JP2023523716A - Vaccine formed by conjugation of virus and antigen - Google Patents

Abstract

Disclosed herein are methods of forming compounds and exemplary stable compounds that have the properties of conjugate compounds at refrigeration or room temperature, and in some embodiments, the methods involve combining an antigen and a virus in a conjugation reaction. mixing particles to form a conjugate mixture whereby the conditions and steps for forming these products are relevant to the use of the conjugate mixture as a vaccine, e.g. It enables use as a vaccine against a variety of pathogens, including for the treatment of diseases caused by viruses (including SARS-COV2).

Description

This international patent application claims the benefit of, and priority to, non-provisional U.S. patent application Ser. A non-provisional U.S. patent application that is a continuation-in-part of, and claims the benefit of and priority to, non-provisional U.S. patent application Ser. No. 16/709,063, which claims the benefit of and priority to U.S. Patent Application Serial No. 16/437,734, which is a non-provisional application filed June 11, 2019; It also 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 U.S. Provisional Patent Application No. It claims the benefit of and priority to application Ser. incorporate into

(reference to sequence listing)
The instant application contains a Sequence Listing in electronic form, the entire contents of which are incorporated herein by reference. The sequence listing text file filed with the filing contains a computer readable format file “Sequence Listings, PCT_ST25” which was created on March 5, 2021 and is 10,826 bytes in size.

(Technical field)
Embodiments described herein include the use of multi-set processes to produce highly purified recombinant viruses as antigen carriers, and still further various embodiments include novel coronaviruses, e.g. SARS-CoV2 (herein referred to as Covid-19 or SARS-2) relates to the production of vaccines using purified virus and purified antigens, including vaccines aimed at preventing disease.

Viruses contain nucleic acid molecules within a protein coat and replicate only inside living cells of other organisms. A wide range of viruses, often considered harmful, are capable of infecting all kinds of organisms (eg humans, livestock and plants). Not without positive aspects, however, there is growing interest in using viruses for a range of therapeutic purposes, including, but not limited to, vaccine production, gene therapy and cancer therapy. However, in order to study viruses, understand their structure, and adapt viruses for use as molecular tools, disease therapeutic vectors and carriers, any cellular debris that interferes with the intended function of the virus, The virus must first be purified to remove macromolecular fibres, organelles, lipids and other impurities.

Once purified, viruses are suitable for several uses. Important to the present disclosure is the conventional notion of using viruses (considering 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 substance that, when properly delivered to an organism, is capable of producing an immune response in that organism by stimulating antibody production through binding with antibodies within the organism that match the molecular structure of the antigen. is a molecule. To give a few examples, recombinant antigens are derived from recombinant DNA cloned into vectors by known techniques and then introduced into specific host cells (e.g., bacteria, mammalian cells, yeast cells and plant cells). produced. The recombinant antigen is then expressed using the translational machinery in the host cell. After expression, the recombinant antigen is recovered and attached to the virus through a covalent bond by a process known as conjugation. After conjugation of an antigen to a virus, the virus can serve as a carrier to deliver the antigen to the organism and activate the immune system's response. In this manner, virus-antigen conjugates can provide therapeutic use. Appropriate virus-antigen conjugation is required for the antigen to activate an immune response that produces antibodies in the host cells of the source organism. Purification of virus and antigen is the basis for this proper conjugation.

Current methods of purifying viruses are generally limited to small biochemical quantities, eg, 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-thaw, or debris is removed by low speed centrifugation by other known methods, and the supernatant is then used for experiments. Infected, untreated organisms are physically disrupted or pulverized and the resulting extract 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 agents that affect the ability to perform experiments and manipulate viruses.

A second example of a conventional purification step is high-speed ultracentrifugation, whereby the virus is pelleted, or further purified through pelleting through low-density sucrose solutions, or between different densities of sucrose solutions. Suspended. Limitations of this method include low yields of purified virus due to limited size and scalability for high-speed separations, and the presence of other host proteins, often co-purified with the virus sample, affecting virus purity. decline.

A third method used to enhance conventional virus purity is density gradient ultracentrifugation. In this method, gradients of cesium chloride, sucrose, iodixanol or other solutions are used for the separation of assembled virus particles or for the removal of gene-free particles. Limitations of this method include the time required for virus purification (often 2-3 days), the limited number of samples, the sample content that can be analyzed at one time (typically 6 per rotor), and the low amount of virus that can be purified ( typically micrograms to milligrams of final product).

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 poor purity, as the product is typically still attached to host proteins, nucleic acids, lipids and sugars, resulting in virus production. Produces significant agglomeration of materials. These limitations undermine the usefulness of the final product for compliance with current Good Manufacturing Practice (cGMP) regulations enforced by the US Food and Drug Administration (FDA).

Current cGMP regulations promulgated by the FDA contain minimum requirements for methods, facilities and regulations used in the manufacture, processing and packaging of drug products. These regulations target product safety and ensure that it has the ingredients and benefits it claims to have. Therefore, for viruses utilized in vaccine production, gene therapy, cancer therapy and other clinical settings, the final virus product must comply with cGMP regulations. If the final viral product does not comply with cGMP regulations, its utility in clinical settings is non-existent or greatly diminished, such as products derived from polyethylene glycol precipitation.

Scalability refers to the process of consistently and reproducibly producing the same product as the quantity of product increases, e.g. from laboratory scale (<0.1 square meters) to systems of at least >20 square meters. means expansion. As noted above, all previously used methods are problematic due to their inconsistency, low scalability (i.e. making products only in biochemical quantities) and non-compliance with cGMP regulations. .

Although plant-based production has attracted attention for large-scale production, there are also significant limitations in their use. Plant-based production systems can provide industrial scale yields at a much lower cost than animal cell production systems (eg, Chinese Hamster Ovary (CHO)). However, certain conventional purification methods (suitable on some scales for non-plant viruses) do not work in the case of 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 primarily produce protein and nucleic acid impurities, plants are also a source of significant 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 cellular organelle impurities. In fact, crude plant extracts often foul the equipment used in the processing and purification of viral and antigenic material obtained from plants (eg, by accumulation of impurities on the separation membranes or media bed readings 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 plant derived protein, virus or other desired "product". Accordingly, 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, and do not produce adequately purified viruses. It is not shown to have done so.

Little progress has been made in terms of virus and antigen purification platforms capable of consistently producing highly purified virus at commercial scale, from grams to kilograms and beyond, and in compliance with cGMP regulations. Such improvements will enable clinical development for tools used in vaccine production, gene therapy, and for cancer therapy. The platform described herein according to multiple embodiments and alternatives, along with other features and advantages outlined herein, meet this and other needs.

To date, seven coronaviruses (CoVs) have been identified as capable of causing human infections, 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 the human population, these three viruses pose significant public health risks and exhibit high mortality: SARS-CoV: 10%, MERS-CoV: 34.4% and SARS-CoV2: 6.1. %. 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, according to the Johns Hopkins Coronavirus Resource Center. Their numbers are growing exponentially, and their epidemic has put a global health and economy in jeopardy. 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 worldwide efforts to find vaccines to control or slow down infections, there are currently no approved antiviral treatments targeting human coronaviruses. Instead, supportive and palliative care remains the primary treatment.

The development of effective SARS-CoV and MERS-CoV vaccines has also met with some success. Many conventional vaccine strategies have been utilized, including inactivated viruses, recombinant attenuated viruses, other live viral vectors, subunit vaccines or individual viral proteins expressed from DNA plasmids or RNA delivery systems. Previous SARS vaccines focused primarily on the spike (S) protein due to its functions in human receptor binding, membrane fusion and viral entry. In addition, the S protein is the major 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 elicited neutralizing antibodies in immunized animals, these conventional vaccines are not effective in humans. It has also been found to raise important safety concerns (eg, by actually enhancing viral infection in many systems). Similarly, while many different SARS and MERS vaccines have been developed and tested, none of them are capable of preventing infection by viral neutralization without the immunopathology associated with full-length or trimerized S protein vaccines. showed defense.

A variety of viral vectors are known as carriers for a wide variety of antigens and are understood to effect effective therapeutic delivery to host organisms such as mammals such as humans. However, such viral vectors tend to vary with respect to the particular antigens delivered and the immune response elicited by different vectors. For example, some viral vectors, such as the ERVEBO® Ebola Zaire Vaccine, are live viruses, which stimulate a response by the host's immune system. However, responses from the viral vector itself to antigens delivered by the viral vector are often distorted, e.g., by exhibiting immune dominance of one antigen that prevents responses upon subsequent administration of the same or similar vaccine blunting the intended immune response. Advantageously, the antigens of the present disclosure do not stimulate subsequent host immune responses, do not exhibit immune dominance of one antigen, and are effective at doses administered subsequent to subsequent vaccines using the same viral vector. It has been found that it can be conjugated to the TMV NtK vector without effect. Also, avoiding such an immune response generated by the viral vector itself is a further advantage because the response for each antigen can be evaluated without considering the viral vector's effect on current and future vaccinations. provides bivalent, trivalent and tetravalent vaccines.

U.S. Pat. No. 7,939,318

Therefore, there is significant potential for an effective and scalable vaccine strategy for the prevention of Covid-19 disease that elicits high levels of neutralizing antibodies and induces sustained and potent neutralizing antibody titers long after immunization. , urgent and global needs exist. The platform described herein according to multiple embodiments and alternatives, along with other features and advantages outlined herein, meet this and other needs. In this regard, vaccines according to the present embodiments have shown strong immune responses in preclinical studies and, as an important advantage over conventional monovalent vaccines, have the ability to conjugate to multiple CoV antigens at once (i.e., multiple valence-binding vaccines), elicit high levels of neutralizing antibodies and have the potential to induce long-lasting and potent neutralizing antibody titers after immunization.

In some embodiments according to the present disclosure, a virus purification method comprises recovering viral material from a source organism comprising at least one virus and removing cellular debris from the at least one virus, thereby removing at least one characterization of the two viruses and concentration of the isolated and characterized viruses in a filtration device comprising, in some embodiments, a membrane with a pore size not exceeding a predetermined limit selected by the user. and treating the concentrated virus by subjecting it to a series of isolation procedures and harvesting the virus after each isolation procedure, wherein at least one The separation procedures include ion exchange chromatography to separate host cell contaminants from viruses, and at least one separation procedure separates residual impurities from viruses by at least size differences between impurities and impurities and one or more chromatographies. Including multi-mode chromatography, which separates from viruses based on chemical interactions that occur with ligands. In some embodiments, the plant is Nicotiana benthamiana and Lemna minor, as non-limiting examples, and is the source organism for recombinant expression of the virus. When the source organism is a plant, recovery may involve 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 is a non-plant host, such as, but not limited to, organisms such as bacteria, algae, yeast, insects or mammals.

In addition, 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 particle may be one or several, including, but not limited to, rod-shaped viruses, icosahedral viruses, enveloped viruses, and one or more fragments thereof. or all viruses and/or 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, for example, but not limited to, bacteria, algae, yeast, Non-vegetative hosts such as insects or mammals.

Advantageously, the multiset process performed according to various embodiments described herein produces highly purified virus or recombinant antigen or both on a commercial scale. Various steps are used to improve upstream purification processes, such as plant virus enrichment processes. In some embodiments, size exclusion chromatography, as well as other features, are used to produce purified recombinant viruses and recombinant antigens. Accordingly, various embodiments described herein provide one or more viruses and one or more antigens suitable for the preparation of one or more vaccines of conjugated viruses and antigens.

With respect to viruses, rod-shaped plant viruses (e.g., tobacco mosaic virus (i.e., "TMV)") and icosahedral plant It is now possible to purify viruses (eg red clover mosaic virus). Embodiments herein have enabled the purification of TMV and red clover mosaic virus, representing two structurally distinct viruses in terms of 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, TMVs are approximately 18 nm in diameter and 300 nm in length, are composed of approximately 2,131 copies of a 17.5 kDa coat protein, and are rigid to helically encapsidate genomic RNA at a ratio of 3 nt per coat protein. It contains 2160 capsid proteins with TMV virions that are rod-shaped particles. The TMV NtK genome is 6,407 nucleotides and is predicted to be encapsidated by 2,135 coat proteins. Although the following description is not intended to limit the viral carriers for use with the present embodiments to any particular one, further description and characterization of suitable TMV-NtK intermediates and such viral coat proteins are US 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 sca ffolds for display of protein antibodies for vaccine applications”, Virology 2006 348(2):475-88. Considering the diversity associated with different virus types, the process of the present invention is based on two structurally distinct viruses, allowing the virus to pass through filtration while retaining unwanted cellular debris. made it possible. In use, by controlling operating parameters, all types of viruses are transferred to the filtrate while chlorophyll/cell debris is retained, allowing the tangential flow (TFF) system to be efficiently controlled without excessive or untimely contamination. It is possible to continue to operate effectively. A further TFF step is designed to retain viruses while transferring small proteins to the filtrate, and a double chromatography step eliminates both large and small viruses while eliminating host cell proteins, host cell DNA, endotoxins and plant matter. Controlled to trap polyphenols.

Based on the successful purification of red clover mosaic virus and TMV, it is believed that the virus purification platform according to multiple embodiments and its alternatives can efficiently purify a wide array of virus particles, including: a variety of genetic material ( double- and single-stranded DNA viruses and RNA viruses), geometries (e.g. rod-shaped, curved rod-shaped, and icosahedral) and families (Caulimoviridae, Geminiviridae) , Bromoviridae, Closteroviridae, Comoviridae, Potyviridae, Sequiviridae, Tombusviridae.

