (1:1 formulation monovalent vaccine) or 1440 μg (8:1 formulation tetravalent vaccine) of TMV NtK carrier in a 0.5 mL dose (as non-limiting examples). Figure 48 shows the biodistribution of TMV vRNA in tissues by organ and dose group 8 days after a single injection. In Figures 47 and 48, "LOQ" refers to limit of quantification and "LOD" refers to limit of detection.

[0273] As expected, the two formulations showed consistent distribution patterns. In Figures 47 and 48, the highest levels of QIV were quantifiable in the injection site skeletal muscle tissue throughout the study, with the amount decreasing over time for both formulations. After skeletal muscle, QIV was most highly detected in the spleen and draining lymph nodes throughout the study, indicating that QIV migrated to these tissues after immunization. Transient levels were detected in the liver, heart, and testes at 24 hours post-dose, and were subsequently below the LOQ for both formulations. The 8:1 formulation showed low, transient signals in blood, lung, and thymus tissues 24 hours after immunization, which were below the limit of quantification (LOQ) by 3 days after immunization, likely due to the 32-fold higher amount of TMV NtK contained in that formulation. No quantifiable QIV residues were detected in brain or kidney tissues for either QIV formulation.

[0274] Thus, in all formulations, the vaccine was observed to be transported from the injection site to immune organs, with no accumulation observed in non-target organs. Similarly, TMV-specific Q-RT-PCR signals were observed to be dose-dependent when measured at the injection site, with rapid clearance from all non-target organs. Thus, detection in immune system organs suggests a "depot" effect (i.e., sustained release of antigen at the injection site) and sustained stimulation of the immune system.

[0275] Examples 14(g) and 14(h) - Toxicity Studies As previously described in Table 51 and as described below in Table 59, several toxicity studies were performed according to GLP requirements to evaluate the potential toxicity of the QIV vaccine and to support the clinical use of the QIV vaccine for the prevention of disease caused by infection with influenza virus (as non-limiting examples). Two repeat-dose toxicity studies in New Zealand White rabbits were performed with two formulations of QIV differing in the content of TMV NtK carrier conjugated to purified recombinant HA antigen according to several embodiments and alternatives. As described in more detail below, the studies showed no treatment-related or toxicologically significant clinical findings, supporting the safe use of the TMV NtK:HA conjugate in human trials.

[0276] [Table 69]

[0277] As shown in Table 59, two repeat dose GLP toxicity studies were performed in rabbits with different virus to antigen ratios. In some embodiments, the QIV vaccine was administered annually as a single intramuscular injection. To investigate the effectiveness of this approach, a number of human doses plus one (N+1) strategy was used with a second dose administered 28 days after the first dose. The study included intended human high doses of QIV vaccine at each conjugation level of TMV NtK carrier molecule (1:1 and 8:1 formulations), as well as high doses of TMV NtK carrier alone.

[0278] In a repeat dose toxicity study of QIV vaccine in a 1:1 ratio of TMV NtK carrier, HA antigen, the seasonal influenza vaccine candidate was administered intramuscularly twice (once daily) to male and female New Zealand White rabbits on study days 1 and 29 to evaluate the reversibility of effects after a 28-day recovery period. As shown in Table 60, each group consisted of 10 rabbits / sex / group. On study day 30 (1 day after the last dose), 5 rabbits / sex / group were sacrificed, and on study day 57 (28 days after the last dose), 5 rabbits / sex / group were sacrificed.

[0279] [Table 70]

[0280] Test articles were administered by intramuscular (IM) injection on study days 1 and 29. For each injection, animals in each group received 0.5 mL of control article (Group 1), low dose vaccine (Group 2), or high dose vaccine (Group 3) using a 25-gauge needle attached to a plastic 1 mL syringe (as non-limiting examples). Administration sites were relatively large muscle masses behind the hind legs, which were shaved or re-shaven (as necessary). On each day of injection, administration sites were swabbed with alcohol and allowed to dry completely for a minimum of 10 minutes prior to administration. IM administration sites alternated between hind legs, with the right hind leg being used for the first administration.

[0281] Five rabbits / sex / group were euthanized on Study Day 31 (2 days after the last dose) and the remaining study animals (4 / sex / group) were kept under observation and euthanized on Study Day 57 (28 days after the last dose).Study endpoints included morbidity / mortality, physical examination, clinical signs of toxicity, inoculation site (Draize) reactivity scoring, body weight, body weight change, food consumption, temperature, ophthalmology, clinical pathology (clinical chemistry, hematology, coagulation), organ weights, immunogenicity analysis, gross and microscopic pathology at necropsy.

[0282] All study rabbits survived until scheduled necropsy. No treatment-related or toxicologically significant clinical findings or inoculation site reactogenicity were observed. No treatment-related or toxicologically significant effects were observed in body weight, body weight change, food consumption, body temperature, ophthalmology, clinical chemistry, hematology, and organ weights.

[0283] Fibrinogen levels were generally elevated in the vaccine-treated groups on the second day after dosing (p<0.05 or <0.01). The elevation of fibrinogen in these groups was considered to be related to the vaccine treatment but was considered an expected (inflammatory) response after treatment with an immunogenic substance. At the end of the 28-day recovery period, fibrinogen was no longer elevated (p>0.05) (a reversible effect).

[0284] Mixed cellular infiltrates in the left sciatic nerve and injection site (side of the last intramuscular dose on study day 29) were common microscopic findings seen in intramuscular vaccine toxicity studies. This lesion was reversible in the sciatic nerve but did not completely resolve at the injection site at the end of the 28-day recovery period. This lesion was not considered adverse, but was an expected finding consistent with administration of an immunogenic agent.

[0285] In conclusion, intramuscular administration of QIV vaccine at doses of 15 or 45 μg was well tolerated in two injections given once every 4 weeks (study days 1 and 29). None of the findings observed resulted in adverse or limited toxicity and were considered to be of minimal toxicological significance following administration of an immunogenic substance (e.g., seen in only one gender, reversible, transient, no changes in organ function, etc.) and / or were expected findings (e.g., increased fibrinogen and mixed cell infiltrates at the injection site or sciatic nerve).

[0286] Example 14(h) - Repeated Dose Toxicity Study In a repeat dose toxicity study of QIV vaccine in an 8:1 ratio of TMV NtK carrier, HA antigen, the seasonal influenza vaccine candidate was administered intramuscularly twice (once daily) to male and female New Zealand White rabbits on study days 1 and 29 to evaluate the reversibility of effects after a 28-day recovery period. As shown in Table 61, each group consisted of 8 rabbits / sex / group. On study day 30 (1 day after the last dose), 5 rabbits / sex / group were sacrificed, and on study day 57 (28 days after the last dose), 5 rabbits / sex / group were sacrificed.

[0287] [Table 71]

[0288] Test articles were administered by IM injection on study days 1 and 29. For each injection, animals in each group received 0.5 mL of control article (Group 1), low dose vaccine (Group 2), or high dose vaccine (Group 3) using a 25-gauge needle attached to a plastic 1 mL syringe (as non-limiting examples). Administration sites were relatively large muscle masses behind the hind legs, which were shaved or re-shaven (as necessary). On each day of injection, administration sites were swabbed with alcohol and allowed to dry completely for a minimum of 10 minutes prior to administration. IM administration sites alternated between hind legs, with the right hind leg being used for the first administration.

[0289] Four rabbits / sex / group were euthanized on Study Day 31 (2 days after the last dose) and the remaining study animals (4 / sex / group) were kept under observation until euthanasia on Study Day 57 (28 days after the last dose).Study endpoints included morbidity / mortality, physical examination, clinical signs of toxicity, inoculation site (Draize) reactogenicity scoring, body weight, body weight change, food consumption, temperature, ophthalmology, clinical pathology (clinical chemistry, hematology, coagulation), organ weights, immunogenicity analysis, gross and microscopic pathology at necropsy.

[0290] All study rabbits survived until scheduled necropsy. No treatment-related or toxicologically significant clinical findings or inoculation site reactogenicity were observed. No treatment-related or toxicologically significant effects were observed on body weight, body weight change, food consumption, temperature, ophthalmology, clinical chemistry, hematology, organ weights, or gross and microscopic pathology.

[0291] Fibrinogen levels were generally elevated in the treatment groups on the second day after administration (p<0.01). The increase in fibrinogen in these groups was considered to be related to the treatment but was considered an expected (inflammatory) response after treatment with an immunogenic agent. No increase in fibrinogen was observed during the recovery phase (reversible effect) (p>0.05).

[0292] Thus, IM administration of the QIV vaccine candidate or inactivated TMV NtK at a dose of either 45 μg of each HA antigen (180 μg total HA + 180 μg TMV NtK) or 1440 μg of each HA antigen was well tolerated in two injections (study days 1 and 29) once every 4 weeks. Findings in these GLP toxicity studies did not result in adverse or limited toxicity and were considered to be of minimal toxicological significance (e.g., seen only in one gender, reversible, transient, no changes in organ function, etc.) and / or were expected findings following administration of an immunogenic substance (e.g., elevated fibrinogen). No significant toxicological issues were noted with either the 1:1 or 8:1 (TMV NtK:HA antigen) formulations of the QIV vaccine in these studies.

[0293] Furthermore, robust antigen-specific immunogenic responses during two GLP toxicology studies (Examples 14(g) and 14(h)) were also measured based on ELISA, HAI, and neutralizing antibody titers in rabbits receiving low and high doses of QIV vaccine test article. Figure 49 shows total anti-HA IgG ELISA analysis from rabbit serum samples after QIV vaccine immunization on days 1 and 29. As shown in Figure 49, immune responses peaked after the second dose of QIV vaccine, as expected from immunization of naive animals. Anti-influenza titers against all four influenza antigens (as non-limiting examples, Michigan H1N1, Singapore H3N2, B / Colorado, and B / Phuket) were measured in serum samples after injection on day 29. Furthermore, up to 90-fold higher mean anti-influenza titers (than controls) were detected on study days 42, 49, and 57.

[0294] As shown below in Table 62, HAI titers were detected in most animals on test days 42, 49, and 57 (by way of non-limiting example) for the A / Michigan / 45 / 2015 (H1N1), A / Singapore / INFIMH-16-0019 / 2019 (H3N2), and B / Colorado / 06 / 2017 viruses. In contrast, HAI titers for the B / Phuket / 3073 / 2013 virus were generally seen in four animals or less on test days 42, 49, and 57 (by way of non-limiting example).