Non-limiting viruses for which the embodiments described herein are believed to be successful include: Badnaviruses (e.g. Commelina yellow spot virus), Caulimoviruses (e.g. cauliflower mosaic virus), SbCMV-like virus (e.g. soybean wilt mottle virus), CsVMV-like virus (e.g. cassava vein mosaic virus), RTBV-like virus (e.g. Leistungro rod-shaped virus), petunia leaf vein transmembrane-like virus (e.g. petunia vein transmissibility virus), Mastrevirus (subgroup I geminivirus) (e.g. corn streak virus), and Curtovirus (subgroup II geminivirus) (e.g. beet curly top virus) and Begomovirus (subgroup III geminiviruses) (e.g. bean golden mosaic virus), Alfamovirus (e.g. alfalfa-mosaic virus), Ilarvirus (e.g. tobacco streak virus), Bromovirus (e.g. Brom-mosaic virus), Cucumovirus (e.g. cucumber mosaic virus), Closterovirus (e.g. beet yellow virus), Crinivirus (e.g. lettuce infectious yellow virus), Comovirus (e.g. cowpea mosaic virus), Fabavirus (e.g. fava 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. Leiss Tunglo spherical virus), Carmovirus (e.g. carnation spotted virus), Dianthovirus (e.g. carnation ringspot virus), Macromovirus (e.g. Machlomovirus (e.g. maize wilt spot 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 foliage mosaic virus), Furovirus (e.g. soil-borne wheat dwarf virus), Hormone Hordeivirus (e.g. wheat spotted leaf mosaic virus), Idaeovirus (e.g. raspberry yellowing virus), Luteovirus (e.g. barley yellow dwarf virus), Marafivirus ( corn rayado finovirus), Potexviruses (e.g. potato virus X and clover mosaic virus), Sobemoviruses (e.g. southern bean mosaic virus), Tenuiviruses (e.g. rice streak virus) viruses), Tobamoviruses (e.g. tobacco mosaic virus), Tobraviruses (e.g. tobacco stem gangrene virus), Trichoviruses (e.g. apple-chlorotic-leaf spot virus), Timoviruses Tymovirus (eg turnip yellow mosaic virus) and Umbravirus (eg carrot spot virus).

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 in some variations of this embodiment production of plant-based viruses is included, the embodiments described herein are directed to production of plant viruses. It is not limited to manufacturing or refining. In some embodiments, the virus purification platform begins by growing plants in a controlled growth chamber, infecting the plants with a replication-competent virus, disrupting the cells with a disruptor, and This is done by recovering the virus by removing the plant fibers from the liquid.

In some embodiments, the purification step involving plant-based and non-plant-based viruses comprises concentrating the clarified extract using a tangential flow system, wherein cassette pore size, transmembrane pressure and membrane The load of clarified extract per square meter of surface area is controlled. Transmembrane pressure (TMP) is the pressure difference between the upstream and downstream sides of a separation membrane and is calculated based on the following formula: ((feed pressure + retentate pressure)/2) - filtrate pressure. To ensure viral ceramic transfer and produce a clarified extract, in some embodiments, the feed pressure, retentate pressure and filtrate pressure are each controlled to obtain the appropriate TMP. . The clarified extract is further concentrated by an ion exchange column volume and washed with an ion exchange chromatography equilibration buffer. In some embodiments, a Capto Q ion exchange column is equilibrated, loaded with feed, 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.

In some embodiments related to plant-based viruses, an extraction buffer is added prior to removing chlorophyll and other larger cellular debris such as macromolecular fibers, organelles, lipids, etc. using tangential flow ceramic filtration. Added. In some embodiments, ceramic filtration promotes retention of chlorophyll from plant hosts, cell debris and other impurities while optimizing virus passage. In plant-based or non-plant-based viruses, this approach facilitates scalability of the process by allowing the desired target (virus or antigen) to pass through filtration while impurities are retained as retentate water. In addition, parameters such as transmembrane pressure, ceramic pore size and biomass loaded per square meter are all controlled to ensure ceramic movement of the virus and produce a clarified extract. Ceramic TFF systems have high scalability, parameters such as TMP, crossflow velocity, pore size and surface area can be easily scaled up or down to accommodate larger amounts of biomass. Additional ceramic modules can easily be added into the system. The feed, retentate, and filtrate pressures can also be controlled to maintain efficient cross-flow velocities 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 allows virus to pass through at smaller and larger scales. Ceramic TFF systems are capable of using highly efficient cleaning chemicals such as nitric acid, bleach and sodium hydroxide which are cleaning tested to meet GMP and/or cGMP requirements is.

Purification methods and alternatives according to embodiments, and other methods for developing scalable, high-throughput methods for purifying viruses, whether plant-based or non-plant-based, include: At least one separation using multimode chromatography that separates remaining impurities from viruses based at least on size differences between the virus and impurities and chemical interactions that occur between the impurities and one or more chromatography ligands. Use procedures. For example, it is within the scope of embodiments to perform at least one separation procedure with a Capto® Core 700 chromatography resin (GE Healthcare Bio-Sciences). Capto® Core 700 "beads" are octylamine ligands designed to have both hydrophobic and positively charged properties to trap molecules of specific size (e.g., 700 kilodaltons (kDA)). including. Because certain viruses are fairly large (e.g. >700 kDa) and the bead exterior is inert, the Capto® Core 700 allows purification of viruses by size exclusion, where the desired material (virus or antigen ) passes as filtrate and impurities are retained as retentate.

Again, in some embodiments, 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 a multimode chromatography column to recover virus in the void volume of the column. After washing the column to baseline, it is 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.

The purified virus is filter-sterilized, eg, by diafiltration, and stored.

With respect to antigens, practice of some embodiments of the inventive antigen purification platform described herein has resulted in 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/Singapore) Kansas), B/Colorado, B/Phuket, RBD-Fc121 (receptor binding domain (RBD, S1 domain) of SAR-2 spike glycoprotein fused to human IgG1 Fc domain (hereinafter "RBD-Fc121" is referred to below). (refers to the amino acid sequence of the SAR-2 spike protein as described in detail) and RBD-Fc139 (hereinafter "RBD-Fc139" refers to a different amino acid sequence of the SAR-2 spike protein), etc. of recombinant antigens were produced and purified.Antigens for various embodiments herein may be from many sources, including conventional antigens such as bacteria, yeast, insects, mammals, etc. It can be produced using recombinant protein manufacturing strategies, or using plant-based expression methods.

In some embodiments, the antigen production platform begins with growing plants in a controlled growth chamber, infecting the plants for recombinant antigen replication, using a crusher, followed by a screw press. This is done by removing the fibers from the aqueous liquid 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. Feed pressure, filtration pore size, clarifier and biomass loaded per square meter of membrane surface are controlled to facilitate filter movement of antigens, whether plant-based or non-plant-based antigens. . Descriptions of various (but non-limiting) process controls suitable for large-scale purification of viruses and antigens are provided in more detail in the Examples section.

In some embodiments, the clarified extract is then concentrated by a tangential flow system, as are plant-based and non-plant-based antigens. During this optional step, factors such as cassette pore size, transmembrane pressure and defined extract load 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. Antigen fractions are then collected in elution and prepared by cobalt-immobilized metal affinity chromatography (IMAC). The IMAC is equilibrated, loaded with feed, then washed with equilibration buffer and eluted. Eluted fractions are diluted, pH checked, and then loaded onto a multimode 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 controlled. Finally, the antigen is concentrated and diafiltered with a saline buffer. The recombinant antigen is sterile filtered and then stored.

Furthermore, according to various embodiments disclosed herein, the following monovalent formulations were efficiently conjugated: H7 rHA to TMV, H1N1 to TMV (Influenza A/Michigan), H3N2 to TMV ( Influenza A/Singapore), B against TMV/Colorado, B/Phuket against TMV, RBD-Fc121 against TMV (SARS-2), and RBD-Fc139 against TMV (SARS-2). According to various embodiments herein, bivalent formulations of TMV against two influenza B viruses (B/Colorado and B/Phuket) were also efficiently conjugated, as well as H1N1 (Influenza A/Michigan), Tetravalent conjugates of TMV to H3N2 (Influenza A/Singapore), B/Phuket and B/Colorado were also made. A "quadrivalent" influenza vaccine is 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 tetravalent vaccines are the most common because they could beneficially provide broader protection against circulating influenza viruses by adding other B viruses. target. As used herein, the term "multivalent" vaccine refers to one or more antigens conjugated to the 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 the source organism, multiple embodiments and alternatives. to generate an immune response according to Accordingly, the disclosure herein provides compositions comprising arrays of virus-protein conjugates, including virus-antigen conjugates. In some embodiments, the virus of choice is TMV or any of a number of viruses identified and/or indicated by the teachings herein. Further, in some embodiments, the antigen can be a protein such as, but not limited to, influenza hemagglutinin antigen (HA), including, but not limited to, influenza mediating viral infection as a soluble form of HA. Those listed in this paragraph, such as proteins present on the surface of viruses. In some embodiments, 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 a major thrust for the prevention of various influenza infections. Since the antigenicity of HA, ie the immunogenicity of HA, is related to conformation, trimerization of HA is known to be more beneficial than the monomeric form in terms of eliciting an immune response. .

In some embodiments, conjugation begins with concentration and diafiltration of the purified antigen and virus in a mildly acidic buffer. Antigen and virus are combined on a molar basis and then mixed. A freshly prepared water-soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (also known as EDC), is added to the mixture and mixed on a molar basis. Chemical reagents for converting carboxyl groups to amine-reactive N-hydroxysulfosuccinimide esters, such as Sulfo-NHS from ThermoFisher, are then added on a molar basis. The reaction is continued until a pre-determined stopping time. The reaction is then quenched by the addition of, for example, an amine group (e.g., a liquid containing free amines), and then on the mixture diluted to the target concentration in accelerating the reaction (e.g., EDC, Sulfo-NHS). Any chemical linkers used are removed by a multimode chromatography step or diafiltration. In some embodiments, complexed and purified viral particles modified with proteins and antigens may 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.

In some embodiments, purified virus-antigen fusions may be derived from genetic fusions in addition to the various embodiments disclosed herein. Antigen and viral structural proteins (located in the coat) form a single continuous open reading frame. In some embodiments, the antigen-coat protein is produced in the plant in reading frame such that the coat protein is self-assembled into viral particles. Plant material is then harvested and virus particles are purified according to embodiments disclosed herein. Viral particles modified with fusion-coat proteins can then be used as vaccines and/or diagnostic tools according to various disclosed embodiments.

Some viruses (eg, 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 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 pentamers or other structural subunits of the virus. A therapeutic agent (eg, a chemotherapeutic agent) is then added to the buffer and allowed to diffuse into the relaxed viral particles. By changing the pH again, the virus particles compact and break up the porous structure that together packages the pentamers or structural subunits, preventing chemical diffusion of the virus particles. The plant material is then harvested, the viral particles are purified, and the therapeutic agent-containing viral particles are used for drug delivery according to embodiments disclosed herein.

Accordingly, embodiments and alternatives include production of one or more highly purified viruses. Further embodiments and alternatives involve the production and/or purification of recombinant antigen. Further embodiments and alternatives include conjugation of purified antigens and viruses for use as vaccines. Indeed, in preclinical studies, TMV-platform vaccines produced according to embodiments described herein stimulated effective immune responses against several pathogens, including viral and antimicrobial systems. Furthermore, vaccines produced on the TMV-platform of the present invention demonstrate the ability to readily conjugate (ie, as multivalent conjugate 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). Virus purification may be performed alone according to this embodiment. Similarly, production or purification of recombinant antigen may be performed alone according to this embodiment. Optionally, different aspects of these multiple embodiments may also be combined, wherein combining embodiments may include one or more sources, among other ways of practicing these embodiments. Starting with an organism, producing one or more viruses and one or more antigens, then purifying such viruses and antigens, and then conjugating between at least one antigen and at least one virus Including forming a vaccine.

The international application to which this disclosure relates contains one or more drawings in color finish, some of which would be virtually pointless or impossible to be shown in black and white finish, examples of which include: For example, without limitation, protein structures and RBD-Fc fusions in FIGS. 51(A) and 51(B), microscopic images in FIG. 61, and FIGS. 71(A)-(C), 79(A). (B) and 81(A)-(B). For publication, Applicant provides drawings in black and white where appropriate to represent color images, which does not add subject matter.

The drawings and embodiments described herein are illustrative of alternative structures, aspects and features of embodiments and alternatives of the invention disclosed herein, which Neither the scope of the embodiments nor the alternatives should be understood as limiting. It is further understood that the drawings described and provided herein are not necessarily to scale and that the embodiments are not limited to the exact arrangements, depictions and instrumentalities shown.