[0295] [Table 72] TIFF2025076429000091.tif36166

[0296] Figure 50 shows the measurement of microneutralization GMT titers throughout the GLP toxicity study with the 8:1 QIV formulation for each virus in the QIV vaccine. Table 63 below also shows the percentage of animals with detectable microneutralization titers at study day 49 for both GLP toxicity studies (i.e., both 1:1 and 8:1 QIV formulations). As shown in Figure 50 and Table 63, neutralization titers against these same four viruses were also consistently measured (by way of non-limiting example) in rabbits administered low and high dose vaccines on study days 42, 49, and 57, with the highest titers seen against the A / Michigan / 45 / 2015(H1N1)pdm09 and A / Singapore / INFIMH-16-0019 / 2019(H3N2) viruses.

[0297] [Table 73]

[0298] Thus, the studies discussed in this example demonstrate that the QIV vaccine consistently produces robust immune responses after intramuscular administration across three species (mice, ferrets, and rabbits). The primary measure of immunity was the generation of HAI antibody titers, a recognized serum biomarker of protection against influenza infection. Humoral immune responses were primarily detected after the second (booster) immunization in immunologically naive animals. Immunogenicity to the vaccine hemagglutinin antigen was dependent on conjugation to the TMV NtK carrier. A QIV vaccine prepared with an 8:1 ratio of TMV NtK carrier to recombinant HA antigen was shown to be desirable. In a disease challenge model, immunization of ferrets with QIV significantly reduced viral loads in animals subsequently challenged with homologous H1N1 and H3N2 strains as assessed from nasal wash samples. This reduction in viral load was equal to or greater than that of the licensed comparator. Immunization ameliorated clinical signs of morbidity associated with the challenge virus.

[0299] The study also investigated the distribution and safety of the QIV vaccine. No edema or injection site reactions were detected in any study. Biodistribution studies with QIV (measuring RNA from the TMV NtK carrier) found that outside the muscle at the injection site, QIV was measured in the spleen and lymph nodes at all time points tested, indicating that TMV viral RNA was relatively stable or decreased slowly in these organs, providing a potential mechanism of action and antigen presentation to the immune system. In the repeat dose toxicity study, the only finding was a reversible increase in fibrinogen levels from clinical chemistry profiles in the treatment groups, usually on the second day after dosing, an expected finding for an immunological agent. No other treatment-related or toxicologically significant effects were observed for body weight, body weight change, food consumption, temperature, ophthalmology, clinical chemistry, hematology, organ weights, or gross and microscopic pathology.

[0300] In conclusion, the data supports that the progression of TMV NtK conjugates to human clinical studies offers clear advantages over currently licensed influenza vaccines. Because no significant toxicological findings were observed with the QIV vaccine, it is highly likely that such events will not be observed for any other antigens purified according to the antigen platform conjugated to TMV NtK carriers described herein. Therefore, it is expected that other antigens conjugated to inactive TMV NtK will have similar biodistribution and toxicity profiles and thus be suitable for use in humans.

[0301] Example 15(a)-(c) - Coronavirus vaccine candidate: RBD-Fc121 conjugated to TMV (SARS-2) A Covid-19 vaccine was generated (as a non-limiting example) by forming an antigen by expression of a recombinant version of the RBD SARS-2 spike protein fused to the Fc domain of human IgG1 in Nicotiana benthamiana (Nb) plants. Prior to conjugation, the formed antigen was purified according to the antigen purification platform described herein according to embodiments and alternatives, and the TMV virions were purified and inactivated according to the virus purification platform described herein according to embodiments and alternatives. The purified recombinant RBD-Fc antigen was then conjugated to purified and inactivated TMV virions according to the teachings of embodiments and alternatives herein. When delivered to a mammalian subject (e.g., human or animal), the RBD-Fc and TMV conjugate displays the SARS-2 spike glycoprotein RBD fused to the human IgG1 Fc domain via chemical conjugation to the TMV virion. As discussed in more detail below, presentation of the RBD-Fc fusion in this embodiment has been demonstrated to enhance Th1 and Th2 responses in all mammalian disease models tested to date.

[0302] In this example, we chose to target the RBD domain of the SARS-2 spike glycoprotein as an antigen for the Covid-19 vaccine because it serves as a binding site for the human ACE-2 receptor and the binding site overlaps with a characterized neutralizing antibody. The SARS-2 spike glycoprotein is found in the S1 subunit at amino acids numbered from about 320 to 520. In FIG. 51(A), the SARS-2 spike trimer is shown as a space-filling model with the RBD circled horizontally and vertically. FIG. 51(B) shows the RBD domain fused to a human 171 allotype IgG1 Fc domain expressed and purified from plants, according to multiple embodiments and alternatives.

[0303] Several considerations were made when selecting the SARS-2 RBD as a fusion partner for developing the RBD-Fc antigen described in this and the following examples. The SARS-2 RBD is the binding site for neutralizing antibodies. Also, as described below, CR3022 is a human mAb isolated from a SARS patient that binds to the SARS-2 RBD domain. CR3022 binding can neutralize both SARS-1 and SARS-2 CoVs. The SARS-2 RBD also presents an ACE-2 (angiotensin II) binding domain.

[0304] According to the methods of this example, a multigene construct was designed and constructed to contain genes encoding the proteins required to synthesize Covid-19 antigens that target the RBD domain of SARS-2. In this example, the following antigen sequences were used for the synthesis of Covid-19 antigens (collectively referred to herein as "RBD-Fc121 constructs"): 1. "(SEQ ID NO:1)" Signal peptide: MGKMASLFATFLVVLVSLSLASESSA 2. "(SEQ ID NO:2)" Amino acids 331-632 of the SARS-2 viral spike:

[0305] [ka] 3. "(SEQ ID NO:3)" Fc hinge: VEPKSCDKTHTCPPCP 4. "(SEQ ID NO: 4) IgG1 171 allotype Fc:

[0306] [ka]

[0307] FIG. 52 illustrates, by way of example only, a TRBO expression plasmid constructed for SARS-CoV2 RBD-Fc fusion peptide (i.e., antigen) production in Nb plants. Following appropriate infiltration and incubation of the plants, antigen extraction and purification was performed as described herein. As illustrated and further discussed herein, a first peptide comprising a receptor binding domain (RBD) of a pathogen (specifically, a coronavirus, and thus the SARS-CoV-2 spike (S) glycoprotein RBD) was fused to a second peptide comprising a fragment crystallizable (Fc) region of an antibody (sequence of amino acids set forth in SEQ ID NO:4, including a human IgG1 Fc domain), and a hinge portion (sequence of amino acids set forth in SEQ ID NO:3) linking the first and second peptides to form a fusion peptide. In Example 15, the RBD comprised the sequence of amino acids set forth in SEQ ID NO:2, and in Example 17, the RBD comprised the sequence of amino acids set forth in SEQ ID NO:8. Please note that the reference to codons in the plasmid in Figure 52 includes both the RBD and Fc regions as well as the hinge portion. The sequence encoding the RBD-Fc antigen was optimized for efficient plant expression. A donor plasmid containing the reference nucleic acid sequence for the fusion peptide was synthesized according to the plasmid design. The antigen donor plasmid and the TRBO expression plasmid were digested with appropriate restriction enzymes. The antigen was ligated into the TRBO expression plasmid and then the colonies were screened to confirm the clones. The successfully ligated expression plasmids (Figure 52) were amplified in E. coli and the DNA was purified. The clones with confirmed presence of antigen vector DNA in the TRBO expression plasmid were used to transform Agrobacterium tumefaciens in preparation for infiltration into Nb plants. Individual colonies were axenically picked, incubated, and 20% glycerol stocks were made from the cultures and stored as antigen master cell banks.As further shown in FIG. 52, the exemplary construct contained the cauliflower mosaic virus (CaMV) 35S promoter, a DNA-dependent RNA promoter set to transcribe the TMV expression vector with a precise 5' end. Additionally, the TMV replicase is composed of 126 kDa and 183 kDa replication-associated proteins. In FIG. 52, "30k" refers to the movement protein produced from a subgenomic promoter at the 3' end of the 183 kDa protein coding region. In the exemplary construct, the antigen gene is transcribed from a subgenomic promoter at the 3' end of the 30 kDa protein coding region, and the 3' untranslated region has a ribozyme to ensure termination of transcription near the authentic 3' end. Additionally, there is an E. coli origin for replication, border regions for the TI plasmid, and other elements for efficient replication in Agrobacterium and insertion into plant cells.

[0308] Thus, a plasmid providing a construct for an antigen conjugable to a virus can be produced according to several embodiments and alternatives herein. Such a construct can include a first and a second coding region encoding a fusion peptide. In a non-limiting manner, the first coding region includes a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:2 above, or as a further example, SEQ ID NO:8 below. The first peptide can include or substantially include a receptor binding domain of a pathogen such as a virus, non-limiting examples of which include coronavirus and influenza virus, as further discussed herein. Also in a similar non-limiting manner, the second coding region includes a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:4 above. The second peptide can be a fragment crystallizable (Fc) region of an antibody capable of binding to an Fc receptor. In some embodiments, the first peptide and the second peptide are linked by a hinge portion encoded by a third coding region. The third coding region can include a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:3 above. In some embodiments, the hinge portion is part of an Fc region. In some embodiments, one or more of the nucleic acid sequences identified herein are part of a heterologous expression system.

[0309] Example 15(a) - Plant expression, purification, and characterization In this example, the RBD-FC121 fusion peptide (hereafter referred to in this example as "RBD-Fc121 antigen") was expressed in tobacco plants, harvested and purified. Plant growth and incubation were performed in a contained and controlled indoor environment, and expression was performed in naive wild-type Nb plants infected with an expression vector for protein replication of the RBD-Fc121 antigen (such as the vector shown in FIG. 52 as a non-limiting example) according to multiple embodiments and alternatives herein. Purification of the RBD Fc-121 antigen was performed according to the antigen purification platform described herein (e.g., as shown in Table 2 and FIG. 8), with the multi-mode ceramic hydroxyapatite (CHT) chromatography column step omitted in this particular example. Thus, for this example, FIG. 51(B) shows the RBD domain fused to the human 171 allotype IgG1 Fc domain expressed and purified from plants according to multiple embodiments and alternatives. The identity of such an exemplary pre-conjugated, SARS-COV2 RBD-Fc purified antigen intermediate can be confirmed by MALDI-TOF mass spectrometry. In some embodiments, the physicochemical properties of the antigen intermediate include a pH of about 7.0±0.4, an osmolality of about 250-350 mOsm / kg H2O, a bioburden of less than 10 CFU / mL, and less than 100 ng / mg of residual host cell protein. The content of inactivated TMV NtK intermediate and RBD-Fc intermediate is incorporated into the conjugation reaction.

[0310] As shown in Figures 54-55, the antigen purification platform according to several embodiments and alternatives successfully purified the RBD-Fc121 antigen, resulting in high yields (>400 mg of RBD-Fc protein) with the antigen pure and stable as RBD-Fc monomer in a GLP compliant manner. Figure 53 includes an SDS page gel showing the purity of the RBD-Fc121 antigen obtained from the conclusion of the purification platform applied to this antigen, lane 1 shows the marker and lane 2 shows the purified RBD-Fc121 protein. The RBD-Fc121 antigen product is highly pure as shown by the clear visible band in lane 2 of Figure 53. Furthermore, the migration of the protein shown in Figure 53 is consistent with the SEC-HPLC report of the free purified RBD-Fc121 shown in Figure 53. In Figure 54, the SEC-HPLC report of the free RBD-Fc121 antigen produced the signal data detailed in Table 64 below.

[0311] [Table 74]

[0312] Table 64 and Figure 54 show that >90% of the purified RBD-Fc121 antigen is in monomeric form. Similarly, impurities were present in the batch at low levels of less than 0.442 EU / mg. Thus, the RBD-Fc121 antigen was successfully and sufficiently purified in compliance with GLP.

[0313] The stability of the RBD-Fc121 antigen is reflected in Figure 55 and Table 65 (below). Figure 55 includes an SDS Page gel of purified RBD-Fc121 antigen 5 weeks after purification. In Figure 55, the lanes include lane 1-marker, lane 2-blank, lane 3-blank and lanes 4-6-RBD-Fc121 antigen 5 weeks after purification. The clear visible bands in Figure 55 indicate that the purified RBD-Fc121 antigen is highly stable. Furthermore, as shown in Table 49 below, the purified RBD-Fc121 antigen maintained its potency for at least 5 weeks after purification based on the ELISA results.

[0314] [Table 75]

[0315] Example 15(a) - Conjugation and Preparation The purified RBD-Fc121 antigen was then conjugated with the TMV NtK carrier. TMV NtK virions with surface lysine residues for efficient conjugation were produced in Nb plants (again, as a non-limiting example). The TMV NtK carrier was purified according to the virus purification platform described herein (e.g., as shown in Table 1 and FIG. 1). According to multiple embodiments and alternatives herein, after purification, the TMV NtK was subjected to micron filtration (e.g., 0.45) and treated with UV inactivation (as discussed in Example 8 herein).

[0316] The purified inactive TMV NtK was then chemically conjugated to the RBD-Fc121 antigen to generate a Covid-19 vaccine according to multiple embodiments and alternatives herein (e.g., as shown in Table 3). As shown in Figure 56, an exemplary conjugation procedure may include at least the following steps: The purified RBD-Fc121 antigen was diafiltered into a 50 mM MES buffered salt (50 mM NaCl) solution, concentrated to a target concentration suitable for conjugation, and subjected to 0.2 micron filtration.

[0317] Purified RBD-Fc121 antigen was conjugated to purified TMV NtK particles using EDC and Sulfo-NHS chemistry with 1 hour of mixing and reaction.

[0318] As indicated above, conjugation can occur at a range of TMV NtK:antigen (mg:mg) ratios, including but not limited to 8:1, 4:1, and 1:1.

[0319] Returning now to this example, RBD-Fc121 antigen and TMV NtK were subjected to a conjugation reaction carried out in EDC (4 mM) and Sulfo NHS (5 mM) at a pH of about 6.0. In this regard, the pH of the conjugation reaction and the pH of the purification step do not need to be the same. The conjugation reaction was quenched with a free amine (Tris base), optionally using a 30 kDa UF membrane, to remove residual EDC, Sulfo NHS and Tris base.

[0320] Therefore, the conjugated drug substance was diafiltered and formulated. Since the conjugate was larger than 0.2 microns, all steps downstream of the TMV NtK UV treatment and antigen 0.2 micron filtration were maintained under sterile conditions.

[0321] At this point, the TMV NtK:RBD-Fc conjugate is ready for drug substance fill and drug product fill (and in the case of Example 15). Suitable delivery mechanisms for the vaccine include liquid vials or lyophilized material that is reconstituted with physiological buffer for injection, with administration of the vaccine by any of the methods described herein.

[0322] To determine the percent conjugation of TMV NtK to the RBD-Fc121 antigen, SV was measured using AUC. As previously described, the fraction between 1-40S indicates the percent RBD-Fc monomer / trimer, and the fraction between 40-2000S indicates the percent TMV NtK:RBD-Fc conjugate, according to multiple embodiments and alternatives.

[0323] In this example, Figure 57 shows the normalized sedimentation coefficient for sample 4 (TMV:RBD-Fc conjugate diluted 28-fold) and Figure 58 shows the normalized sedimentation coefficient for the other sample (TMV:RBD-Fc conjugate diluted 10-fold). Figure 57 shows 100% total virus related material (i.e., viral antigen conjugates) and Figure 58 shows 99.7% total virus related material (i.e., viral antigen conjugates). The results shown in Figures 57 and 58 indicate essentially complete engagement of the RBD-Fc product in the TMV conjugation event.

[0324] Successful conjugation between TMV NtK and RBD-Fc121 antigen was confirmed by SDS-Page analysis. Figure 59 contains a gel of SDS-PAGE of TMV NtK:RBD-Fc conjugates at different times after conjugation. In Figure 59, the lanes are configured as follows: lane 1 - marker, lane 2 - conjugate mixture 0 min, lane 3 - conjugate mixture 5 min after conjugation, lane 4 - conjugate mixture 15 min after conjugation, lane 5 - conjugate mixture 30 min after conjugation, lane 6 - conjugate mixture 45 min after conjugation, lane 7 - conjugate mixture 60 min after conjugation, lane 8 - final conjugate mixture after mixing, lane 9 - TMV:HA conjugate as control, lane 10 - conjugate mixture at 0 min, lane 11 - conjugate mixture 5 min after conjugation, lane 12 - conjugate mixture 15 min after conjugation, lane 13 - conjugate mixture 30 min after conjugation, lane 14 - conjugate mixture 45 min after conjugation, lane 15 - conjugate mixture 60 min after conjugation, lane 16 - final conjugate mixture after mixing, and lane 17 - TMV:HA conjugate as control. Clearly visible bands in lanes 8 and 16, similar to those in lanes 9 and 17, indicate successful conjugation of TMV NtK to the RBD-Fc121 antigen.

[0325] Thus, nearly 100% conjugation of the RBD-Fc121 antigen to TMV NtK was confirmed by AUC and SDS-Page.

[0326] Example 15(b) - In vitro testing To determine the efficacy of the conjugate of TMV NtK to RBD-Fc121 as a Covid-19 vaccine candidate, the binding of the antigen to CR3022 and the ACE-2 receptor, as well as the immune response in mice, were analyzed. As discussed in more detail below, the data show that the RBD-Fc antigen was conjugated to TMV NtK and successfully bound to the human ACE-2 receptor. As further shown in Table 51, the initial immune response stimulated after one vaccination with the TMV NtK:RBD-Fc conjugate supported this observation. Similarly, the RBD-Fc121 antigen was conjugated to the TMV NtK SARS-2 RBD-specific human neutralizing monoclonal antibody (mAb), CR3022.

[0327] To determine the binding potency of the conjugates, an ELISA test was developed to measure RBD potency against both free antigen (i.e., RBD-FC121 alone) and conjugated (TMV to RBD-Fc) using CR3022 mAb for capture. The ELISA test found sera that did not bind to the Fc portion of the antigen, necessitating elimination of background binding. This test was performed by various methods including pre-adsorption and / or binding to the kappa region of the CR3022 light chain. Figure 60 shows CR3022 ELISA data showing the maintenance of RBD conformation between free antigen and TMV:RBD-Fc conjugates formulated at ratios of 8:1 and 4:1 (TMV NtK:antigen), respectively. In Figure 60(A), the ELISA standard curve shows the sensitivity and linearity of binding of SARS-2 spike antigen to the CR3022 antibody. In Figure 60(B), the TMV:RBD-Fc conjugate is referred to as "TMV:RBD-Fc". Figure 60(B) shows strong and dose-dependent binding of CR3022 to both the RBD-Fc121 antigen and the formulated TMV vs. RBD-Fc121 conjugate. Thus, Figure 60 shows that the RBD-Fc121 antigen is more than 5-fold more reactive to CR3022 compared to the commercial control SARS spike and RBD reagents. This data also suggests that the purified RBD-Fc121 antigen maintains essential conformational epitopes in a more favorable manner compared to conventional commercial reagents.

[0328] ACE-2 serves as a cell surface receptor for the spike protein of SARS-CoV2 during invasion of respiratory epithelial cells. To analyze the ability of the RBD-Fc121 antigen to bind to the ACE-2 receptor and induce a protective immune response against SARSCoV2 infection, quantitative and functional ACE-2 binding was performed using two-color confocal microscopy and colocalization and competitive binding assays on Vero e6 cells to assess both the colocalization of the ligand with ACE-2 and the relative affinity of the RBD-antibody complex for ACE2 compared to angiotensin II.

[0329] Vero cells are derived from the kidneys of African green monkeys and are commonly used in cell testing. Vero e6 cells are a subclone of Vero76 and display a range of virus susceptibility. Here, we compared the binding ability of the natural agonist, angiotensin II, to RBD-Fc binding by analyzing the concentration-dependent ability of blocking the binding of an ACE-2 specific antibody (ab15348, a homologue of angiotensin II) to its receptor on live Vero e6 cells. In performing these tests, recombinant H7 strain influenza HA protein produced in plants was used as a control. Multiple concentrations of each antigen-antibody complex, ranging from 0.04 μg / ml to 10 μg / ml, were incubated with Vero e6 cells and compared to angiotensin II, the natural ligand of ACE-2, for its ability to bind to the receptor. Figure 62(A) is a graph showing ligand colocalization events, where FAM-angiotensin II bound to Vero e6 cells as expected, with an average of 452 particles per field and a 2.79-fold increase over the non-specific H7 control (n=10 analyses). (Considering Figures 61, 62(A) and 62(B), angiotensin II refers to ab15348.) As shown in the figure, RBD-Fc121 antigen bound to Vero e6 cells in a concentration-dependent manner, with a 2.58-fold increase at a concentration of 10 μg / ml (n=10 analyses). In contrast, in the figure, binding of recombinant influenza HA-H7 remained at an average of 122.5 particles per field with no significant change at any concentration. Figure 61 visually illustrates the ligand co-localization and competitive binding between angiotensin II and RBD-Fc121 antigen in Vero e6 cells as discussed in Figure 62(A). The images in Figure 61 were obtained using confocal microscopy at the following ACE-2 specific antibody concentrations: 10 μg / ml, 3.3 μg / ml, 1.1 μg / ml, and 0.12 μg / ml (scale bar equals 15 μm). In Figure 61, red indicates binding of angiotensin II to Vero e6 cells and green indicates co-localized binding of RBD-Fc121 antigen to these cells. Binding of RBD-Fc121 antigen to Vero e6 cells was 2.58-fold higher than the H7 HA control and occurred in a concentration-dependent manner.Thus, increased binding of the RBD-Fc antigen (green) was observed to correlate with decreased detection of angiotensin II binding (red). Higher concentrations (n=10) of both angiotensin II and RBD-Fc121 antigen correlated with a direct and significant decrease in detectable levels of ACE-2 receptor binding (compared to lower concentrations), whereas recombinant influenza HA-H7 did not correlate with a direct decrease with increasing concentration.