FIG. 1 is a flow diagram showing steps in a particular virus purification platform within the scope of the present disclosure, according to multiple embodiments and alternatives. FIG. 2 is a purified icosahedral red clover mosaic virus according to embodiments and alternatives. FIG. 3 is a Western blot analysis of the purification of icosahedral red clover mosaic virus according to several embodiments and alternatives. FIG. 4 is a purified icosahedral red clover mosaic virus according to embodiments and alternatives. FIG. 5 is a Western blot analysis of the purification of icosahedral red clover mosaic virus according to several embodiments and alternatives. FIG. 6 is a purified rod-shaped tobacco mosaic virus according to embodiments and alternatives. FIG. 7 is a Western blot analysis of the purification of purified rod-shaped tobacco mosaic virus according to several embodiments and alternatives. FIG. 8 is a flow diagram showing the steps of the antigen production platform, according to several embodiments and alternatives. FIG. 9 is a Western blot analysis of several steps of the antigen production platform, according to several embodiments and alternatives. FIG. 10 is a Western blot analysis of several steps of the antigen production platform, according to several embodiments and alternatives. Figure 11 is a Western blot analysis of several steps of the antigen production platform, according to several embodiments and alternatives. Figure 12 is a Western blot analysis of purification of various antigens by the antigen production platform, according to several embodiments and alternatives. FIG. 13 is an illustration of conjugation of recombinant antigens to viruses, according to several embodiments and alternatives. FIG. 14 is an SDS-PAGE analysis of conjugation of antigens to viruses according to several embodiments and alternatives. FIG. 15 is an SDS-PAGE analysis of conjugation of antigens to viruses according to several embodiments and alternatives. FIG. 16 is an SDS-PAGE analysis of conjugation of antigens to viruses according to several embodiments and alternatives. FIG. 17 is a size exclusion-high performance liquid chromatography (SEC-HPLC) report of free TMV products, according to several embodiments and alternatives. FIG. 18 is a SEC-HPLC report of a 15 minute conjugation between virus and antigen according to several embodiments and alternatives. FIG. 19 is a SEC-HPLC report of a 2 hour conjugation between virus and antigen according to several embodiments and alternatives. FIG. 20 is a Western blot analysis of conjugation between virus and antigen, according to several embodiments and alternatives. Figure 21 is a graph showing infectivity of virus treated with various levels of UV irradiation, according to several embodiments and alternatives. FIG. 22 is an illustration of some steps of a platform for conjugation of recombinant antigens to viruses, according to several embodiments and alternatives. FIG. 23 is an SDS-PAGE analysis of conjugation of antigens to viruses according to several embodiments and alternatives. FIG. 24 is a negative staining transmission electron microscopy (TEM) image of recombinant antigen, according to several embodiments and alternatives. FIG. 25 is a negative staining TEM image of viruses according to embodiments and alternatives. FIG. 26 is a negative staining TEM image of recombinant antigens conjugated to other recombinant antigens along with added virus, according to several embodiments and alternatives. FIG. 27 is a negative staining TEM image of recombinant antigen conjugated to virus at a ratio of 1:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 28 is a negative staining TEM image of recombinant antigen conjugated to virus at a ratio of 1:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 29 is a negative staining TEM image of recombinant antigen conjugated to virus at a ratio of 4:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 30 is a negative staining TEM image of recombinant antigen conjugated to virus at a virus to recombinant antigen ratio of 16:1, according to several embodiments and alternatives. FIG. 31 is a normalized sedimentation coefficient distribution for antigens, according to several embodiments and alternatives. FIG. 32 is a normalized sedimentation coefficient distribution of viruses according to several embodiments and alternatives. FIG. 33 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a ratio of 1:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 34 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a ratio of 1:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 35 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a ratio of 1:1 virus to recombinant antigen, according to several embodiments and alternatives. FIG. 36 is a normalized sedimentation coefficient distribution of recombinant antigen conjugated to virus at a ratio of 4:1 virus to recombinant antigen, according to several embodiments and alternatives. 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 several embodiments and alternatives. FIG. 38 is a scatter plot of antigen-related titers in source organisms after administration of viral antigen products at various virus-to-recombinant ratios, according to embodiments and alternatives. Figure 39 is a geometric mean study showing antigen-related titers in source organisms after administration of viral antigen products at various virus-to-recombinant ratios, according to several embodiments and alternatives. Figure 40 is an SDS-PAGE analysis of purified recombinant antigens according to several embodiments and alternatives. FIG. 41(A) shows a graph of stability data for QIV conjugates at two different temperature ranges. FIG. 41(B) shows a graph of stability data for QIV conjugates at two different temperature ranges. FIG. 42 is a graph of immunogenicity of tetravalent vaccines in mice, according to several embodiments and alternatives. FIG. 43 is an illustration of immunogenicity and challenge studies of tetravalent vaccines in ferrets, according to several embodiments and alternatives. FIG. 44 is an illustration of virus titers in nasal washes after virus challenge in ferrets, according to several embodiments and alternatives. FIG. 45 is an illustration of virus titers in nasal washes after virus challenge in ferrets, according to several embodiments and alternatives. Figure 46 is an illustration of the immunogenicity of monovalent vaccines produced in mice at various virus to recombinant antigen ratios, according to embodiments and alternatives. FIG. 47 is an illustration of TMV vRNA in tissues over time after injection of a tetravalent vaccine, according to embodiments and alternatives. FIG. 48 is an illustration of TMV vRNA in tissues 8 days after injection of a tetravalent vaccine, according to embodiments and alternatives. FIG. 49 is an illustration of total anti-influenza titers based on ELISA analysis from rabbit serum samples after injection of tetravalent vaccine, according to several embodiments and alternatives. FIG. 50 is an illustration of neutralization titers measured in rabbits after injection of a tetravalent vaccine, according to embodiments and alternatives. FIG. 51(A) is an illustration of the protein structure of the Covid-19 spike trimer in a spatial stereo model with the receptor binding domain circled in horizontal and vertical views. Figure 52 is an illustration of an expression plasmid containing a recombinant Covid-19 antigen construct, according to several embodiments and alternatives. Figure 53 is an SDS-PAGE analysis of purified recombinant Covid-19 antigen according to several embodiments and alternatives. FIG. 54 is an SEC-HPLC report of purified recombinant Covid-19 antigen according to several embodiments and alternatives. Figure 55 is an SDS-PAGE analysis of purified recombinant Covid-19 antigen according to several embodiments and alternatives. FIG. 56 is an illustration of some of the platform steps for conjugation and drug substance loading of recombinant Covid-19 antigen to virus, according to multiple embodiments and alternatives. FIG. 57 is a normalized sedimentation coefficient distribution of recombinant Covid-19 antigen conjugated to virus, according to several embodiments and alternatives. FIG. 58 is a normalized sedimentation coefficient distribution of recombinant Covid-19 antigen conjugated to virus, according to several embodiments and alternatives. FIG. 59 is an SDS-PAGE analysis of virus-conjugated recombinant Covid-19 antigens according to several embodiments and alternatives. Figure 60(A) is an ELISA standard curve showing binding of recombinant Covid-19 antigen to Covid-19 human neutralizing monoclonal antibodies. FIG. 60(B) illustrates various vaccine (sometimes referred to herein as vaccine candidates) options according to embodiments and alternatives described herein. Graph showing binding to Covid-19 human neutralizing monoclonal antibodies. FIG. 61 consists of confocal microscopy images showing co-localization of recombinant Covid-19 antigen to ACE-2 specific antibodies, according to several embodiments and alternatives. FIG. 62(A) is a graph showing co-localization of native agonist and recombinant Covid-19 antigen to ACE-2 specific antibodies, according to several embodiments and alternatives. FIG. 62(B). Co-localization of native agonist and recombinant Covid-19 antigen to ACE-2-specific antibody in the presence of a co-localization control, according to embodiments and alternatives. 3 is a graph showing conversion. FIG. 63(A) Preimmune and day 2, day 28 after first administration with an exemplary vaccine at increasing dilution levels and varying concentrations of RBD-Fc antigen (with or without adjuvant). Figure 10 is a graph showing the results of a plaque neutralization assay of pooled serum samples on days 1 and 42 (boosted on day 14). FIG. 63(C) Pre-immune and day 2, day 28 after first dose with an exemplary vaccine at increasing dilution levels and varying concentrations of RBD-Fc antigen (with or without adjuvant). Figure 10 is a graph showing the results of a plaque neutralization assay of pooled serum samples on days 1 and 42 (boosted on day 14). FIG. 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 according to the same dilutions. FIG. 64(B) provides graphs further examining the neutralizing titers produced in the 15 and 45 g groups (but without adjuvant) on days 28 and 42 according to the same dilutions. FIG. 65(A) is a graph showing the immune response of animals immunized with recombinant Covid-19 antigens and controls. FIG. 65(B) is a graph showing the immune response of animals immunized with virus-conjugated recombinant Covid-19 antigen (without adjuvant). FIG. 65(C) shows the immune response of animals immunized with adjuvanted Covid-19 antigen conjugated to virus. Figure 65(D) compares IgG titers recognizing Covid-19 antigen (black) and RBD-His (grey). FIG. 66(A) is a graph showing IgG isotype analysis of individual sera from animals immunized with virus-conjugated recombinant Covid-19 antigen, control and adjuvant, according to several embodiments and alternatives. be. FIG. 66(A) shows the Th1 responses (IgG2a and IgG2c) associated with the assay. FIG. 66(B) is a graph showing IgG isotype analysis of individual sera from animals immunized with virus-conjugated recombinant Covid-19 antigen, control and adjuvant, according to several embodiments and alternatives. be. FIG. 66(B) shows the Th2 response (IgG1) associated with the analysis. FIG. 66(C) is a graph showing IgG isotype analysis of individual sera from animals immunized with virus-conjugated recombinant Covid-19 antigen, control and adjuvant, according to several embodiments and alternatives. be. FIG. 66(C) is a stacked plot comparison of relative antibody responses of IgG2 vs. IgG1 by ELISA analysis. FIG. 67 is a graph showing cell viability after incubation with SARS2 virus in mouse serum, according to embodiments and alternatives. FIG. 68 is a graph showing immune response titers and time series comparisons of animals immunized with virus-conjugated recombinant Covid-19 antigens and controls, according to several embodiments and alternatives. FIG. 69 graphically depicts virus neutralization titers after vaccination of mice with varying concentrations of an exemplary vaccine. FIG. 70(A) is a graph showing IFNγ ELISpot analysis of animals immunized with virus and recombinant Covid-19 antigen. FIG. 70(B) is a graph showing IFNγ ELISpot analysis of animals immunized with TMV:RBD-Fc vaccine without adjuvant. FIG. 70(C) is a graph showing IFNγ ELISpot analysis of animals immunized with TMV:RBD-Fc vaccine and adjuvant. FIG. 71(A) provides a graph of survival rate. FIG. 71(B) shows clinical symptoms. FIG. 71(C) provides post-challenge weights of subjects post-infection compared to control groups observed based on administration of various concentrations of RBD-Fc vaccine. FIG. 72 is a graph showing VaxArray® analysis of mouse sera obtained from mice immunized (with or without adjuvant) with various TMV:RBD-Fc vaccine doses, according to embodiments and alternatives. be. FIG. 73(A) provides a graph of experimental results from analysis of specificity and relative titers of sera from animal studies when compared to different capture antigens by VaxArray®. FIG. 73(B) provides graphs of experimental results from analysis of specificity and relative titers of sera from animal studies when compared to different capture antigens by VaxArray®. Figure 74 is a graph of titers as measured by VaxArray on day 42 serum from individual animals compared to individual and pooled neutralizing titers. FIG. 75(A) is a graph showing results on survival of macrophages exposed to SARS-2 spike protein with nucleocapsid and added non-invention antibody as an indicator of antibody enhancement of the disease. FIG. 75(B) is a graph of antibody-containing mouse sera generated from TMV:RBD-Fc vaccines according to embodiments and alternatives after exposure to SARS-CoV2 nucleocapsid and spike proteins. . FIG. 75(C) shows statistical differences applicable to the graphs of FIGS. 75(AB). Figure 76 is an SDS-PAGE analysis of purified recombinant Covid-19 antigen according to several embodiments and alternatives. FIG. 77 is an SEC-HPLC report of purified recombinant Covid-19 antigen according to several embodiments and alternatives. Figure 78 shows a sequence alignment of the receptor binding domains of spike proteins of SARS-2 and other related coronaviruses. FIG. 79(A) provides graphs of data for the mouse and ferret challenge studies, in which subjects were administered an exemplary vaccine at different rates with and without adjuvant. FIG. 79(B) provides a graph of data for a mouse challenge study, in which subjects were administered an exemplary vaccine at different rates with and without adjuvant. FIG. 80 provides a graph of vaccine tolerance based on weight change over time after administration of an exemplary vaccine in the absence of infection. FIG. 81(A) provides a graph of weight gain and survival data from a rabbit challenge study, in which subjects were administered an exemplary vaccine at different rates, with and without adjuvant. FIG. 81(B) provides graphs of weight gain and survival data from a rabbit challenge study, in which subjects were administered an exemplary vaccine with and without adjuvant at different rates.

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

Virus Production and Purification Table 1 and FIG. 1 illustrate the steps of the virus purification platform according to several embodiments and alternatives.

This purification platform is designed for commercial scalability and compliance with cGMP regulations and utilizes one buffer throughout the entire purification process. According to embodiments and alternatives, the virus purification platform steps are performed in conjunction with plant expression. However, even for non-plant viruses (unless the context clearly relates to plants, e.g. plant fiber removal references), the subsequent steps of aerial tissue collection and cell disruption also apply, as described below. be.

According to several embodiments and alternatives described herein, viral expression is accomplished by methods appropriate to the particular host. In some embodiments, virus-based gene delivery to a plant host is achieved by a modified TMV expression vector that recombinantly forms virus in tobacco plants. One such available option is the GENEWARE® platform described in US 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, with a short recovery period (e.g., less than 21 days) after inoculation. Express various viruses. Tobacco plants inoculated with viral genes express specific viruses in infected cells, which are extracted upon harvesting. In the methods described herein, inoculation is performed by manual inoculation on the surface of the leaves, mechanical inoculation on the plant bed, high pressure spraying on the leaves or vacuum infiltration, as examples selected by the user. .

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

In some embodiments, the growth of Nicotiana benthamiana wild-type plants is performed in a controlled growth room. Plant cultivation is controlled by cycles of irrigation, light and fertilization. Plants are grown in soilless media and temperature is also controlled throughout the process.

At a suitable number of days after sowing (DPS), eg 23-25 DPS, the plants are infected with viral replication. After infection, the plants are irrigated only and controlled via light cycle and temperature for a certain number of days after infection (DPI) depending on the virus type.

Plants are closely examined for height and signs of infection, and aerial tissues are collected.

For virus recovery/cell disruption, comminution with a grinder configured with optimized blade/sieve size, followed by removal of residual cellulosic plant fibers from the aqueous liquid (e.g., in one example, by a screw press). done by removal.