[0330] Figure 62(B) shows that adding a colocalization control reduces all specific binding, demonstrating reduced ACE-2 antibody binding in the presence of both angiotensin II and RBD-Fc121 antigen. Note that binding of H7 HA did not affect detection of ACE-2 by the monoclonal antibody at any concentration. These figures also show that the RBD-Fc121 antigen effectively competes with angiotensin II for ACE-2 receptor binding on Vero e6 cells and colocalizes with these cells with similar affinity and specificity as angiotensin II.

[0331] Thus, Figures 60-62 show that the RBD-Fc121 antigen maintains functional conformation and activity through binding of CR3022 and ACE-2. Binding to ACE-2 shows similar affinity and specificity to the natural agonist angiotensin II, suggesting that the conformation of the spike protein RBD in the recombinant RBD-Fc121 fusion peptide complex is equivalent to that observed in the native SARS-2 spike protein. This data indicates that the RBD-Fc121 antigen presents the correct structure required to induce the production of neutralizing antibodies against the RBD of the Covid-19 spike protein.

[0332] To further evaluate the ability of RBD-Fc121 antigen (both free and TMV-conjugated) to neutralize viral infection by blocking cell entry at the ACE-2 receptor as well as stimulate immune responses, pooled serum samples were analyzed pre-immunization, 12, 28, and 42 days post-boost using a SARS-CoV2 plaque neutralization assay. Neutralization titers were calculated by determining the dilution of serum that reduced 50% of plaques. A standard 100 PFU dose of SARSCoV-2 was incubated with two-fold serial dilutions of serum samples for 1 h. The virus-serum mixture was then used to inoculate Vero e6 cells for 60 min, the cells were covered with EMEM agar medium + 1.25% Avicel, incubated for 2 days, and plaques were counted after staining with 1% crystal violet in formalin. As Figures 63(A)-(D) show, pre-immune sera showed no evidence of interference with the SARS-CoV2 plaque neutralization assay (Figure 63(A)). Day 12 sera showed a slight trend towards inhibition at the lowest dilution of 1 / 20, but was not statistically different from the vehicle control (Figure 63(B)). By day 28 (14 days after the second vaccination), a statistically significant reduction in titers was observed for the 15μg neat (non-vaccinated) and 15μg+CpG groups at the 1 / 20 dilution. However, the 45μg neat and 45μg+CpG groups were significant at the 1 / 320 dilution (Figure 63(C)). By day 42 (28 days after the second vaccination), the 15μg neat group showed significant neutralization titers at the 1 / 640 dilution and the 15μg+CpG group at the 1 / 320 dilution. Neutralization titers increased at 1 / 5120 dilution for 45mcg neat and at 1 / 1280 dilution for 45μg+CpG (Figure 63(D)).

[0333] Figure 64(A)-(B) further highlights the increase in neutralizing titers for the 15 and 45 μg groups. This study combines neutralizing antibody titers elicited by the TMV:RBD-Fc121 vaccine in two mouse preclinical studies measured by CPE neutralization assay and geometric mean titer (GMT) using the PRNT50 method with the data shown in Table 68 below, which shows no dose-response and no additional induction of neutralizing titers by inclusion of CpG compared to the neat vaccine group shown in Figure 64(A). As further summarized in Table 52, sera at day 42 (28 days after vaccination, 2 days after vaccination) were tested using a standard cytopathic effect (CPE) neutralization assay tested with SARS-CoV2. The CPE neutralization assay begins with titrating dilutions of heat-inactivated serum samples mixed in duplicate with 100 TCID50 of SARS-CoV-2 and incubated for 1 hour. The serum / virus mixture was then added to Vero e6 cells and incubated for 3 days at 37°C and 5% CO2. After incubation, the cell monolayers were fixed and stained with crystal violet to assess the presence of virus-induced CPE. The virus neutralization titer of each sample is reported as the reciprocal of the highest dilution that prevented CPE in 50% of the wells. The results obtained from the CPE neutralization assay of individual serum samples from all groups correlate well with the results of pooled sera obtained in the SARS-CoV2 plaque assay. The groups showing the highest neutralization titers, in descending order, were TAP COVID-19 45μg neat (GMT=2702), 45μg+CpG (GMT=1280), 15μg (GMT=676), 15μg+CpG (338), RBD-Fc antigen alone (GMT=294) and PBS vehicle (<64). Group correspondence is apparent, indicating a dose-response and no measurable contribution of CpG to driving enhancement of neutralizing titers.

[0334] Example 15(c) - In vivo testing The TMV NtK:RBD-Fc121 Covid-19 vaccine described above was evaluated for immunogenicity in two parallel evaluations in female C57BL / 6 mice. As shown in Figure 65(A)-(D), pooled sera were tested for total IgG anti-RBD reactivity by ELISA, using recombinant COV2 RBD-His as a capture antigen, to compare immune responses between groups.

[0335] In the first evaluation, 10 animals were used per group, and 5 animals per group were sacrificed on day 14 to have enough serum for extensive testing. The remaining 5 animals per group were boosted on day 14 and sacrificed on day 28. As used herein, the term "boost" or "booster" or other similar synonyms thereof refer to a second administration of vaccine at the same dose as the first. In the second evaluation, 5 animals were used per group, and were vaccinated with prime and boost vaccines on days 0 and 14, respectively, and all animals were bled on days 0, 12, 14, 28, and terminal. In both evaluations, the study endpoints included measurement of antigen-specific geometric mean antibody titers induced in mice, SARS-2 neutralization titers, and antibody isotype analysis. The study is outlined in Table 66 below and includes various doses, each vaccine (purified TMV NtK only, RBD-Fc only, TMV:RBD-Fc conjugate), and compares the same dose both with and without CpG oligodeoxynucleotide (CpG) as an adjuvant. CpG was added to the vaccine formulation so that the concentration of antigen was the same as in the neat (non-adjuvanted) vaccine formulation per 100 mcL injection. In some embodiments, monophosphoryl lipid A (MPLA) and / or SE-M were utilized as adjuvants to enhance the subject's immune response to the vaccine. "SE-M" is a type of stable emulsion that typically uses a TRL4 Toll-like receptor agonist as an adjuvant.

[0336] [Table 76] TIFF2025076429000098.tif176166

[0337] Figure 65(A) shows the immune response (ng / mL) stimulated by the RBD-Fc antigen only 12, 28 and 42 days after the first vaccination. Figure 65(B) shows the immune response (ng / mL) stimulated by the TMV:RBD-Fc conjugate at doses of 15mcg and 45 neat (non-adjuvanted) at 12, 28 and 42 days after the first vaccination. Figure 65(C) shows the immune response (ng / mL) stimulated by the adjuvanted TMV:RBD-Fc conjugate (15mcg) and TMV:RBD-Fc conjugate (45mcg+CpG) vaccines at 12, 28 and 42 days after the first vaccination. Figure 65(d) shows a comparison of IgG titers recognizing the RBD-Fc antigen (black) and RBD-His (grey) from sera taken at day 42. The relative proportion of responses to RBD-His is shown above each stacked plot member as a percentage of the total RBD-Fc response.

[0338] Baseline serum antibody levels were very low, less than 100 ng / mL, for RBD-His and RBD-121-Fc antigens. Furthermore, no significant responses were measured in PBS vehicle control animals, demonstrating that unconjugated RBD-Fc antigen produced a limited antibody response and that conjugation to the TMV NtK carrier was necessary to generate a robust immune response.

[0339] As shown in Figures 65(B) and 65(C), all TMV:RBD-Fc vaccine groups showed measurable antibody responses by day 12. A dose response was observed when comparing RBD-Fc antigen alone with the TMV:RBD-Fc conjugate in Figures 65(A), 65(B) and 65(C). Additionally, a strong boost was observed from day 12 to day 28 after the second vaccination. The TMV:RBD-Fc conjugate groups showed increased antibody titers from day 28 to day 42, while the CpG adjuvanted vaccines showed consistent (45μg+CpG) or reduced titers (15μg+CpG). The non-adjuvanted TMV:RBD-Fc conjugate groups showed similar quantified titers as the CpG adjuvanted vaccine, with all sera analyzed simultaneously against a single capture protein.

[0340] As shown in Figure 65(D), the relative immune response to SARS-2RBD as well as the human Fc portion of the antigen on the immune response was tested. Sera on day 42 were analyzed by ELISA using either SARS-2RBD-His or RBD-121-Fc protein as capture agent. ELISA data were measured after 1:100 dilution for PBS / RBD-121-Fc antigen immunization group and 1:1000 dilution for TMV:RBD-Fc conjugate group. Titers recognizing RBD were significantly lower than RBD-Fc antigen, and the percentage of anti-RBD varied from 12 to 35% depending on the TMV:RBD-Fc group (Figure 65(D)). Immune response titers were not significantly different between TMV:RBD-Fc conjugate groups regardless of capture antigen.

[0341] A favorable balance of Th1 / Th2 cytokines produced promotes a safe and effective immune response by balancing pro-inflammatory and anti-inflammatory responses, and considering the importance of Th1 / Th2 balance, IgG isotype analysis was also performed by measuring IgG1 (Th2) and IgG2 (IgG2a+IgG2c; Th1) isotypes for individual sera collected on day 42, and the results are shown in Figure 66. Figure 66(A) shows the Th1 response (IgG2a and IgG2C), Figure 66(B) shows the Th2 response (IgG1), and Figure 66(C) is a stack plot comparison of relative IgG2 to IgG1 antibodies by ELISA. In Figure 66, " * " indicates p<0.05 compared with other groups.

[0342] As expected, the PBS and RBD-Fc antigen groups showed lower overall IgG2 antibody responses compared to all TMV:RBD-Fc conjugate groups. All TMV:RBD-Fc conjugate groups differed from each other, with dose and addition of adjuvant significantly improving IgG2 titers. IgG1 titers were significantly higher in the non-adjuvanted TMV:RBD-Fc conjugate group compared to all other groups, significantly lower in the CpG adjuvanted group, and comparable to the RBD-Fc alone group. The IgG1:G2 isotype ratio varied between groups, with IgG1:G2 ratios of >1 in the non-adjuvanted TMV:RBD-Fc conjugate group and IgG1:G2 ratios of <0.1 in the TMV:RBD-Fc conjugate + CpG adjuvant group, with predominantly IgG1 isotype titers in the RBD-121-Fc group. In summary, the CpG adjuvant strongly skewed the isotype response to the TMV:RBD-Fc conjugate vaccine toward Th1 type, whereas the unadjuvanted TMV:RBD-Fc conjugate showed a more balanced mix of Th1 / Th2 responses. The unconjugated protein stimulated an almost entirely Th2 response.

[0343] In the first evaluation above, SARS-2 neutralization titers were measured using the Vero E6 cell viability test from day 14 samples. Figure 67 shows the mean cell viability following co-incubation of SARS-2 virus with mouse serum, where the mean cell viability as a percentage of total cells is plotted against serum dilution. The data shown in Figure 67 shows a significant increase in viability observed in the TMV:RBD-Fc121 vaccine groups (45mcg alone, 15 and 45mcg + CpG) compared to the control groups (vehicle alone and antigen alone). The geometric mean neutralization titers were calculated for each group and are shown in Table 51.

[0344] [Table 77]

[0345] The data in Table 67 show measurable neutralizing titers induced in mice after prime vaccination with adjuvanted and neat (non-adjuvanted) TMV:RBD-Fc121 vaccines. Figure 69 is a graph showing serum neutralizing activity based on titers after vaccination of mice against SARS CoV-2. The data shown in Figure 69 represents the log(2) transformed endpoint serum dilution that inhibited CPE by 50%. The dotted and dashed lines represent the starting dilution used for post-prime and post-boost assays, respectively. (*p<0.05, 2-way ANOVA, Tukey's multiple comparison, GraphPad Prism 8.4.2). The data in Figure 69 further support that exemplary TMV:RBD-Fc vaccines (including but not limited to TMV:RBD-F121 vaccines) can induce measurable neutralizing titers after a single vaccine with or without adjuvant. After the second vaccine boost, these titers are significantly increased at both the 15 and 45 μg concentrations. With reference to Figures 71(A)-(C), the exemplary TMV:RBD-Fc vaccine confers protection to the organism after exposure to SARS-CoV2. Five groups of subjects received either a control (PBS) or the exemplary vaccine at various levels of RBD-Fc concentration (CpG adjuvant only, 15 μg no adjuvant, or 45 μg no adjuvant) or no immunization. Survival (Figure 71(A)), clinical symptoms (Figure 71(B)), and post-challenge weight (Figure 71(C)) were assessed and graphed. Clinical symptom data was based on body temperature and physical appearance, the latter scored on a scale of 0-3: 0=normal; 1=mild, lack of grooming; 2=moderate, coarse hair, nasal / ocular spitting; 3=severe, labored breathing, lack of response to stimuli, and human euthanasia. Even without adjuvant, subjects receiving the 5 μg and 45 μg concentrations fared best for survival, the latter showing 100% survival 12 days after infection.

[0346] In the second evaluation, recombinant RBD protein (6-His fusion) was used as the capture protein and antigen-specific antibodies were measured and expressed as ng IgG bound / mL of pooled group serum. Figure 68 shows response titers measured after vaccine 1 (prime; day 12) and vaccine 2 (boost, day 28) induced in mice with TMV:RBD-Fc121 vaccine. Samples represent analysis of pooled serum from 5 animals in each group, and the capture antigen is recombinant RBD-6-His protein. As shown in Figure 68, measurable responses were observed on day 12 (post-vaccine 1-prime vaccination) for TMV:RBD-Fc121 vaccine mixed with 45mcg neat or CpG adjuvant. After vaccination 2 on day 28 (indicated by "boost" in Figure 67), similar titers were shown for TMV:RBD-Fc121 vaccine mixed with 45mcg neat or CpG antigen groups. Moreover, these titers appear to be significantly higher than those induced by the TMV:RBD-Fc121 vaccine, either neat at 15mcg or mixed with CpG antigen. In FIG. 68, all TMV:RBD-Fc121 groups are significantly higher than the immune response induced by the RBD-Fc antigen alone. These studies demonstrate the dose-dependence of the TMV:RBD-Fc121 vaccine and reveal that the adjuvant does not significantly contribute to enhancing the immune response to the RBD-Fc121 antigen when conjugated to inactivated TMV particles. Furthermore, as shown in FIG. 68, the TMV:RBD-Fc121 vaccine induced response levels of over 600mcg / mL when provided in a 45mcg dose.

[0347] Using a SARS-2 plaque assay, sera were tested for the generation of virus neutralizing antibody (Nab) titers in immunologically naive animals. Nab titers were detectable after a single immunization but increased significantly to <4,000 GMT after a second immunization (booster immunization). As shown in Table 68 below, two different neutralization assay methods were used, showing comparable titers and providing high validity to the results. It should be noted that the adjuvant did not increase Nab titers in any of the experiments. The data support the efficacy of as little as 15mcg of the TMV:RBD-Fc121 vaccine (showing Nab titers of approximately 200 after the first dose, >4,000 after the second dose in the first study, and >600 titers in the second study).

[0348] [Table 78] TIFF2025076429000101.tif66166

[0349] Further analysis of mouse sera demonstrated a balanced Th1 / Th2 immune response elicited by the TMV:RBD-Fc121 vaccine, with immunogenicity to the vaccine being highly dependent on conjugation to the TMV NtK carrier. In one in vitro model to evaluate the potential of the TMV:RBD-Fc vaccine to induce antibody-enhanced disease (ADE), sera containing neutralizing antibody titers of ≥2700 showed no evidence of enhanced SARS-CoV-2 entry into macrophages. This indicates that the vaccine strategy stimulates Nab titers without promoting ADE.

[0350] In addition, Th1 immune responses were explored through cell stimulation analysis. In this study, groups of 5 mice were immunized on days 1 and 14 with either 15 μg or 45 μg antigen doses, with or without CpG, administered subcutaneously once in a total volume of 50 mcL (PBS with buffer). Serum was collected on days 0, 12, 18 and 42 and the extent of antibody response to vaccine antigens was evaluated as total titers against target antigens and compared to PBS immunized sera or pre-immune sera. The unconjugated protein RBD-Fc was used as a target for IgG evaluation as well as RBD-HIS to evaluate antigen responses against total antigen and RBD (spike S1 domain) components. Approximately 6 weeks after the first immunization, vaccinated mice were euthanized, terminal serum was collected and spleens were collected for IFNγ ELISpot analysis. A summary of the study is shown in Table 69.

[0351] [Table 79]

[0352] Spleens from two animals in each group were harvested on day 42, single cell suspensions were generated, and the cells were stimulated with SARS-CoV-2 RBD-HIS protein for 36 hours and the number of IFNγ-secreting cells was measured. The results are shown in Figure 70, where Figure 70(A) shows IFNγ ELISpot analysis of vehicle and RBD-Fc only, Figure 70(B) shows IFNγ ELISpot analysis of unadjuvanted TMV:RBD-Fc vaccine, and Figure 70(C) shows IFNγ ELISpot analysis of CpG-adjuvanted TMV:RBD-Fc vaccine.

[0353] As shown in Figure 70, only cells recovered from mice vaccinated with the TMV:RBD-Fc vaccine with CpG adjuvant induced statistically significant numbers of IFNγ secreting cells at day 42 (4 weeks after the second immunization), with comparable numbers in both the 15 and 45 μg dose groups (Figure 70(C)). This data supports the IgG isotype analysis showing that the TMV:RBD-Fc vaccine with CpG adjuvant co-delivery stimulates a Th1 response in mice.

[0354] Additionally, VaxArray® SeroAssay analysis of mouse sera immunized with various TMV:RBD-Fc vaccine doses, with or without adjuvant, was performed to assess relevance of binding to heterologous coronavirus antigens. In Figure 72, nCoV antigen (i) is the complete SARS-CoV2 spike protein, (ii) is the S1 (RBD) domain, SARS (i) is the complete SARS-1 spike, as are MERS (i) and HKU1, each produced in a mammalian system. Relative titers are expressed as fold enhancement of signal over the reported control. Strong reactivity was observed to both SARS-2 antigens and apparent cross-reactivity to SARS-1 antigens. As shown in Figure 72, no binding was observed to the derivative MERS or HKU1 spike proteins. Thus, the results in Figure 72 show that antisera from mice with neutralizing titers recognized (i.e., exhibited cross-reactivity with) heterologously produced (mammalian cell) SARS-2 spike protein, RBD and SARS-1 spike protein.

[0355] The specificity and relative titers of sera from the last bleed (day 42) were analyzed by VaxArray®. Nine different capture antigens were used, including eight heterologous antigens for SARS-CoV2 vaccine components: SARS-CoV2 spike (produced in insect cells), SARS-CoV2 spike (produced in mammalian cells), RBD(S1)-human Fc produced in plants (homologous antigens), SARS-CoV2 RBD(S1) (produced in mammalian cells), SARS-CoV2 S2 domain (produced in insect cells), SARS-CoV2 S1-sheep Fc (produced in mammalian cells), SARS-CoV1 S1-rabbit Fc (produced in insect cells), MERS spike (produced in mammalian cells); and HKU1 spike (produced in mammalian cells). VaxArray® assays were performed with individual sera from each group and read at exposures of 100, 350 and 700 ms. The reactivity against each antigen by each serogroup is shown in Figures 73(A) and 73(B). Figure 73(A) shows the reactivity measured against each capture antigen. Figure 73(B) shows the comparison of reactivity against specific capture antigens of each test group. After 100 ms exposure, reactivity of vaccine sera against all capture antigens was observed except for SARS-CoV2 S2 domain, MERS and HKU1 spike protein. However, significant reactivity was observed for SARS-COV1 S1 domain. Different overall reactivity was observed with complete and S1 capture antigens from SARS-CoV2 origin. In terms of overall reactivity, strong reactivity was observed from 45μg+CpG, 45μg neat, 15μg neat, 15μg+CpG and RBD alone in descending order, while sera from PBS-treated animals showed no reactivity. Furthermore, Figure 74 shows further correspondence when correlating the individual animal responses measured by CPE seen in Table 52 with the microneutralization titers of the plaque population measured by VaxArray®. In Figure 74, the lower titers seen in animals 1 and 3 immunized with CoV2 RBD-Fc, animal 2 with 15 μg TAP or animal 3 with 15 mcg+CpG all indicate low antibody titers and low neutralization titers. On the other hand, higher titers as indicated by absolute numbers and inability to be reduced by dilution indicate higher neutralization values.The overall correlation between intergroup titers and group neutralization titers corresponds to the absolute numbers of individual titers, GMTs and pooled serum values.