Add a suitable extraction buffer (e.g., 200 mM sodium acetate, pH 5.0, see step 201 in FIG. 1 as a non-limiting example) to the resulting extract at a buffer:tissue=1:1 ratio. do. Pilot scale chlorophyll and large cellular debris removal uses tangential flow (TFF) ceramic filtration (1.4 microns/5.0 microns). 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 virus passage through the ceramic. In some embodiments, the 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, such as 1.2 micron glass fiber filtration.

The ceramic filtrate is further clarified through the use of glass fiber depth filtration (step 203 in FIG. 1 as a non-limiting example).

The clarified extract is concentrated by a TFF system (from Sartorius AG). Cassette pore size (100-300 kDa), appropriate TMP as described herein, and load of clarified extract per square meter of membrane surface are controlled.

The clarified extract is concentrated on an NMT 2X ion exchange column volume with ion exchange chromatography equilibration buffer (200 mM sodium acetate, pH 5.0, shown in step 204 of FIG. 1 in a non-limiting example), washed twice. A Capto Q ion exchange column is equilibrated with 200 mM sodium acetate, pH 5.0 (step 205 in FIG. 1 is shown as a non-limiting example) for 5 column bed volumes, 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 a high salt concentration.

Flow-through and wash fractions are collected, combined, and prepared for multimode Capto® Core 700 chromatography. A 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 provides a non-limiting example).

Combine the flow-through and wash fractions from the Capto Q ion exchange chromatography, load it onto the column and recover the virus in the void volume of the column. The column is washed to baseline and stripped with high conductivity sodium hydroxide. Loading ratio, column bed height, residence time and chromatography buffer are all controlled. Formulation and concentration of virus (step 208 in FIG. 2) is performed in some embodiments by a TFF system (eg, Sartorius AG system). Pore size (30-300 kDa), appropriate TMP described herein, loading per square meter of membrane surface area and pore material are all controlled. The virus is concentrated to a suitable concentration, eg 10 mg/ml, and in some embodiments diafiltered with a suitable buffer, eg sodium phosphate. The formulated virus is sterilized and properly stored. In some embodiments, sterilization is through PES filters.

All examples provided herein describe virus production, virus purification, antigen production, antigen purification and virus-antigen conjugation according to some or all of the various aspects of multiple embodiments and alternatives. It is provided in an illustrative sense. These examples are non-limiting and merely illustrate features of several alternative embodiments herein.

Example 1 - Purification of icosahedral red clover mosaic virus. Show purification. Similarly, Western blotting in FIG. 5 shows successful purification of the icosahedral red clover mosaic virus illustrated in FIG. Both viruses were purified according to embodiments described herein. Target proteins were extracted from the tissue according to known detection techniques. Sample proteins were then separated using gel electrophoresis based on their isoelectric point, molecular weight, charge or various combinations of these factors. Samples were then loaded in various lanes of the gel, with lanes provided as "ladders" containing mixtures of known proteins of known molecular weight. For example, lane 12 in FIG. 3 functions as a ladder. A voltage was then applied to cause different proteins to migrate through the gel at different velocities based on the factors mentioned above. Separation of different proteins into visible bands occurred within each lane, as provided in Figures 3 and 5, respectively. Pure product was characterized by a distinct visible band by Western blotting and is characterized in these figures.

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 different steps in the virus purification platform. In FIG. 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 FIG. 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.

After the final step has been 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.

Example 2 Purification of Rod-Shaped TMV FIG. 6 shows purified rod-shaped TMV, and FIG. Figure 1 illustrates the virus purification platform used in obtaining TMV. Similar to Figures 3 and 5, Figure 7 shows the purity of the virus product after completion of various steps of the virus purification platform of the present invention. After the final purification step, the product obtained is a highly purified virus product consistent with the distinct visible band in lane 13 of FIG. Exemplary, prior to such conjugation, the identity of intermediate TMV-NtK can be confirmed by mass spectrometry, eg, MALDI-TOF mass spectrometry. In some embodiments, the physiochemical properties of the intermediate TMV-NtK are pH of about 7.0-7.4, osmolality of about 15-45 mOsm/kg H ₂ O, 100 CFU (colony forming units )/mL or less and less than 100 ng/mg of residual host cell-derived protein.

Thus, the virus purification platform of the present invention efficiently purifies any virus to which the inventors have applied these methods, including icosahedral and rod-shaped viruses, and the platform is reproducible and consistent. are expected to purify any (if not all) types of viruses on a commercial scale.

Recombinant Antigen Production and Purification Table 2 and FIG. 8 illustrate the steps of the antigen purification platform according to several embodiments and alternatives.

This purification platform is designed for commercial scalability and compliance with cGMP regulations and utilizes one buffer throughout the entire purification process. According to several embodiments and alternatives, the steps of the antigen purification platform are as follows.

Growth of Nicotiana benthamiana wild-type plants in a controlled growth chamber. Plant growth is controlled by cycles of irrigation, light and fertilization. Plants are grown in soilless media and temperature is controlled throughout the process. After an appropriate number of DPS, eg 23-25 days, the plants are infected for protein replication of the selected antigen. Once attached, the protein is sufficient for retention in the ER of transgenic plant cells. After infection, the plants are irrigated only and controlled by light cycle and temperature for an appropriate number of days post-infection, eg 7-14 days, depending on the type of antigen. Plants are closely examined for height and signs of infection, and aerial tissues are collected.

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

A suitable extraction buffer is added to the resulting extract at a suitable ratio, eg, a 1:1 buffer:tissue ratio, or a 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 cellular debris involves the use of filtration. Add Celpure 300 at a rate of 33 g/L and mix for 15 minutes. Feed pressure (<30 PSI), filtration pore size (0.3 microns), clarifier (Celpure 300) and biomass loaded per square meter of membrane surface are all controlled to ensure antigen passage.

The clarified extract is concentrated by a TFF system (eg Sartorius AG system). In some embodiments, the cassette pore size (eg, 30 kDa), the appropriate TMP described herein and the load of clarified extract per square meter of membrane surface area are controlled.

The clarified extract is concentrated and washed seven times with a suitable ion exchange chromatography equilibration buffer (eg 50 mM sodium phosphate + 75 mM NaCl, pH 6.5). A Capto Q ion exchange column was equilibrated with 50 mM sodium phosphate + 75 mM NaCl, pH 6.5 for 5 column volumes, loaded with feed, washed with equilibration buffer, and eluted/desorbed the column at high salt. .

Antigen fractions are collected in the elution and subjected to preparation on Cobalt IMAC chromatography. IMAC is equilibrated with 50 mM sodium phosphate + 500 mM sodium chloride, pH 8.0 for 5 column volumes, loaded with feed, washed with equilibration buffer, and eluted using imidazole.

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

The antigen is then concentrated to a suitable concentration, eg 3 mg/ml, and diafiltered through a suitable buffer (eg phosphate buffered saline, pH 7.4). The formulated antigen is sterilized and properly stored. In some embodiments, sterilization is through PES filters.

Figures 9, 10 and 11 illustrate various steps of the antigen purification platform according to several 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. indicates

Examples 3, 4, 5 and 6 - H5 rHA, H7 rhA, WNV rDIII and LFV rGP1/2
As shown in FIG. 12, multiple embodiments and alternatives of the antigen purification platform efficiently purified H5 rHA, H7 rhA, WNV rDIII and LFV rGP1/2. Figure 12 shows two images resulting from the antigen purification platform:
Left image contains SDS Page gel showing purity of viral vector TMV NtK (NtK is an abbreviation for N-terminal lysine) and influenza antigens, right image shows immunoreactivity to West Nile and Lassa fever antigens. Including western blot. Each antigen product is of high purity, as indicated by the distinct visible bands in FIG. Thus, multiple embodiments and alternatives of the antigen purification platform were used in a manner that also complies with cGMP regulations to consistently purify each type of antigen at commercial scale. Likewise, this platform will reproducibly purify virtually (if not all) any type of antigen.

Production of Recombinant Antigen-Virus Conjugates FIG. 3 illustrates the steps of conjugation of recombinant antigens, according to several embodiments and alternatives.

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.

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.

Chemical reagents for converting carboxyl groups to amine-reactive N-hydroxysulfosuccinimide esters, such as ThermoFisher's Sulfo-NHS, are formulated in purified water to a molar concentration of 0.1M.

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

A freshly prepared water-soluble carbodiimide (eg, EDC) is added to the mixture and mixed on a molar basis.

A chemical reagent (eg, Sulfo-NHS) to convert carboxyl groups to amine-reactive esters is added within 1 minute of EDC addition on a molar basis. The conjugation reaction is initiated and allowed to continue for a pre-determined mixing stop time, eg, 4 hours, and controlled at room temperature.

Reactions are quenched by the addition of free amines and chemical linkers (eg EDC and Sulfo-NHS) are removed by multimode chromatography steps such as Capto® Core 700 or diafiltration into phosphate buffered saline. . According to several embodiments and alternatives, residual impurities are filtered out by the conjugation reaction (sometimes referred to herein as conjugate (referred to as a mixture).

The conjugate mixture is diluted to the target concentration. At this point the viral antigen conjugate is prepared for use as a purified vaccine/drug substance. Suitable delivery mechanisms for vaccines include lyophilized materials containing liquid vials or 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.

Example 7 - Conjugation of H7 rHA to TMV Figure 13 provides an illustration of the conjugation of recombinant antigen (meaning "vaccine antigen") to virus, in which light and dark shaded ovals are shown. represents the scope of conjugation of the vaccine antigens represented in the examples. Light shading represents free virus, while dark shading represents antigen conjugated to the viral coat protein. Also, as shown in FIG. 13, some viruses contain proteins arranged in a coat around the RNA genome. For example, the viral vector TMV NtK contains an N-terminal lysine that functions 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 provide amine-targeted conjugation (e.g., antigen to virus) of the protein for binding of recombinant antigen. Strengthen your presentation. Relating to the discussion of radius measurements herein, viral radius is greatly increased following conjugation of recombinant antigen to viral coat protein. In some embodiments, modifications are made when attempting to modify the presentation of those residues by enveloped viruses.

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

SEC-HPLC reports also showed successful conjugation of H7 rHA to TMV according to embodiments of the conjugation platform of the present invention. Figure 17 shows the SEC-HPLC report of the free TMV product. The signal data detailed in Table 4 below were obtained from the SEC-HPLC report of the free TMV product in FIG.

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. Signal data detailed in Table 5 were obtained from the SEC-HPLC report in FIG. 18 after H7 rHA was conjugated to TMV for 15 minutes.

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. Signal data detailed in Table 6 below were obtained 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.

As illustrated in Figures 19 and 20, the SEC-HPLC report showed 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 several embodiments and alternatives, the SEC-HPLC report indicates that the conjugation reaction achieved at least about a 50% reduction of the unconjugated, native molecular weight, viral coat protein, and conjugation lasted 4 hours. It shows that about 3% free TMV remained after it happened.

As illustrated in Figure 20, Western blot analysis of the conjugated product showed successful conjugation of H7 rhA to TMV via covalent attachment. FIG. 20 shows a Western blot analysis of various steps of the conjugation platform according to an embodiment of the 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.

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. is necessary. In addition, many regulatory agencies have enacted regulations (eg, cGMP regulations) requiring 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 in biopharmaceutical products remains unexplored and research on its ability to effectively inactivate viruses has been limited.

Therefore, different UV-C conditions (i.e., energy density and wavelength) and different UV-C conditions (i.e., energy density and wavelength) and different UV-C conditions to effectively inactivate and sterilize TMV NtK after virus production and purification and prior to conjugation with recombinant antigen. TMV concentration was evaluated. Although many energy densities were tested, only high levels of energy densities efficiently inactivated TMV NtK. In addition, the success or failure of viral inactivation was found to be concentration dependent, as UV-C irradiation did not effectively sterilize all virus in the sample when the TMV solution was not diluted to the appropriate concentration. rice field. Therefore, TMV solutions must be diluted appropriately to allow UVC irradiation to interact with and effectively inactivate each virus.

As shown in FIG. 21, various amounts of UVC irradiation (energy densities from 300 J/m ² to 2400 J/m ² ) were tested on Nicotiana tabacum to assess infectivity. As shown in Figure 21, lesions were reduced to zero after a UVC energy dose of 2400 J/ ^m2 , thus indicating good inactivation of the virus. In addition, a number of higher energy doses were also tested and it was found that good inactivation of TMV NtK also occurred at energy densities from 4800 J/m ² to 5142 J/m ² .

According to several embodiments and alternatives, the virus inactivation step (after purification and before conjugation) is as follows.

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

Filtration of the TMV solution at 0.45 micron to remove bacteria and any other large species that may interfere with the measurement of UV radiation, another purification step according to this embodiment, is performed just prior to inactivation by UV light. conduct.

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

Inactivated TMV NtK is then prepared for conjugation to recombinant antigen.

These virus inactivation steps are designed for commercial scalability and compliance with cGMP regulations.

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

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

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

Based on this experiment, a model of conjugation (shown in Figure 22) was developed. According to embodiments and alternatives, exposing the virus to a conjugation environment improves the chemical readiness of the virus (referred to herein as "activating," "activating," or "activating"). The conjugation between the purified virus and the purified antigen (denoted as "rHA" in Figure 22) is greatly enhanced by associating the antigen with the antigen. In some embodiments, viral activation is achieved by formulating the virus at an acidic pH prior to the conjugation reaction to concentrate positive charges on the viral surface. In some embodiments, the activating step comprises 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 acidic pH activates the virus by charging the lysines of the coat protein. As a result of this activation step in the conjugation environment, positive charge concentrations on the viral surface (as shown in Figure 22) via amine groups and viral clustering are available for conjugation with the carboxyl terminus of the recombinant antigen. Be prepared.

The virus activation step according to embodiments and alternatives is in contrast to conventional approaches in which 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 the positive charge on the viral 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.