[0356] Further data on survival are shown in Figures 79(A)-(B), respectively. The former shows information from a mouse challenge study on survival, and the latter shows information from a ferret challenge study on survival. In the 14-day mouse study (Figure 79(A)), subjects (other than the placebo group) were vaccinated with HA7 pandemic influenza vaccine (i.e., TMV:H7 antigen vaccine with or without CpG adjuvant) at an 8:1 or 1:1 ratio at or near the time of infection. Subjects receiving the adjuvant had a 100% survival rate through day 14. Figure 79(B) shows information on the results of the ferret challenge study. Again, this was done with a TMV-H7 conjugate with or without CpG adjuvant. Based on virus titers collected from nasal turbinates, the results showed that the vaccine improved recovery rates with lower titers in the adjuvanted group compared to non-adjuvanted.

[0357] Mouse studies have also tested a mouse macrophage line (Raw 264.7) lacking the ACE-2 binding domain and found that the anti-spike RBD neutralized virus killing and did not promote antibody-dependent enhancement of infection in mouse macrophages. In humans, ACE-2 serves as a cell surface receptor for the spike protein of SARS-CoV2 during invasion of respiratory epithelial cells. Concurrently, neutralizing antibodies against the receptor-binding domain of this spike protein have been recovered from SARS-CoV and found in patients in humans, mice and other organisms that appear to be protective against infection by SARS-CoV2. However, it has also been shown that non-neutralizing antibodies may enhance the severity of SARS-CoV infection and hinder successful vaccine development for coronaviruses, including SARS-CoV1 and SARS-CoV2.

[0358] The mouse study compared the ability of specific antibodies, including those generated by the subject TMV:RBD-Fc vaccine, to enhance antibody-dependent enhancement of SARS-CoV2 infection. Untreated 264.7 cells were grown in 96-well plates in VGCM and allowed to adhere overnight. Cells were washed extensively before addition of serum or antibodies. Test antibodies (or pooled serum) were incubated simultaneously for 1 hour to facilitate viral inactivation by neutralizing antibodies prior to incubation with macrophages. Following this incubation, media containing both antibodies and SARS-CoV2 was added to the macrophages and incubated for 48 hours, at which point viability was assessed through addition of Cell Titer Glo (Promega) and based on luminescence output.

[0359] As further shown in Figure 75(A-B), non-neutralizing antibodies against SARS-CoV2 nucleocapsid and spike proteins enhanced virus entry into mouse macrophages, a cell type that does not express ACE2 on the cell surface and is normally resistant to infection. The viability of macrophages exposed to virus in the absence of antibodies averaged 91.5% of cell viability control at 48 hours post-infection (n=10). Figure 75(A), Figure 75(B) 1st and 5th bars. In the presence of anti-nucleocapsid antibodies raised against 6.25ug / ml of whole spike protein, viability decreased to an average of 55.69%, with the greatest concentration-dependent decrease above 1ug / ml. Figure 75(A). In the presence of polyclonal antibodies raised against 6.25ug / ml of whole spike protein, viability decreased to an average of 59.78%. Figure 75(A). In contrast, pooled mouse sera from the study in Example 15(b) against 6.25 μg / ml TAP COVID-19 vaccine showed high neutralizing titers without observable antibody-dependent enhancement as indicated by a mean viability of 125.56% compared to cell viability control. Statistical differences were determined using one-way ANOVA with Tukey's P-values ​​and the results are shown in Figure 75(C). Thus, while non-neutralizing antibodies (against nucleocapsid or spike proteins) promoted virus entry into macrophages, antibodies generated from the TMV:RBD-Fc vaccine did not show this effect.

[0360] Thus, the in vivo test shows that the TMV:RBD-Fc121 vaccine induces measurable and statistically significant SARS-2 neutralizing antibody titers over the control. The TMV:RBD-Fc121 vaccine of this example shows a strong, vigorous and promising immune response. In addition to IgG, Immunoglobulin M (IgM) is another indicator of vaccine efficacy. In vaccinated subjects, IgM levels can be assessed by ELISA and neutralizing antibody titers. In general, IgM antibody levels are expected to rise after vaccine administration, typically for a period of about several weeks, e.g., about two weeks. Thus, testing by ELISA or neutralizing antibody titers or both on days 0, 7, 14 and 28 can be performed after administration of the Covid-19 vaccine according to this embodiment, although other periods can be used. Such testing after such administration is expected to confirm an increase in its levels between days 0 and 14, which serves as an indicator of IgM production and immunogenic response to vaccine administration. IgM levels are then predicted to decline by day 28 as IgG antibody titers increase. It is also noted that the entire vaccine manufacturing process, from initial sequencing of the Covid-19 antigens to final production of cGMP vaccine for sterility and release of the drug product, can be accomplished within an 8 week time frame, a significant advantage over traditional vaccine production methods that typically take more than 6 months.

[0361] To assess potential adverse reactions to vaccination, the exemplary TMV:RBD-Fc121 vaccine described herein was tested for its effect on animal weight. Virus-naive female mice (n=5) were vaccinated with an 8:1 TMV to antigen ratio at the concentrations shown in Figure 80, and the weight of each animal was recorded weekly after vaccination. The data graphed in Figure 80 represents animals administered Vaccine 1 (Day 0) and Vaccine 2 (Day 14), with the study terminating at Day 28. As shown in Figure 80, all subjects gained weight.

[0362] In a separate challenge study, mice were administered different ratios of the exemplary vaccine with or without adjuvant and evaluated against the control group for post-challenge weight (Figure 81A) and survival (Figure 81(B)). As shown in these figures, the vaccine group performed significantly better than the placebo in both categories over a 12-day period.

[0363] Furthermore, as described in Example 14, studies with the QIV vaccine showed very high potency in experimental animals for H1, H3, H5 and H7 influenza models, with no significant toxicological findings observed. This data, as well as the role of inactivated TMV as a carrier, suggest similar potential for the Covid-19 vaccine disclosed herein due to similar structural components and development platform. As discussed in Example 16 below, the Covid-19 vaccine disclosed herein exhibits high stability at room temperature and under room temperature conditions for at least 6 months.

[0364] Thus, through the practice of certain embodiments herein, conjugable antigens suitable for conjugation to carriers can be produced. The teachings herein contemplate or are directed to antigens that can be conjugated to viral particles, viral particles, vaccines, constructs and other compositions of matter, as well as all methods used in making them. Such antigens can be recombinant antigens, which generally include a fusion peptide having a first peptide that includes a receptor binding domain of a pathogen (a coronavirus in a non-limiting example) and a second peptide that includes a fragment of an antibody that can bind to an Fc receptor, the crystallizable (Fc) region. In some embodiments, the first peptide and the second peptide are linked by a hinge portion. Optionally, the hinge portion may be a portion of the Fc region. In some embodiments, the hinge portion includes the amino acid sequence set forth in SEQ ID NO:3. Exemplary coronaviruses from which the receptor binding domain can be obtained include, but are not limited to, SARS-CoV-1, SARS-CoV-2, and MERS.

[0365] Referring now to Figure 51(A), an exemplary receptor binding domain for SARS-CoV-2, the receptor binding domain is contained in the S-1 subunit of the coronavirus spike protein and includes contact residues located in the range of about 289 to about 662. In some embodiments, the second peptide can include an Fc domain of an IgG1 antibody, such domain comprising the amino acid sequence set forth in SEQ ID NO:4, as described herein.

[0366] Consistent with the teachings herein, in some embodiments, a vaccine is produced that includes at least one conjugable antigen and a carrier that includes a viral particle as described according to embodiments and alternatives of the present disclosure. In some embodiments, such a viral particle is a virus, such as TMV. The first peptide of the conjugable antigen may include an amino acid sequence as set forth in SEQ ID NO:2, or the amino acid sequence may be an amino acid sequence as set forth in SEQ ID NO:8. The first peptide may include a contact residue located in the range of about 289 to about 662 of the S-1 subunit of the coronavirus spike protein, which contacts an ACE-2 receptor on cells of a mammalian subject to which such a vaccine is administered after the antigen is released from the viral particle carrier following administration. For the RBD-FC121 antigen, the contact residue may be located in the range of about 301 to about 662 of the S-1 subunit. For the conjugable antigen referred to herein as RBD-FC121 antigen, the first peptide lacks the amino acid sequence shown in SEQ ID NO:5, whereas the first peptide of the RBD-Fc139 antigen was found to contain this sequence. In both cases, when the conjugable antigen of the vaccine is released in a mammalian subject having cells containing one or more ACE-2 receptors, at least one conjugable antigen binds to one or more ACE-2 receptors. In some embodiments, the viral particles used to produce such vaccines have surface lysine residues that are chemically associated with the fusion peptide resulting from the conjugation reaction, more specifically the first peptide.

[0367] Consistent with the teachings herein, in some embodiments, the vaccine according to the teachings herein is multivalent and includes at least one conjugable antigen having a receptor binding domain of influenza A virus and at least one conjugable antigen having a receptor binding domain of a coronavirus. Other possible combinations are within the scope of the present embodiments, and the combinations provided herein are exemplary and non-limiting. In some embodiments, the vaccine according to the teachings herein is multivalent and includes at least one conjugable antigen having a receptor binding domain of influenza B virus and at least one conjugable antigen having a receptor binding domain of a coronavirus. Alternatively, the vaccine according to the teachings herein is multivalent and includes at least one conjugable antigen having a receptor binding domain of influenza A virus, at least one conjugable antigen having a receptor binding domain of influenza B virus, and at least one conjugable antigen having a receptor binding domain of a coronavirus. Various ratios can be selected and expressed as the mass ratio of virus particles:antigen (i.e., the mass ratio of virus particles to at least one conjugable antigen) as follows: Optionally, this ratio can range from about 1:1 to about 8:1, more specifically, 8:1. Optionally, CpG may be included with a vaccine in accordance with the teachings herein as an adjuvant to enhance the immune response of the mammalian subject to the vaccine.

[0368] Example 16 - Stability of coronavirus vaccine candidates under refrigerated and room temperature conditions: TMV:RBD-Fc121 vaccine Similar to the stability of the QIV vaccine discussed in Example 13, both the RBD-Fc121 antigen and the TMV:RBD-Fc121 vaccine demonstrated consistency and stability under refrigerated and room temperature conditions for at least six months. In this Example, the stability of the RBD-Fc121 antigen, as well as the stability of the TMV:RBD-Fc121 vaccine, was measured using the tests discussed in Example 13.