In studies of successful conjugation with TMV, good conjugation generally occurs when the dynamic light scattering (DLS) measured viral radius increases by at least 2.75-fold during the activation step. was observed (see Table 9A compared to Table 9B). In general, as shown in these tables, successful TMV conjugations (eg, discussed in Table 9C) were characterized by increased DLS radii from about 70 nm to about 195 nm or more.

Based on successful conjugations utilizing viral activation, we developed a platform for conjugating purified antigens to purified viruses. According to several embodiments and alternatives, the steps for preparing purified antigen for conjugation are as follows:
To ensure pH control of the conjugation reaction, purified antigen is formulated in the reaction buffer just prior to starting the reaction.

Prior to conjugation, purified antigen is stored in slightly neutral to basic pH phosphate-buffered saline.

Depending on the nature of the molecule, the target pH for antigens is typically pH 5.50-6.50.

To facilitate conjugation to virus, storage buffer is exchanged with MES/NaCl buffer by ultrafiltration using acidic pH. Also increase the protein concentration above 3 mg/mL.

The conjugation reaction is then initiated within 4 hours of completion of antigen preparation to prevent destabilization of protein structure.

According to embodiments and alternatives, the steps for preparing purified virus for conjugation are as follows:
After storage at neutral pH, virus is activated at acidic pH prior to conjugation. For a good reaction, the virus is formulated in a pH 7.4 phosphate buffer to a pH 5.50 acetate buffer for a minimum of about 18 hours and a maximum of about 72 hours before starting the conjugation reaction. In some embodiments, the virus is formulated in pH 7.4 phosphate buffer to pH 4.50 acetate buffer for a minimum of about 18 hours and a maximum of 72 hours prior to initiation of the conjugation reaction. It was observed that when viruses were stored at acidic pH for more than 72 hours, self-associations between viruses were formed that made the viruses less soluble and reduced conjugation efficiency.

Tables 9A and 9B further demonstrate that increasing the radius of the virus (in this case TMV) as measured by DLS advances the activation step. Specifically, Table 9A presents data from increased DLS radii of TMV after activation and before successful conjugation with the antigens listed in the right column. "Folding radius increase" is the TMV radius after activation divided by a typical TMV radius of about 70 nm at neutral pH. Conversely, Table 9B presents data in which the DLS radius of TMV was increased after initiating the activation step and prior to unsuccessful attempts at conjugation with the antigens listed in the right column. In Tables 9A and 9B, the left column represents the standard radius of TMV rods under neutral pH and typical storage conditions, ie before activation.

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 time of the conjugation reaction using DLS after the virus was activated using acidic pH. As shown in Table 9C, molecular radius is one indicator of successful coating of viral rods by antigenic molecules.

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

Example 10 - TEM Imaging for Conjugation with Different Ratios of Purified Virus to Purified Antigen The desired conjugation reaction between purified virus and purified antigen is represented by the following equation: :
Virus + antigen → virus/antigen (Formula 1)

However, antigens are known to be prone to self-conjugation and may not provide the desired activity, as shown in the following formula:
Virus + antigen → virus/antigen + antigen/antigen (Formula 2)

Self-conjugation of purified antigens is a challenge for successful vaccine development because the antigen-antigen conjugates are not removed during the size chromatography step, resulting in minimal or reduced immune responses.

To address this self-conjugation issue, various experiments were performed to determine how unreacted antigen and antigen conjugates are consumed. Antigens were first capped by exposing them to a self-conjugation inhibiting reagent. We expected this conventional approach to be successful, but it failed because the reaction occurred too quickly.

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

In the various designations listed herein, "KBP-VP" refers to TMV Antigen Presentation and is provided for reference purposes only. FIG. 24 is a TEM image of Sample 1 (Free HA, Lot 19UL-SG-001) at 52,000× magnification and 200 nm scale bar. In FIG. 24, the sample contained small spherical arrowheads (indicated by arrow A), 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 that fits the ordered aggregation of HA, matching the natural trimeric conformation. In addition, the particles were well dispersed with minimal clumping.

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 (arrows A) of sizes varying in length from about 125 nm to about 700 nm and width from about 18 nm to about 20.5 nm were observed. These dimensions are consistent with the size and shape of the TMV particles. In addition, a central ~4 nm channel is observed in the rods (arrow B), which is a known feature of TMV. The 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 rod-shaped particles in the background. These spherical particles (arrow C) did not resemble individual HA trimers.

FIG. 26 is a TEM image of Sample 3 (HA:HA self-conjugate, TMV NtK spiked, Lot 19UL-SG-004) at 52,000× magnification and 200 nm scale bar. In FIG. 26, rod-shaped particles are observed, varying in length from about 25 nm to about 885 nm, width from about 18 nm to about 20.5 nm (arrow A), and inner channel of about 4 nm in the middle (arrow B). rice field. The rods were either completely undecorated or sparsely decorated with small protein 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 rods (arrow D). Figure 26 shows large clumps of HA particles, but the TMV appeared identical to the unconjugated TMV (shown in Figure 25) as expected.

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 are observed, varying in length from about 50 nm to about 1000 nm or more, width from about 18 nm to about 20.5 nm (arrow A), and an inner channel of about 4 nm in the middle (arrow B). rice field. The particle rods were similar in size and shape to the conjugated TMV observed in Figure 28, with the exception that most rods were heavily decorated with small protein densities on their surface ( Arrow C). Some of the small, proteinaceous particles were also seen in the background and were not associated with rods (arrow D). Sample 5, shown in Figure 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 brought back to pH 5.50 at the start of the conjugation reaction. For the batches shown in Figures 28-30, virus was formulated directly at pH 4.50 and maintained overnight prior to conjugation.

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 vary in length from about 65 nm to about 720 nm, width from about 18 nm to about 20.5 nm (arrow A), and inner channel of about 4 nm in the middle (arrow B). observed. The particle rods were similar in size and shape to the free TMV NtK (Sample 2) observed in FIG. However, in contrast to the unconjugated virus shown in Figure 25, the particle rods observed in Figure 28 were decorated with moderate protein densities (Arrow C). These densities were irregular in shape and size and appeared to be randomly associated with the surface of the rods with no apparent pattern. Some of the small, proteinaceous particles were also seen in the background and were not associated with rods (arrow D).

FIG. 29 is a TEM image of Sample 6 (TMV:HA, 1:4 ratio, Lot 19UL-SG-002) at 52,000× magnification and 200 nm scale bar. In FIG. 29, rod-shaped particles fluctuate with a length of about 25 nm to about 1000 nm or more, a width ranging from about 18 nm to about 20.5 nm (arrow A), and an inner channel of about 4 nm in the middle (arrow B). observed. The particle rods observed in Figure 29 were similar in size to the previously conjugated samples, but the density of small proteins (arrow C) varied from moderate to sparse levels of surface modification. fluctuated up to Some of the small, proteinaceous particles were also seen in the background and were not associated with rods (arrow D).

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 with a length of about 30 nm to about 1000 nm or more, a width of about 18 nm to about 20.5 nm (arrow A), and an inner channel of about 4 nm in the center (arrow B). The particle rods observed in Figure 30 were similar to the previous conjugated samples in terms of overall morphology. However, the rods were only sparsely decorated with protein (arrow C) or not at all. Small, proteinaceous particles were seen slightly in the background and were not associated with rods (arrow D).

Figures 24-30 show that a 1:1 ratio showed complete rod decoration, a 4:1 ratio showed medium decoration and a 16:1 ratio showed sparse decoration. indicates In other words, a 1:1 ratio generated viral rods with heavy antigenic decoration (i.e., greater density) of HA antigens, while a 16:1 ratio produced less antigenic decoration of HA antigens on each rod. viral rods were generated with a (ie, lower density). HA-HA self-conjugation was observed as a by-product of the conjugation reaction, mainly in a 1:1 ratio reaction. Furthermore, TEM images as well as reaction analysis by SDS-PAGE showed less free HA or HA-HA conjugate 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 efficiency of conjugation of exclusively HA to TMV rods was overall higher in the 16:1 ratio, but with a lower density of HA per rod than in the 1:1 reaction.

Example 11 - Centrifugal Sedimentation Velocity Analysis of Different Conjugation Conditions Sedimentation Velocity ("SV") Measured in an Analytical Ultracentrifuge ("AUC") Provides Information on Protein Heterogeneity and Aggregate Association State The ideal way. Specifically, aggregates or different oligomers can be detected on the basis of different sedimentation coefficients. This method also detects aggregates or other minor constituents at levels of 1% by weight or less. Furthermore, SV provides high quality quantification of the relative abundance of species and provides accurate sedimentation coefficients in any aggregate.

To determine the content of unreacted self-conjugated HA and HA occupancy on TMV NtK by different conjugation conditions, total signals associated with centrifugation of free antigen, free virus and various TMV:HA ratios were analyzed. Measured using SV-AUC. The following samples and descriptions are shown in Table 12:

These stocks were shipped refrigerated (not frozen) and then stored at 2-8°C until analysis. Corning 1×PBS was used as a blank reference for sample dilution. Samples for sedimentation velocity were prepared by diluting sample 1 1:1 and samples 2-7 1:3 in 1X PBS. These dilutions were made to include the total absorbance of the samples within the linear range of the absorbance detection system.

Method: Diluted samples were loaded into a cell with a 2-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 in an analytical rotor, loaded into an analytical ultracentrifuge and cooled to 20°C. The rotor was then spun at 3000 rpm and the samples were scanned (280 nm) to confirm proper cell loading. For samples 2-7, the rotor had a final spin speed of 9,000 rpm. Scans were recorded at this rotor speed (every 3 minutes) for approximately 11 hours (250 total scans for each sample) as fast as possible. For sample 1 (free HA), the rotor was at 35,000 rpm and scans were recorded every 4 minutes for 5.3 hours. Schuck, P.; (2000), "Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling", Biophys. J. The data were then analyzed using the c(s) method described in 78, 1606-1619. This method was used to directly fit the raw scans to derive the sedimentation coefficient distribution, while modeling the effects of diffusion on the data to enhance resolution.

Results and Discussion: High-resolution sedimentation coefficient distributions for samples 1-7 are shown in Figures 31-37. In these figures, the vertical axis indicates concentration and the horizontal axis indicates separation based on sedimentation coefficient. Each distribution was normalized by setting the total area under the curve to 1.0 (100%) to ensure that the area under each peak represented the proportion of that species. Data analysis was pushed as samples 2-7 contained material that precipitated over a wide range of sedimentation coefficients, covering species that precipitated as rapidly as 2000 Svedburg units (S), whereby the horizontal axis was Log scale. To compensate that log scaling can distort the visible region of the peaks, the vertical axis is multiplied by the sedimentation coefficient so that the relative peak areas are properly scaled. Data for sample 1 (free HA) are conventionally presented using a linear sedimentation coefficient scale.

FIG. 31 is the normalized sedimentation coefficient distribution for Sample 1 (HA alone, Lot 19S-G-001). Due to the much smaller size of the free antigen than the virus, this sample was run at a much higher rotor speed (35,000 rpm) than samples 2-7 (9,000 rpm) to characterize the proper particle size distribution. bottom. As shown in Figure 31, Sample 1 is somewhat uniform, showing a main peak of 73.7% at 8.967S. This was the expected result for the HA antigen only sample. The width of the main boundary and this sedimentation coefficient mean that this main peak species has a molar mass of about 222 kDa, which roughly corresponds to the expected ∼70 kDa monomer of HA. can be shown to correspond to mers. It is physically absurd that this sedimentation coefficient corresponds to a monomer, instead the main peak corresponds to an oligomeric state larger than the monomer. As highlighted in the SEC HPLC data of HA3 Singapore release in Table 13 below, >90% of HA was identified in the trimeric state, with 50% for 3 of the 4 samples analyzed. There was super trimer formation.

As also shown in Figure 31, seven minor peaks of faster sedimentation than the main peak were detected, which together represent 6.2% of the absorbance of all sediments. Presumably, those two peaks represent product aggregates rather than higher molecular weight impurities. The major aggregate species of 12.4S (4.25%) is sedimenting 1.4 times faster than the monomer, falling in the range of 1.4-1.5 typically observed for the dimer. ratio. The ratio suggests that this species is a dimer of the main peak material (probably a hexamer of about 70 kDa monomer), and its sedimentation coefficient suggests that it is highly elongated of the main peak material, or It could suggest a partially open trimer (probably a nonamer of about 70 kDa monomers).

In Figure 31, the next peak at 15.3S (0.96%) precipitates 1.7 times faster than the monomer, suggesting a trimer of the main peak material. 30.9S. No absorbance of any higher sedimentation coefficient was detected. Three minor peaks of 2.8S (2.81%), 4.5S (12.44%) and 6.0S (4.94%) were also detected that settled more slowly than the main peak. . Among these minor peaks, the 4.5S peak most likely corresponds to the antigen monomer.

Figure 32 is the normalized sedimentation coefficient distribution for sample 2 (free TMV NtK, Lot.18HA-NTK-001). As shown in Figure 32, no sedimenting material was detected at about 60S. This sample appeared very heterogeneous, with the richest peak being the sedimentation of 229S (30.9%). A second richest 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, with the largest sedimentation coefficient seen in this Example 11. It is unclear what the partially split peak from about 60S to 2000S represents.

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 settle. This was established by increasing the rotor speed to 35,000 RPM after completion of each run in order to pellet all remaining material. This material was not observed in either free antigen or free TMV NtK samples. However, this material did not precipitate and thus did not affect the measured size distribution results.

FIG. 33 is the normalized sedimentation coefficient distribution for Sample 3 (Lot 19 UL-SG-004 at a 1:1 ratio of TMV to HA). As shown in Figure 33, the results for 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 with 9.9S may correspond to the main peak observed with the free HA sample (shown in Figure 32). The smaller peak diversity may reflect the HA-HA self-conjugation event.