[0369] As described above, RBD-Fc121 antigen was purified and produced according to multiple embodiments. In this example, the stability of the purified RBD-Fc121 antigen was then analyzed. The following tables show the stability data and storage efficacy of the purified RBD-Fc121 antigen measured at the time of release and at various times after filling into vials and storing under refrigerated conditions (2°C to 8°C) and room temperature (22°C to 28°C). In these tables, "HMW" is an abbreviation for high molecular weight.

[0370] [Table 80]

[0371] [Table 81]

[0372] According to several embodiments and alternative production and purification methods, Tables 70 and 71 show that the purified RBD-Fc121 antigen is highly stable and potent.

[0373] Similarly, when the same purified RBD-Fc121 antigen is conjugated to TMV, according to multiple embodiments and alternatives, the stability profile and storage potency remain consistent. The following table shows stability data for the TMV:RBD-Fc121 vaccine (at a TMV to antigen ratio of 8:1) at the time of release and at various times after filling into vials and storage under refrigerated conditions (2° C.-8° C.):

[0374] [Table 82] TIFF2025076429000106.tif46168

[0375] The following table shows the stability data for the TMV:RBD-Fc121 vaccine (TMV to antigen ratio 8:1) at various times upon release and after filling into pouches and storage under refrigerated (2° C.-8° C.) and room temperature (22° C.-28° C.) conditions:

[0376] [Table 83]

[0377] [Table 84]

[0378] Tables 72 and 73 show that the TMV:RBD-Fc121 vaccine exhibited strong stability measurements for at least six months under refrigerated conditions. Similarly, Table 74 shows that the TMV:RBD-Fc121 vaccine produced according to embodiments and alternatives exhibited strong stability measurements for at least six months at room temperature storage (22°C to 28°C). This is in contrast to conventional SARS-2 vaccines, which in most circumstances require storage in ultra-low temperature freezers (e.g., -20°C to -70°C) or under refrigerated conditions, or are stable at room temperature for no more than a few hours. Furthermore, the purification and formulation process according to embodiments and alternatives stabilizes the RBD-Fc121 antigen itself, far beyond the stability limits of conventional approaches.

[0379] Example 17 - Coronavirus vaccine candidate: Plant expression, purification, characterization and conjugation to TMV of RBD-Fc139 (SARS-2) In addition to the RBD-Fc121 antigen, it is anticipated that any type of viral antigen, including other Covid-19 antigens, can be purified and conjugated to inactivated TMV and used as an effective vaccine candidate according to multiple embodiments and alternatives. In some embodiments, the following antigen sequence (referred to herein as the "RBD-Fc139 construct") is used to synthesize the Covid-19 antigen: 1. (SEQ ID NO:1) Signal peptide: MGKMASLFATFLVVLVSLSLASESSA 2. (SEQ ID NO:5) Amino acids 319-330 of the SARS-2 virus spike: RVQPTESIVRFP 3. (SEQ ID NO:6) Amino acids 331 to 591 of the SARS-2 viral spike:

[0380] [ka] 4. (SEQ ID NO:7) Tobacco etch virus NIA cleavage sequence: enlyfqg 5. (SEQ ID NO: 3) Fc hinge: VEPKSCDKTHTCPPCP 6. (SEQ ID NO: 4) IgG1 171 allotype Fc:

[0381] [ka]

[0382] In one embodiment of the RBD-FC139 construct, the following antigen sequences are used for the synthesis of Covid-19 antigens: (SEQ ID NO: 8) Amino acids 319 to 591 of the SARS-2 viral spike:

[0383] [ka]

[0384] A multigene construct was designed and constructed to contain genes encoding the proteins necessary to synthesize the RBD-Fc139 antigen, which also targets the RBD domain of SARS-2 in a manner similar to RBD-Fc121. According to several embodiments and alternatives, the RBD-Fc139 construct was ligated into a TRBO vector (as a non-limiting example) and subsequent colony screening confirmed clones. The constructed expression plasmid containing the RBD-Fc139 construct, similar to the plasmid shown in Figure 52, was amplified and then purified for use in generating a master cell bank.

[0385] According to multiple embodiments and alternatives, the RBD-Fc139 antigen is expressed in plants and purified using the antigen purification platform described herein. As shown in Figures 76-77, according to multiple embodiments and alternatives, the antigen purification platform successfully purifies pure and stable antigen and RBD-Fc139 in high yield as RBD-Fc monomer in a manner that complies with GLP regulations. Figure 76, derived from the conclusion of the antigen purification platform, includes an SDS-page gel under both reducing and non-reducing conditions showing the purity and successful purification of the RBD-Fc139 antigen. In Figure 76, the lanes are as follows: Lane 1 - 11.8ug purified antigen under reducing conditions, lane 2 - blank, lanes 3-5 - 1.5ug purified antigen under reducing conditions, lane 6 - blank, lane 7 - marker, lane 8 - blank, lanes 9-11 - 1.5ug purified antigen under non-reducing conditions, lane 12 - blank and lane 13 - 11.8ug purified antigen under non-reducing conditions. Clear visible bands indicate that the RBD-Fc139 antigen preparation is highly pure.

[0386] Figure 77 is a SEC-HPLC report of free RBD-Fc139 antigen, which produced the signals detailed below in Table 75. 100% area under the peak indicates that 100% of the RBD-Fc139 antigen is in monomeric form.

[0387] [Table 85]

[0388] Thus, the SDS-page gel and SEC-HPLC reports of the free RBD-Fc139 antigen confirm that the antigen purification platform used according to multiple embodiments and alternatives successfully purified the RBD-Fc139 antigen.

[0389] In parallel with the production of Covid-19 antigens, TMV NtK virions with surface lysine residues for efficient conjugation are produced in plants and purified according to the virus purification platform described herein. After purification, TMV NtK is subjected to micron filtration and immediately UV inactivation treatment. The RBD-Fc139 antigen is then conjugated to the inactivated TMV via the surface exposed lysine residues using the conjugation of recombinant antigen embodiments described herein.

[0390] The TMV:RBD-Fc139 conjugate according to this embodiment herein is also currently being investigated as a vaccine candidate for Covid-19 disease. However, based on the success of the tetravalent vaccine and the strong immune responses with the initial TMV:RBF-Fc121 conjugate, the TMV:RBD-Fc139 conjugate is also expected to be a promising Covid-19 vaccine.

[0391] It is readily understood that the teachings herein are consistent with a number of embodiments with a wide selection of alternative ways to carry out the embodiments. Thus, without limitation, in an embodiment directed to an antigen, referred to herein as embodiment A, the fusion peptide is formed from a first peptide comprising a receptor binding domain of a pathogen and a second peptide comprising a fragment crystallizable (Fc) region of an antibody capable of binding to an Fc receptor. Within the scope of embodiment A, the antigen in an embodiment referred to herein as embodiment B further comprises a hinge portion linking the first and second peptides. The portion of the Fc region, more specifically the hinge portion referred to herein as embodiment C, may comprise the amino acid sequence set forth in SEQ ID NO: 3. Within the scope of embodiment A, the pathogen in an embodiment referred to herein as embodiment D is a coronavirus having a receptor binding domain, more specifically the coronavirus referred to herein as embodiment E is selected from the group consisting of SARS-CoV-1 and SARS-CoV-2. In an embodiment within embodiment E, herein designated embodiment F, the coronavirus receptor binding domain comprises contact residues located in the range of about 289 to about 662 of the S-1 subunit of the coronavirus spike protein, which contact an ACE-2 receptor on a cell of a mammalian subject, or in an embodiment herein designated embodiment G, the contact residues are located in the range of about 301 to about 662 of the S-1 subunit. In an embodiment within embodiment F, herein designated embodiment H, the coronavirus receptor binding domain lacks the amino acid sequence set forth in SEQ ID NO:5.

[0392] The coronavirus within the scope of embodiment D, in embodiments herein referred to as embodiment I, is a Middle East Respiratory Syndrome coronavirus. The second peptide within the scope of embodiment A, in embodiments herein referred to as embodiment J, is an Fc domain of an IgG1 antibody, more specifically, in embodiments herein referred to as embodiment K, in which the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:4, and the first peptide comprises the amino acid sequence set forth in either SEQ ID NO:2 or SEQ ID NO:8. Thus, the antigen may be implemented (i.e., made, formed, designed, used, etc.) according to embodiment A, as more fully described herein. In implementing the embodiments described herein, if desired, the antigen described herein may be implemented by incorporating any one or more of embodiment A of embodiments B, C, D, E, F, G, H, I, or J, and the embodiments may be directed to compounds, methods, and genetic constructs in implementing any one or more of these alternative embodiments. Similarly, embodiments may be directed to a vaccine comprising an antigen, a method of forming an antigen, or a genetic construct useful for forming an antigen as described in embodiment A, B, C, D, E, F, G, H, I, or J, in combination with any preceding embodiment, further comprising a carrier comprising a viral particle, which in some embodiments is a virus, more specifically a tobacco mosaic virus, and wherein the fusion peptide is chemically associated with lysine residues on the surface of the carrier.

[0393] Further, in a non-limiting embodiment, herein referred to as embodiment K, the vaccine comprises a carrier comprising a viral particle having an influenza hemagglutinin antigen (HA) and a surface lysine residue, wherein the HA is chemically associated with the surface lysine residue. Within embodiment K, in an embodiment herein referred to as embodiment L, the viral particle releases at least one antigen in a mammalian subject having cells comprising one or more ACE-2 receptors, wherein the at least one antigen binds to the one or more ACE-2 receptors. Within embodiment K, in an embodiment herein referred to as embodiment M, the viral particle is a virus, and more specifically, in an embodiment herein referred to as embodiment N, the virus is tobacco mosaic virus. Within any of embodiments K, L, M, or N, in an embodiment herein referred to as embodiment O, the vaccine is multivalent, the HA is selected from the group consisting of type A HA and type B HA, and the vaccine further comprises at least one antigen having a coronavirus receptor binding domain, wherein the at least one antigen having a coronavirus receptor binding domain is chemically associated with a surface lysine residue. In embodiments within embodiment O, herein designated embodiment P, the HA comprises two or more type A hemagglutinin antigens (HA) and two or more type B HA. Also within embodiment O, herein designated embodiment Q, an additional feature found in any one or more of embodiments A, B, C, D, E, F, G, H, I, or J is incorporated into the coronavirus element of the vaccine. Further, in embodiments herein designated embodiment R, the ratio of viral particles to at least one antigen (by weight) in a vaccine within embodiment Q is in the range of 1:1 to 8:1, more particularly in the embodiment designated embodiment S, the range is 8:1.