Figure 34 is the normalized sedimentation coefficient distribution for Sample 4 (Lot 18 KBP-VP-SG-002 at TMV to HA = 1:1 ratio) and Figure 35 is the distribution for Sample 5 (TMV to HA = 1 Distribution of normalized sedimentation coefficients for lot 19UL-SG-001) at :1 ratio. 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, the low resolution at this rotor speed makes it difficult to comment on the differences observed for free antigen samples (1-40S). Nonetheless, FIGS. 34 and 35 show that there is more total signal from 40S-2,000S (indicative of virus-associated material) than sample 3. FIG.

Figure 36 is the normalized sedimentation coefficient distribution for Sample 6 (Lot 18 19 UL-SG-002 at TMV to HA = 4:1 ratio) and Figure 37 is for Sample 7 (TMV to HA = 16:1 Distribution of normalization factor for lot 19UL-SG-003) in ratio. Figure 36 shows 91.1% virus-related material (ie, virus-antigen conjugates) and Figure 37 shows 99.4% virus-related material (ie, virus-antigen conjugates).

The results of the virus-antigen normalized sedimentation coefficient distributions shown in FIGS. 33-37 are shown in Table 14. As mentioned above, according to embodiments and alternatives, the fraction between 1-40S represents the percentage of HA monomer/trimer and the fraction between 40-2000S represents TMV NtK- Percentage of HA conjugates is shown.

The results in Table 14 show that the 1:1 ratio has more HA self-conjugate and HA product compared to the 4:1 and 16:1 ratios. Furthermore, increasing the TMV:HA ratio results in virtually complete association of the HA product at the TMV conjugation event (approaching almost 100% conjugation in sample 7).

According to embodiments and alternatives, the amount of HA in the conjugation reaction was reduced by increasing the TMV NtK to HA ratio from 1:1 to 16:1 by (1) Example 10 and (2) the amount of self-conjugation and unreacted HA events, as shown in FIGS. 31-37 and Table 14; and (3) the association (percentage) of HA to TMV increases compared to self-conjugation and unreacted HA events, as shown in FIGS.

Example 12 - Immune Response in Mice To determine the immune response following administration of a viral antigen conjugate of the invention, mice were administered the conjugate 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 administered to most animals on days 0 and 14 of the study (control animals were administered buffer alone, TMV alone, or HA alone). Administered vaccines 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, a third on Days 28, 42, and 90, and then , the samples were subjected to a hemagglutination inhibition (HAI) assay.

Based on this assay, none of the animals produced a measurable response on Day 7 or Day 14 with either vaccine. However, early reactions were observed in some animals on day 21. Specifically, 10/27 animals responded to the H1N1 vaccine (Influenza A/Michigan/45/2015 (H1N1pdm09)) at low levels (only 1 of them >80 HAI titer). . Also, 22/27 responded to the H3N2 vaccine (Influenza A/Singapore/INFIMH-16-0019/2016) at low levels (only 2 of them >80). On day 28, the number of animals within this cohort measurably responding to the H1N1 vaccine was 8/29, with a single animal having an 80 HAI titer and all others lower. For the H3N2 vaccine, the number of measurable responses was 14/29 and one animal had an 80 HAI titer and all others were lower.

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 received vaccines against influenza B viruses (B/Colorado/06/2017 (V) and B/Phuket/3073/2013 (Y), respectively). Influenza B virus and the corresponding HA immunogens are known to not give rise to HAI titers in mice with efficiency and efficacy as HA type A immunogens, so, as expected, none of these animals No response was detected on day 2.

To further evaluate the system of the present invention for appropriate virus-to-antigen ratios, apart from the immune response studies described above, humoral immune responses in mice were tested with influenza A and influenza B antigens, along with controls, as described below. Various TMV:HA conjugate ratios (ie, 1:1, 4:1, 16:1) of both antigens were evaluated after vaccination. In this way different conjugation ratios and their effect on the immune response were tested. Vaccinated mice were administered 15 mcg of HA by injection into 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 groups of mice (12 mice per group) and agents administered, each The right column of the table shows the ELISA antibody (Ab) titer results.

Figure 38 is a scatter plot related to Table 16, showing a graphical analysis of H3:HA antibody titers after administration of vaccine at ratios of 0, 1:1, 4:1, and 16:1 (TMV:HA). offer. Figure 39 also shows geometric mean studies of antigen-associated Ab titers using recombinant H3 antigen (Table 17) as a coating or captured H3 virus (Table 17) as a capture protein that binds anti-influenza A H3 antigen antibodies. The results of With respect to density (surface area of TMV occupied by HA), the three ratio trends are 1:1 (highest charge density) > 4:1 > 16:1 (lowest charge density), as demonstrated by TEM and AUC analysis. density). In these figures representing ELISA results obtained with H3 antigen, the highest immune response was observed with the lowest density conjugate. That is, the trend of immune response was 16:1>4:1>1:1, which was opposite to the density trend. Therefore, it was surprisingly found that at these ratios of TMV:HA, lower conjugation densities tended to provide better immune responses. Explanations for this surprising finding that antigenicity does not correlate with maximal HA conjugation events include: (1) when the density is relatively low, there is more homogeneity of the antigen, as well as unreacted or self-conjugated; (2) resulting in more efficient processing of conjugated antigens and more conserved/uniform antigen conformation; and (3) (as an example) TMV rods exhibit more antigen presentation. The ability to stimulate cells to migrate to the injection site, to process adherent antigens, or some combination of these factors. Note, however, that the presence of TMV particles alone does not replace the need for conjugation (see, eg, Tables 14 and 15).

In addition to influenza A H3 antigen, influenza B antigen (B-Phuket HA) was also tested using the binding propensity of recombinant influenza B Phuket antigen and its corresponding antibody. Table 17 below shows the results of this portion of the study that were not shown to exhibit 16:1>4:1>1:1 based on mean ELISA Ab titer results.

Still, a ratio of 16:1 showed the highest average antibody titers. Therefore, it is reasonable to expect the same relationship between density and immune response to apply to testing for influenza B antigen (B-Phuket HA). Thus, similar to the results of the H3 antigen, the immune response is higher in the formation of lower density conjugates. There is also reason to believe that the 4:1 ratio conjugation reaction did not proceed as well as reactions at other ratios, possibly due to anomalies during conjugation, and electron microscopy was also superficially examined for this sample. No centrifugation analysis was performed. In any case, the data here show an immune response at all three ratios. The fact that immune responses were seen at multiple ratios underscores the robustness of the system in not being bound to any one particular ratio. This flexibility seen with certain TMV conjugate vaccines is likely due to the fact that other antigens were conjugated to TMV in addition to the H3 and H1 antigens included in these studies, and to other viruses in addition to TMV. A further indication is that the system works well when a carrier is used as the carrier.

With respect to clinical utility, products conjugated according to any of the multiple embodiments and alternatives described herein include, but are not limited to, Examples 7, 9, 10, 11, and 12. Delivery of purified antigens via purified viruses, such as the viral antigen conjugates described, can be utilized as vaccines. Still further, embodiments of the present disclosure can be made from any virus-protein conjugate composition, the conjugation of which is provided herein, in any number of forms (e.g., , vials). In this regard, embodiments provide that such vaccine products may be administered by syringe or spray via routes such as, but not limited to, subcutaneous, intramuscular, intradermal, and nasal, and, to the extent clinically compatible, by mouth. Including those suitable for delivery in unit dosage forms provided to a subject, such as oral administration and/or topical administration. As a non-limiting example, without compromising the breadth and scope of the embodiments herein, the size of TMVs (typically 18 nm x 300 nm) and their rod-like shape are similar to antigen presenting cells (APCs). , thus enhancing T cell (such as Th1 and Th2)-mediated immunity, including cellular responses, and serving to provide adjuvant activity to surface-conjugated subunit proteins. This activity is also stimulated by viral RNA/TLR7 interactions. As a result, the combined effects of vaccine uptake directly stimulate APC activation. Humoral immunity is typically balanced between IgG1 and IgG2 subclasses via subcutaneous and intranasal delivery. Responses upon mucosal vaccine delivery also include substantial systemic and mucosal IgA. Cell-mediated immunity is also very robust and induces antigen-specific secretion similar to live viral infection responses. Whole-antigen fusions allow epitope processing of natural cytotoxic T lymphocytes (CTL) without regard to human leukocyte antigen (HLA) distribution.

The broad (humoral and cellular) and enhanced (amplified and potent) immune responses associated with the multi-set purification platform according to the present embodiments induce little or no cellular or humoral immunity; In sharp contrast to the subunit proteins tested without TMV conjugation. These immune response implications suggest that, according to embodiments of the present invention, vaccines generated via multiset platforms promote highly protective responses as single-dose vaccines, compared to other conventional vaccine platforms. to provide speed and security not provided by Indeed, the conjugation platform has been shown to combine in a wide range of ratios, dose well at various doses, and act on a wide range of viruses and proteins (including antigens), again demonstrating the robustness of the system. show gender. Further advantages of the multiset platform for producing vaccines in embodiments of the present invention include a proactive stimulation approach for systemic immune protection against pathogen challenge, where the platform extracts antigen domains from disease pathogens. (including viral glycoproteins or non-secreted pathogen antigens) and the platform serves as an effective vaccine platform against both viral and bacterial pathogens.

In addition to advantages for vaccination, plant virus particles purified via the multiset platform according to embodiments of the present invention can be formulated for various drug delivery purposes. These various objectives may include: 1) immunotherapy: by conjugation of therapeutic antibodies to the surface of viral particles and their delivery to enhance their cytotoxic effects; Therapy: by loading specific nucleic acids for introduction for genetic modification into specific cell types and 3) drug delivery: by loading chemotherapeutic agents into viral particles for delivery to target tumors.

To give a simple example of the many advantages of the methods discussed herein, in a multiset platform according to several embodiments, purified virus is first exposed to a pH shift as discussed above. It can be utilized as a drug delivery tool by swelling by heating. The virus in this state is then incubated with a concentrated solution of a chemotherapeutic agent, such as doxorubicin, and the pH is then returned to neutral, thereby returning the virus to its pre-swelling state, thereby removing the chemotherapeutic agent molecule. is captured. The viral particles can then be delivered to the organism by delivery mechanisms such as, but not necessarily limited to, injection for targeted therapy of tumors.

Thus, the above description includes (i) plant-based production and purification of viruses, (ii) plant-based production and purification of antigens, and (iii) therapeutically useful explants as vaccines and antigen carriers. and (iv) delivery of therapeutic vaccines comprising purified virus and purified antigens.

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 very sensitive to temperature changes. F. Coenen et al., Stability of influenza sub-unit vaccine. Does a couple of days outside the refrigerator matter? As discussed in Vaccine 24 (2006), 525-531, influenza vaccines generally cannot be stored at room temperature (ie, about 25° C.) for 5 weeks and become inactive. F. Of all the influenza vaccines discussed in the Coenen paper, only one vaccine was stable at room temperature for 12 weeks. In many situations this is a significant problem for other types of vaccines as well as for individual specific components (eg intermediates) of vaccines. Therefore, vaccines generally must be refrigerated throughout the supply chain from the moment of production by the vendor until administration, often referred to as the "cold chain".

While in the refrigerated environment, the majority of vaccines remain stable for the typical 78-week stability target period. However, absolute requirements for the cold chain are often difficult to guarantee in developing countries, leading to widespread vaccine losses and thus limiting vaccine availability worldwide. It is a problem. Many efforts have been made to create vaccines with room temperature stability, but as discussed in the literature, these efforts have been unsuccessful. In addition, cold chains are very costly to maintain for manufacturers and for the physicians and organizations that receive, store and administer vaccines to populations. Therefore, it is critical to increase vaccine stability and enhance vaccine antigen stability to reduce reliance on the cold chain and ensure that vaccines retain potency until administration, and the global is necessary. With regard to the stability of the antigen itself, ie, as an intermediate ready for conjugation with a suitable viral carrier, there is the advantage that antigen stability can be maintained after production and purification and prior to conjugation. These advantages include, but are not limited to, the ability to manufacture the antigen at a separate manufacturing facility or at a different time than the production and purification of the viral carrier. If you can do that, you can be more flexible in your supply chain. In addition, improving stability can extend the shelf life of vaccines, which facilitates stockpiling vaccines in preparation for potential pandemics and reduces vaccine loss under inappropriate conditions. To prevent. The scope of the present embodiments, along with other features and advantages outlined herein, meet these and other needs. In doing so, the purification and conjugation platform of the present invention will advance the stability of protein-viral conjugates under both refrigerated and room temperature conditions.

There are several methods of measuring antigen quality and vaccine stability, for example: (1) protein concentration as measured by the BCA protein assay (protein converts Cu ²⁺ to Cu ⁺¹ in alkaline solution; (2) preservative efficacy as measured by VaxArray® antibody array binding (utilizing a multiplex sandwich immunoassay); (3) SDS-Page single (4) pH as a measure of physical contaminant properties, and, where possible, (5) size exclusion chromatography to characterize the multimeric structure of the antigen, to determine the purity of one migrating band. In some embodiments, a bioburden test is also performed to analyze the presence of bacterial or fungal contamination (in the tables below, "TAMC" is an abbreviation for Total Aerobic Microbial is an abbreviation for total yeast/mold count). Additionally, a vaccine is considered unacceptable for use if it fails the BCA protein assay, the VaxArray® test, or the SDS-Page analysis. In other words, if a vaccine fails any one of these three tests, it is unacceptable for use and is inactive. The VaxArray® efficacy test is used to assay 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 VaxArray® assays related to influenza viruses and vaccines are described by 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 epidemic, VaxArray® potency assays for testing coronaviruses, vaccines and vaccine intermediates are commercially available.

Accordingly, the five studies referred to in the paragraph above 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 table below shows the stability data and storage efficacy measured at release and at various times after filling into vials and storage under refrigerated conditions (2-8° C.). As used herein, initial concentration or completeness refers to a compound, conjugate mixture, drug product, vaccine, etc. and the date of release is 21 C.F. F. R. Part 11 and ICH Q1A Stability Testing of New Drug Substances and Products, Revision 2 (November 2003), and references cited in the latter document, and the entire contents of all of the above for any purpose. incorporated herein by reference to .

Tables 18-23 show that the purified free antigen exhibits 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 significant (as is typically observed) measurements there was no deviation. However, other antigens such as B/Colorado and H1N1 (A/Brisbane), and to a lesser extent H3N2 (A/Kansas) and B/Phuket, under these conditions degrade, lose trimers, or showed loss of other important properties. For example, Figure 40 is an SDS-PAGE analysis of purified B/Phuket after one month under refrigerated conditions. In Figure 40, there is a degraded band of lower molecular weight than the intact band of approximately 60 kDA, indicating that the purified B/Phuket antigen has been degraded. As expected, the data in Tables 18-23 and Figure 40 show that different proteins exhibit different stabilities under refrigeration conditions.

When the same purified antigen is conjugated to TMV, according to several embodiments and alternatives, the stability profile and preservative efficacy change. In some embodiments, the methods of the invention enhance measures of stability of conjugated compounds comprising proteins and viral particles that activate the viral particles and then combine the viral particles and antigens in the conjugation reaction. to form a conjugate mixture, whereby the conjugate compound has enhanced stability when placed in a non-refrigerated environment and after a period of at least 42 days after the date of release. An exemplary storage temperature is at least 20°C. Enhanced stability can be measured by comparing the stability of the conjugate mixture to the stability 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 as measured by BCA or other suitable method, a difference of at least 10% between the concentration of conjugated compound and the concentration of antigen alone is within the scope of this embodiment. is within. Similarly, when the measure of stability is antigen integrity as measured by SDS-PAGE, SEC-HPLC or other suitable method, at least a 10% difference between the integrity of the conjugated compound and the integrity of the antigen alone. % difference is within the scope of this embodiment. Similarly, if 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, the potency and A difference of at least 30% from the potency of the antigen alone is within the scope of this embodiment.

Therefore, the table below shows several monovalent formulations (TMV to antigen ratio 1:1) at release and at various times after filling in vials and storing under refrigerated conditions (2°C to 8°C). ) provides stability data for:

In addition, the table below shows several monovalent formulations (TMV to antigen ratio 8:1 ) provides stability data for:

The table below shows some other results at various times upon release and after filling in vials and storing under refrigerated conditions (2°C-8°C) or room temperature conditions (22-28°C). provides stability data for a titer formulation (8:1 TMV to antigen ratio):

In each of the conjugates listed in Tables 24-39, purity, pH, protein concentration, and preservative efficacy were maintained under refrigerated conditions through at least 6 months of storage, and for the others through at least 12 months of storage. be. Furthermore, polydiversity is also consistent over this time frame. Polydiversity refers to the particle size variability in the composite product, and generally the better the product, the lower the polydiversity. Similarly, purification and conjugation platforms according to multiple embodiments and alternatives have successfully maintained the stability and potency of different antigens, utilized different conjugation ratios, and exemplified their versatility.

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

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

Further, the potencies of the tetravalent conjugates described in Tables 42A, 42B, 43A and 43B are shown in Figures 41A and 41B, respectively. FIG. 41(A) is a graph showing QIV stability (referred to in the figure as routine stability) at freezing temperatures of 2-8° C. from Tables 42A and 42B. FIG. 41(B) is a graph showing the QIV stability (referred to in the figure as accelerated stability) in the high temperature range of 22-28° C. from Tables 43A and 43B. As shown in Figures 41A and 41B, there was a statistically significant increase 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. No significant difference was observed.

In addition, at a TMV to antigen ratio of 8:1, the following tetravalent conjugates produced according to several embodiments and alternatives were refrigerated (2-8°C) and room temperature (22-28°C) conditions. showed high stability under both: unlike the tetravalent conjugates discussed in Tables 42-43 above, the following tetravalents were conjugated to modified TMV NtK carriers with four different HA which includes the following antigens: H1 (Brisbane), H3 (Kansas), B/Y (Phuket), B/V (Colorado).

Tables 40-45 and Figures 41A and 41B demonstrate the consistency and stability of the tetravalent conjugates for at least 3-12 months under both refrigerated and room temperature conditions with respect to protein concentration, preservative efficacy, pH and appearance. show that you maintain your sexuality. The data also demonstrate that the purification and conjugation platform, according to multiple embodiments and alternatives, successfully conjugated and increased stability over a wide range of antigens and various conjugation ratios.

Table 46 provides the percent change in preservative potency for various antigens listed in Tables 41A and 41B by comparing initial potency to preservative potency at specified time points.

Thus, as shown in Table 46, when the conjugates were placed in a non-refrigerated environment, the preservative potency after 30 days was at least 70% of the initial potency 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 potency, and the preservative efficacy of the conjugate mixture was at least 75% at the end of at least 180 days. .

The following table shows the stabilizing effect of the embodiments described herein by comparing release conditions for the same protein conjugated to TMV and purified recombinant antigen according to several embodiments and alternatives. is exemplified. Additionally, the stability of the purified antigen and TMV after 6 months under refrigerated conditions (4°C-8°C) was assessed by analyzing protein concentration, potency, SDS-page purity and pH as follows. Comparisons were made between the same conjugated antigens:

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

Further, Tables 41A-45 and Figure 41B demonstrate that tetravalent conjugates produced according to several embodiments and alternatives are strongly stable at room temperature storage (22°C-28°C) for at least 6 months or 24 weeks. indicates that it indicates a measure of sexuality. Compared to conventional vaccines, which exhibit an average stability of about 5 weeks at room temperature (as discussed in the F. Coenen paper, supra), vaccines according to multiple embodiments and alternatives are more stable than conventional influenza vaccines. At least 5-fold higher and several-fold longer stability than purified antigen. Thus, formulation and conjugation processes according to multiple embodiments and alternatives stabilize highly labile antigens such as B/Colorado, H3N2 (A/Singapore), H1NI (A/Michigan), and B/Phuket. It extends the stability of other antigens, such as , well beyond the stability limits of free antigens and conventional vaccines.

Examples 14(a)-14(h) - Testing Immunogenicity of Quadrivalent Influenza Vaccines and Safety, Efficacy and Utility of Embodiments Disclosed herein for Protection Against Seasonal Viral Challenges To demonstrate, several preclinical studies were conducted using a tetravalent seasonal vaccine candidate (referred to in this example as "QIV vaccine"). The vaccines used in the trials were manufactured according to multiple embodiments and alternatives disclosed herein. Eligible QIV vaccines include the 2018/2019 North American seasonal influenza vaccine strains recommended by the World Health Organization, Centers for Disease Control and Prevention, conjugated to inactivated TMV NtK, and the FDA's Vaccines and Related Biological Products Advisory Committee (VRBPAC), (A/Michigan/45/2015 (H1N1)pdm09, A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Phuket/3073/2013 (B Yamagata lineage), and B/ The following influenza HA antigens from Colorado/06/2017 (B Victoria strain) were included: In some embodiments, a phosphate buffered solution with 0.01% thimerosal as a preservative (as a non-limiting example) The four vaccine-antigen conjugates are blended together in a single injectable tetravalent vaccine formulation.As discussed in more detail below, these studies are disclosed herein. The embodiments demonstrated enhanced immunogenicity of the recombinant hemagglutinin protein antigen, as determined by various measurements and assays, including hemagglutination inhibition and neutralizing antibody titers. The study described in this example is useful for the QIV seasonal vaccine because challenge with a viral strain homologous to the vaccine strain HA antigen has shown levels of protection in all mammalian disease models tested to date. Unless otherwise stated, the content of inactivated TMV NtK and HA intermediates incorporated into the conjugation reaction was calculated as mg:mg (i.e. weight ( wt))) basis, As a non-limiting example, a QIV vaccine conjugated at an 8:1 TMV NtK:HA ratio (mg:mg basis) of the drug substance intermediate was used. Subsequent testing showed a favorable humoral response.

Table 51 provides a summary of studies performed on QIV vaccines according to this example. "GLP" refers to the CPMP Note on Guidance for Preclinical Pharmaceutical and Toxicological Testing of Vaccines (CPMP/SWP/465/95) and the World Health Organization Guidelines for the Nonclinical Evaluation of Vaccines (WHO Technical Report Series, No. 927). ), the entire contents of both of which are incorporated herein by reference. See the table below for further discussion of various aspects of the study.

As summarized in Table 52, immunogenicity studies in BALB/c mice were performed using monovalent and tetravalent preparations, respectively, preferably to assess formulation ratios, and to evaluate injection site reactions and toxicity clinically. Monitored for symptoms.

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

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 described in Armitage and Berry, Statistical Methods in Medical Research, 2nd Ed. (1987), pages 31-33, the entire contents of which are incorporated herein by reference:
GM={x ₁ x ₂ x ₃ . . . . . xn} ^1/n (equation 3)

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 (before the second vaccination), antibody titers were below detectable levels in all groups except: With an HAI titer of 10 against /Singapore/INFIMH-16-0019/2016 (H3N2), 2/5 mice in group 7 (30 μg tetravalent vaccine) were tested against A/Singapore/INFIMH-16- It had a titer of 10 against 0019/2016 (H3N2). In Table 37, the HAI antibody titer was the reciprocal of the highest dilution of serum that inhibited hemagglutination with 4 HA units of virus.

On day 14, HAI titers were detected against H3N2 virus at the high (30 μg) dose only in the monovalent and QIV vaccine groups. HAI titers against A/Michigan/45/2015 (H1N1) in the tetravalent vaccine and A/Singapore/INFIMH-16-0019/2016 (H3N2) in both the monovalent and tetravalent vaccines were dose dependent at day 28 increased significantly. HAI titers were detectable in some animals at antigen doses as low as 1.5 μg, with the majority of animals developing antibody titers at 7.5 μg per HA antigen. No detectable titers were produced by mice vaccinated with monovalent vaccines against the other three strains tested. To a lesser extent, the QIV vaccine also induced HAI titers in a subset of mice to B/Colorado/06/2017. There were no detectable HAI titers generated against the Yamagata strain B/Phuket/3073/2013 component.

In summary, based on HAI assay data, the monovalent formulation vaccine induced a detectable humoral immune response against H3N2 virus. The QIV formulation induced a detectable humoral immune response against 3 of the 4 antigens. The induced immune responses to influenza H1N1 and H3N2 antigens seen in mice vaccinated with monovalent and QIV vaccine formulations were dose dependent. As shown in Table 37, H1N1 GMT ranged from 20 to 279 (increased dose-dependently) and H3N2 GMT ranged from 20 to 52. In addition, no adverse clinical signs or injection reactions were observed with monovalent or QIV vaccination.

As summarized in Table 54, immunogenicity studies in naive mice (BALB/c mice) over time showed that the tetravalent vaccine by assessing the immunogenicity of The purpose of this study was to confirm the results from previous studies, shown in Table 53, to compare the immunogenicity of QIV to a vaccine in which the same antigen was not conjugated to a TMV NtK carrier, and to demonstrate the efficacy of immunization over 90 days. The purpose was to analyze the persistence of responses. The vaccines in the table below were conjugated at a 1:1 ratio of TMV NtK:HA antigen.

Figure 42 shows QIV vaccine induction of H1N1 and H3N2 hemagglutination inhibition (HAI) titers in mice over time. Data include geometric mean titers (GMT) of BALB/c mice receiving 15 μg QIV vaccine per HA. GMT values were calculated using the following formula:
GM={x ₁ x ₂ x ₃ . . . . . xn} ^1/n (equation 3)

Mice with titers <10 were assigned a titer of 5 for GMT calculations according to Armitage and Berry, supra.

As shown in Figure 42 and Tables 55 and 56 below, HAI titers were only in animals that received the QIV vaccine (referred to as "KBP-VP H1NI" and KBP-VP H3N2 in Figure 42) and H1N1 and H3N3 antigen only. No detectable immune response was generated against the unconjugated antigen. Titers were below detectable levels by day 21 for A/Michigan/45/2015 (H1N1) and A/Singapore/INFIMH-16-0019/2016 (H3N2). HAI titers remained detectable on days 28 and 42 and were highest on day 90. Moreover, the humoral response continues to increase for at least 90 days, a feature not known in conventional influenza vaccines. This study further supports the efficacy of conjugating antigen to the TMV NtK carrier in generating an immune response, as antigen alone had no detectable response.

Similar to HAI titers, virus neutralizing (VN) titers were only observed in animals receiving the QIV vaccine. VN titers against A/Michigan/45/2015 (H1N1) and A/Singapore/INFIMH-16-0019/2016 (H3N2) were also not observed until day 21, as shown in Table 57. VN titers remained detectable on days 28 and 42 and were highest on day 90.

In the study outlined in Table 54, mice were also monitored for clinical signs and injection site reactions. At necropsy, organ weights were measured and gross necropsy and tissue histopathology were performed. No test article-related gross findings were observed in animals necropsied on Day 21 or Day 90.

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

In conclusion, the QIV vaccine elicited detectable humoral immune responses based on HAI and virus neutralization assays. The strongest responses were to 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 demonstrate that the QIV vaccine is safe and immunogenic in mice.

Example 14(c) - Immunogenicity and Challenge Studies in Ferrets Immunogenicity and challenge studies with QIV vaccines were also performed and licensed in ferrets, which are the most representative animal model for influenza infection. Vaccine efficacy was assessed via reduction in viral load after challenge relative to the vaccine comparator. Blood for immunogenicity studies was drawn on the following study days: -3, 7, 14, 21, 28, and 42, as indicated in the study design shown in FIG. Animals were challenged on study day 43, nasal washes were performed on study days 45 and 47, and virus titers were analyzed.