[0394] Within any of embodiments K, L, M, or N, the vaccine in an embodiment referred to herein as embodiment T is multivalent, where an influenza hemagglutinin antigen (HA) refers to the first antigen, and the multivalent vaccine comprises a second antigen chemically associated with a surface lysine residue. Within embodiment T, the second antigen in an embodiment referred to herein as embodiment U is an influenza HA other than the first antigen. Within any of embodiments T or U, the multivalent vaccine in an embodiment referred to herein as embodiment V comprises two or more type A hemagglutinin antigens (HA) and two or more type B HA. Within embodiment T, the second antigen in an embodiment referred to herein as embodiment W comprises a coronavirus receptor binding domain. Within embodiment W, the coronavirus in an embodiment referred to herein as embodiment X is selected from the group of SARS-CoV01 and SARS-CoV-2. The multivalent vaccine in an embodiment within embodiment T, W, or X, referred to herein as embodiment Y, comprises two or more type A hemagglutinin antigens (HA) and two or more type B HA. The viral particle in an embodiment within embodiment T, referred to herein as embodiment Z, is a tobacco mosaic virus. Also within embodiment Z, referred to herein as embodiment AA, the multivalent vaccine includes the additional features found in any one or more of embodiments T-Z. Furthermore, in an embodiment referred to herein as embodiment BB, the ratio (by weight) of viral particles to at least one antigen in a vaccine within embodiment AA is in the range of 1:1 to 8:1, more particularly in the range of 8:1 in the embodiment referred to as embodiment CC.

[0395] In one embodiment, referred to herein as embodiment DD, the present invention is directed to a virus-antigen conjugate comprising a virus and at least one antigen, the at least one antigen comprising a first peptide comprising a receptor binding domain of a pathogen, and a fusion peptide comprising a second peptide, the fusion peptide being conjugable to the virus. In an embodiment within embodiment DD, herein referred to as embodiment EE, the conjugate is multivalent and further comprises at least one influenza hemagglutinin antigen (HA) selected from the group consisting of type A HA and type B HA. In an embodiment within embodiment DD or EE, herein referred to as embodiment FF, the conjugate is multivalent and the second peptide is a fragment crystallizable (Fc) region of an antibody capable of binding to an Fc receptor, the Fc region being an Fc domain of an IgG1 antibody. In an embodiment within any of embodiments DD, EE, or FF, herein referred to as embodiment GG, the conjugate further comprises a hinge portion linking the first peptide and the second peptide, the hinge portion comprising the amino acid sequence set forth in SEQ ID NO:3, and the pathogen is a coronavirus having a receptor binding domain. In an embodiment within any of embodiments DD, EE, FF, or GG, and referred to herein as embodiment HH, the first peptide comprises the amino acid sequence set forth in either SEQ ID NO:2 or SEQ ID NO:8. In an embodiment within any of embodiments DD, EE, FF, GG, or HH, and referred to herein as embodiment II, the virus is a tobacco mosaic virus comprising an N-terminal lysine residue. In an embodiment within any of embodiments DD, EE, FF, GG, HH, or II, and referred to herein as embodiment JJ, the pathogen is a coronavirus having a receptor binding domain, the coronavirus receptor binding domain being located on the S-1 subunit of the coronavirus spike protein and comprising contact residues located in the range of about 289 to about 662.

[0396] Further embodiments and uses of the novel subject matter herein In addition to the above antigens, including RBD-Fc antigens for treatment against SARS-CoV2, countless other antigens can be formed according to the multiple embodiments and alternatives described herein. The scope of the description and teachings herein is intended to be limited only according to the claims. For example, a strategy similar to that of Examples 14, 15, and 16 can be used to obtain candidate vaccines from other human-infecting coronaviruses, including acute respiratory syndrome coronavirus (SARS-1) and Middle East respiratory syndrome coronavirus (MERS). As shown in Figure 78, homology domains can be identified in the functional regions of the spike S1 domain that correlate with receptor binding and other essential activities.

[0397] In this regard, Figure 78 shows the receptor binding domains in the spike protein sequence alignment of SARS-CoV-2 and other related coronaviruses. Here, we show the sequence alignment of the interacting (i.e., binding) domains of SARS-CoV-2 (MN938384), Bat-CoV (MN996532 and MG772933) and SARS-CoV (NC004718). The important amino acids described for the interaction with ACE2 and SARS-CoV2 are underlined. (Line (-) = same amino acid, dot (.) = deletion). Figure 78 is from Ortega JT, Serrano ML, Pujol FH, Rangel HR. Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE-2 receptor: An in silico analysis. EXCLI J. 2020;19:410-417, published March 18, 2020, doi:10.17179 / excli2020-1167.

[0398] As seen in the constructs and N-terminal readings of the RBD-Fc121 and RBD-Fc139 antigens, the functional core elements range from the "RVQPT" motif for the RBD-Fc139 construct to the "CGPKK" domain for both the RBD-Fc121 and RBD-Fc139 constructs. Looking more closely at the RBD-Fc121 construct, beyond the core domain there are more extended protein domains that facilitate proper folding. Indeed, this strategy is expected to extend to any type of coronovirus antigen through the following process: 1. Protein homology analysis shown in Figure 78 2. In silico protein folding using existing coronavirus spike models 3. Creation of extended signal peptide-RBD gene fusions that promote efficient cleavage as determined by SignalIP or Phobius 4. Gene fusions with the Fc reading frame as shown by RBD-Fc121 and 139 constructs 5. Expression in plants 6. Purification by Protein A and other methods exemplified in this patent application 7. Conjugation to TMV according to several embodiments and alternatives described herein

[0399] Such vaccines can be made to contain several different diverse antigens and can be used to prevent identified coronavirus pathogens such as SARS-1 or MERS, or can be formulated into a multivalent vaccine with either RBD-Fc121 or 139 antigens to prevent SARS-1, SARS-2 and MERS, as non-limiting examples. According to several embodiments and alternatives described herein, a multivalent TMV conjugate vaccine can be mixed with influenza A and B antigens, for example, in equal or proportional amounts of each TMV-antigen conjugate, and administered in a single immunization. As described herein, a multivalent TMV conjugate vaccine does not show immunodominance by one antigen, which prevents responses to a second or third or fourth antigen. Indeed, as seen in Example 14, both HAI and neutralizing antibodies were generated against all strains in animals immunized with a quadrivalent TMV influenza vaccine. This quadrivalent vaccine can be used to measure protection against multiple individual influenza vaccines.

[0400] Thus, it is expected that the approach described herein, according to multiple embodiments and alternatives, will likely provide a broad and diverse range of antigens on a single viral particle carrier. Illustrated in a non-limiting manner, in one embodiment, a multivalent vaccine is provided that includes two, three, four, five or more different antigens against various viruses and other pathogens in accordance with the teachings herein. At least one antigen can neutralize or stimulate an immune response against one type of virus (e.g., influenza), while at least one other antigen can do the same against another type of virus (e.g., coronavirus or pandemic virus such as HA7). The antigens that occupy a given location on the carrier may be composed of a modeled fusion protein, where the receptor binding domain of the virus is followed by a fusion partner, such as the Fc domain of an antibody from the tail region of an IgG1 molecule. The influenza portion of at least one antigen on a single vaccine may include both influenza A and influenza B. Similarly, the approach may provide at least one antigen against one or more coronaviruses. The flexibility of the approach herein and the broad range of antigen-virus conjugates that can be combined contemplated herein facilitates the ability to manufacture broad-spectrum vaccines at scale, often in as little as a few weeks as opposed to months.

[0401] It is understood that the embodiments described herein are not limited in their application to the details of the teachings and illustrations set forth or shown in the accompanying drawings. Rather, it is understood that the present embodiments and alternatives described and claimed herein can be practiced or used in various ways. It is also to be understood that the words and phrases used herein are for purposes of description and should not be considered as limiting. The use herein of "including," "comprising," "for example," "containing," or "having," and variations of these words, are meant to encompass the items listed thereafter, and equivalents thereof, as well as additional items.

[0402] Thus, the above description of several embodiments and alternatives is intended to be illustrative rather than serving as a limitation on the scope of what is disclosed herein. The description herein is not intended to be exhaustive or to limit the understanding of the embodiments to the precise forms disclosed. It will be appreciated by those skilled in the art that modifications and variations of these embodiments are reasonably possible in light of the above teachings and descriptions.

Claims

1. a first antigen comprising an influenza hemagglutinin antigen (HA); a second antigen, and Carriers containing virus particles with surface lysine residues A multivalent vaccine comprising: A multivalent vaccine, wherein the first antigen and the second antigen are chemically associated with the surface lysine residues.

2. The multivalent vaccine described in claim 1, wherein the second antigen is an influenza HA different from the first antigen.

3. 3. The multivalent vaccine of claim 1 or 2, comprising two or more type A hemagglutinin antigens (HA) and two or more type B HAs.

4. The multivalent vaccine described in claim 1, wherein the second antigen comprises a coronavirus receptor binding domain.

5. The multivalent vaccine of claim 4, comprising two or more type A hemagglutinin antigens (HA) and two or more type B HAs.

6. A multivalent vaccine as described in claim 1, wherein the viral particles are tobacco mosaic virus.

7. 2. The multivalent vaccine of claim 1, wherein when placed in a non-refrigerated environment at storage temperatures for a predetermined period of time, the integrity or concentration of the vaccine at the end of the predetermined period is at least 90% of the initial integrity or concentration of the vaccine, and the predetermined period is at least 42 days after the release date of the vaccine.

8. The multivalent vaccine of claim 7, wherein the storage temperature is at least 20°C.

9. A virus-antigen conjugate comprising a virus and at least one antigen, wherein the at least one antigen comprises a fusion peptide having a first peptide comprising a receptor-binding domain of a pathogen and a second peptide, the fusion peptide being capable of binding to the virus.

10. 10. The conjugate of claim 9, wherein the conjugate is multivalent and further comprises at least one influenza hemagglutinin antigen (HA) selected from the group consisting of type A HA and type B HA.

11. The conjugate of claim 9 or 10, wherein the second peptide is a fragment crystallizable (Fc) region of an antibody capable of binding to an Fc receptor, and the Fc region is the Fc domain of an IgG1 antibody.

12. A conjugate described in any one of claims 9 to 11, further comprising a hinge portion connecting the first peptide and the second peptide, the hinge portion comprising the amino acid sequence set forth in SEQ ID NO: 3, and the pathogen is a coronavirus having the receptor binding domain.

13. The conjugate according to any one of claims 9 to 12, wherein the first peptide comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:

8.

14. The conjugate of any one of claims 9 to 13, wherein the virus is a tobacco mosaic virus comprising an N-terminal lysine residue.

15. The conjugate of claim 9, wherein the pathogen is a coronavirus having the receptor-binding domain, and the coronavirus receptor-binding domain is located on the S-1 subunit of the coronavirus spike protein and comprises contact residues located in the range of about positions 289 to about 662.

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