Briefly, on study days 0 and 14, ferrets N=30 (15M/15F) per group were immunized with one of the following:
1. Placebo buffer as negative control (same volume as Group 4)
2. 3. Fluzone® tetravalent (15 μg per HA, 60 μg total HA) vaccine as licensed comparator. 15 μg QIV per HA antigen (60 μg total HA antigen; 60 μg TMV NtK carrier)
4. 45 μg QIV per HA antigen (180 μg total HA)
After each dose, injection sites and clinical signs were monitored daily for 7 consecutive days. Animals were bled at regular intervals for determination of HAI and neutralizing antibody titers, as shown in FIG. Animals from each dose group were subdivided into two groups (N=12, 6M/6F) and tested on day 43 at A/Michigan/45/2015 (H1N1) pdm09 or A/Singapore/INFIMH-16-0019/ 2016 ( ^H3N2 ) (non-limiting example). Animals were monitored and nasal washes were performed 2 and 4 days after challenge to quantify residual influenza virus titers and assess viral clearance.

Hemagglutination inhibition (HI) against all four viruses tested was detected in ferrets administered either 15 or 45 μg dose levels of QIV and Fluzone® [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 to the A/Michigan/45/2015 (H1N1) virus. Virus neutralizing (VN) titers were detected against all viruses with the following responses in descending order: A/Michigan/45/2015 (H1N1) pdm09, A/Singapore/INFIMH-16-0019/2016 ( H3N2), B/Phuket, and B/Colorado/06/2017 showed the lowest responses.

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

Figure 45 shows nasal wash titers (log10 _TCID50 /mL) after H3N2 challenge, where the QIV vaccine is referred to as "QIV." As shown in Figure 45, after challenge with A/Singapore/INFIMH-16-0019 (H3N2), virus was detected in nasal washes of all challenged ferrets on days 2 and 4 post-challenge. The greatest log reduction in viral titers was observed on day 2 in the QIV vaccine 45 μg dose group (1.0 log10 TCID ₅₀ /mL) when compared to the placebo group, which was statistically lower than placebo. QIV vaccine and Fluzone® produced log reductions of 0.7 log10 TCID50/mL and 0.5 log10 TCID50/mL, respectively, which were not statistically reduced compared to the placebo group (p-value 0.5). 05). As shown in FIG. 45, on day 4, all vaccinated groups, Fluzone® (1.3 log10 _TCID50 /mL), QIV vaccine (15 μg dose, 1.4 log10 _TCID50 /mL), and A similar level of significant log reduction in viral titers was observed for the QIV vaccine (45 μg, 1.2 log10 TCID ₅₀ /mL). In addition, the maximum mean weight loss for the placebo and Fluzone® vaccinated groups was similar at 3.1% and 2.6%, respectively. Animals immunized with QIV had an infection-induced maximum mean weight loss of 2.1% (15 μg QIV) and 1.2% (45 μg QIV).

Regarding testing of immunogenic responses, the QIV vaccine of this example induced a detectable humoral immune response against the four antigens tested in the HI and VN assays. The strong VN response was consistent with the reduction in nasal wash virus titers observed 4 days post-challenge in vaccinated ferrets challenged with A/Michigan/45/2015 (H1N1)pdm09. The log reduction in viral titer observed on day 4 in QIV-vaccinated ferrets was 1.1-1.5 log 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 A/Singapore/INFIMH-16-0019/2016(H3N2)-challenged ferrets, improved protection was observed in the high-dose group on days 2 and 4 post-infection; ), similar log reductions in viral titers were observed, both resulting in viral titers statistically lower than placebo. These data demonstrate that the QIV vaccine is immunogenic and provides ferrets with a level of protection against challenge with H1N1 and H3N2 homologous viruses.

Example 14(d) - Matrix Immunogenicity Study in Mice The purpose of this study was to assess the immunogenicity of the QIV vaccine at 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, fixed HA + increased TMV doses were compared to fixed TMV + decreased HA doses (data not shown).

Eight-week-old female BALB/c mice (5 per group) were treated with the A/Singapore/INFIMH-16-0019/2016 (H3N2) monovalent influenza vaccine on days 1 and 14 of the indicated vaccines. The composition was used to immunize by the subcutaneous route of administration. Mice were bled and sera were taken on days 12, 28, 42 and a final serum on day 60.

Figure 46 shows the immunogenicity of monovalent vaccines obtained at various ratios of TMV NtK:HA antigens. 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 were higher than the benchmark 1:1 ratio group. continued to show significant improvement compared to

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

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

Examples 14(e) and 14(f) - Pharmacokinetic Study A pharmacokinetic study was conducted to evaluate the biodistribution of the QIV vaccine following a single intramuscular injection for 8 days in male New Zealand white rabbits. This test was developed to measure TMV NtK viral RNA extracted from tissue or blood, using a sophisticated RT-qPCR method, the test was prepared with varying amounts of TMV NtK carrier. Two different QIV vaccine formulations were compared. Tissues analyzed were blood, skeletal muscle (injection site), lymph node, spleen, thymus, heart, liver, lung, kidney and testis.

Figure 47 shows that either 45 μg (1:1 formulation monovalent vaccine) or 1440 μg (8:1 formulation tetravalent vaccine) of TMV NtK carrier was incorporated in a 0.5 mL dose (as a non-limiting example)2 A comparison of TMV vRNA distribution over time in tissues measured by RT-qPCR after a single injection of four different QIV formulations is shown. 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 quantitation and "LOD" refers to limit of detection.

As expected, the two formulations showed consistent distribution patterns. In Figures 47 and 48, the highest levels of QIV were quantifiable from injection site skeletal muscle tissue throughout the study period and decreased in amount over time for both formulations. After skeletal muscle, QIV was detected most abundantly in the spleen and draining lymph nodes throughout the study, indicating that QIV translocated to these tissues after immunization. Transient levels were detected in liver, heart, and testis at 24 hours post-dose, and were then below the LOQ for both formulations. The 8:1 formulation detected a low transient signal in blood, lung, and thymus tissue 24 hours after immunization, which was below the limit of quantitation (LOQ) by 3 days after immunization, probably due to its This is due to the 32-fold higher amount of TMV NtK contained in the formulation. No quantifiable QIV residues were detected in brain or kidney tissue with any QIV formulation.

Thus, in all formulations, the vaccine was observed to be transported from the injection site to immune organs and no accumulation was 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. Therefore, detection in immune system organs suggests a "depot" effect (ie, sustained release of antigen at the site of injection) and sustained stimulation of the immune system.

Examples 14(g) and 14(h) - Toxicity Studies Several toxicity studies were conducted according to GLP requirements, as described above in Table 51 and as described in Table 59 below, for QIV vaccines. and supported the clinical use of QIV vaccines for the prevention of disease caused by infection with influenza virus (as a non-limiting example). Two repeated-dose toxicity studies in New Zealand white rabbits were conducted according to embodiments and alternatives using two formulations of QIV with different contents of TMV NtK carrier conjugated with purified recombinant HA antigen. . As described in more detail below, the study showed no treatment-related or toxicologically significant clinical findings, supporting the safe use of TMV NtK:HA conjugates in human studies.

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

In a repeat-dose toxicity study of a QIV vaccine with a 1:1 ratio of TMV NtK carrier to HA antigen, seasonal influenza vaccine candidates were administered to male and female New Zealand white rabbits twice on study days 1 and 29 (once daily). ) administered intramuscularly and evaluated for reversibility of the effect after a 28-day recovery period. As shown in Table 60, each group consisted of 10 rabbits/sex/group. Five rabbits/sex/group were sacrificed on study day 30 (1 day after the last dose) and 5 rabbits/sex/group were sacrificed on study day 57 (28 days after the last dose).

Test substances were administered by intramuscular (IM) injection on days 1 and 29 of the study. At each injection, animals in each group received 0.5 mL of control (Group 1), low dose vaccine (Group 2), or high dose vaccine (Group 2) using a 25 gauge needle attached to a plastic 1 mL syringe. 3) received (as a non-limiting example); The dosing site was a relatively large muscle mass behind the hind leg and was shaved or re-shaved (if necessary). On each day of injection, the dosing site was wiped with alcohol and allowed to dry completely for a minimum of 10 minutes prior to dosing. IM administration sites alternated between hindlimbs, with the right hindlimb used for the first dose.

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 on Study Day 57 (28 days after the last dose). days later) and were euthanized. Experimental 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 pathology at necropsy, and microscopic pathology.

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

Fibrinogen levels were generally elevated in vaccine-treated groups on day 2 post-dose (p<0.05 or <0.01). Elevated fibrinogen in these groups was considered to be associated with vaccine treatment, but an expected (inflammatory) response after treatment with immunogenic agents. Fibrinogen ceased to rise (p>0.05) at the end of the 28-day recovery period (reversible effect).

Mixed cell 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 at the sciatic nerve, but not completely at the injection site at the end of the 28-day recovery period. Although this lesion was not considered harmful, it was an expected finding consistent with administration of an immunogenic agent.

In conclusion, intramuscular administration of QIV vaccine at doses of 15 or 45 μg was well tolerated for two injections (study days 1 and 29) once every 4 weeks. None of the observed findings resulted in adverse or limited toxicity and were considered to be of minimal toxicological significance after administration of immunogenic substances (e.g. hypersensitivity, no change in organ function, etc.) and/or expected findings (eg increased fibrinogen and mixed cell infiltration at the injection site or sciatic nerve).

Example 14(h)—Repeated Dose Toxicity Study In a repeated dose toxicity study of a QIV vaccine with an 8:1 ratio of TMV NtK carrier, HA antigen, seasonal influenza vaccine candidates were administered to male and female New Zealand White rabbits on Day 1 and Two doses (once daily) were administered intramuscularly on day 29 to assess reversibility of the effect after a 28-day recovery period. As shown in Table 61, each group consisted of 8 rabbits/sex/group. Five rabbits/sex/group were sacrificed on study day 30 (1 day after the last dose) and 5 rabbits/sex/group were sacrificed on study day 57 (28 days after the last dose).

Test substances were administered by IM injection on days 1 and 29 of the study. At each injection, animals in each group received 0.5 mL of control (Group 1), low dose vaccine (Group 2), or high dose vaccine (Group 2) using a 25 gauge needle attached to a plastic 1 mL syringe. 3) received (as a non-limiting example); The dosing site was a relatively large muscle mass behind the hind leg and was shaved or re-shaved (if necessary). On each day of injection, the dosing site was wiped with alcohol and allowed to dry completely for a minimum of 10 minutes prior to dosing. IM administration sites alternated between hindlimbs, with the right hindlimb used for the first dose.

The 4 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 on Study Day 57 (28 days after the last dose). days later) were euthanized. Experimental endpoints included morbidity/mortality, physical examination, clinical signs of toxicity, inoculation site (Draize) reactogenicity scoring, body weight, body weight change, food consumption, body temperature, ophthalmology, clinical pathology (clinical chemistry, hematology). organ weights, immunogenicity analysis, gross pathology at necropsy, and microscopic pathology.

All study rabbits survived to the scheduled necropsy. No treatment-related or toxicologically significant clinical findings or inoculation site reactogenicity were observed. Treatment-related or toxicologically significant effects on body weight, body weight change, food consumption, body temperature, ophthalmology, clinical chemistry, hematology, organ weights, gross and microscopic pathology no effect was observed.

Fibrinogen levels were typically elevated (p<0.01) in the treatment groups on day 2 post-dosing. Elevated fibrinogen in these groups was considered treatment-related, but an expected (inflammatory) response after treatment with immunogenic agents. No increase in fibrinogen was observed during recovery (reversible effect) (p>0.05).

Therefore, IM administration of QIV vaccine candidates or inactivated TMV NtK at doses of either 45 μg each HA antigen (total HA 180 μg + TMV NtK 180 μg) or 1440 μg each HA antigen was administered with two injections once every 4 weeks (study day 1). and 29 days) were well tolerated. Findings in these GLP toxicity studies do not result in adverse or limited toxicity and are seen only in one sex, reversible, transient, no change in organ function, etc. of minimal toxicological significance. ) and/or were the expected findings (such as increased fibrinogen) after administration of the immunogenic agent. No significant toxicological problems were observed with either the 1:1 or 8:1 (TMV NtK:HA antigen) formulations of the QIV vaccine in these studies.

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

As shown in Table 62 below, HAI titers were tested against A/Michigan/45/2015 (H1N1), A/Singapore/INFIMH-16-0019/2019 (H3N2) and B/Colorado/06/2017 viruses. Detected in most animals on days 42, 49, and 57 (as non-limiting examples). In contrast, HAI titers against the B/Phuket/3073/2013 virus were commonly seen (as a non-limiting example) in 4 animals or less on test days 42, 49, and 57.

Figure 50 shows the measurement of micro-neutralizing GMT titers throughout the GLP toxicity test 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 on day 49 of both the 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 high in rabbits receiving low and high dose vaccines on study days 42, 49, and 57. It was the highest titer measured (as a non-limiting example) and seen against the A/Michigan/45/2015 (H1N1)pdm09 and A/Singapore/INFIMH-16-0019/2019 (H3N2) viruses.

Thus, the studies discussed in this example demonstrate that QIV vaccines consistently produce robust immune responses after intramuscular administration across three species (mouse, ferret and rabbit). A primary measure of immunity is the production of HAI antibody titers, a recognized serum biomarker of protection against influenza infection. Humoral immune responses were predominantly 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 a TMV NtK carrier to recombinant HA antigen ratio of 8:1 was shown to be desirable. In a disease challenge model, immunization of ferrets with QIV significantly reduced viral load in animals subsequently challenged with homologous H1N1 and H3N2 strains assessed from nasal wash samples. This reduction in viral load was greater than that of a licensed comparator. Immunization ameliorated clinical signs of morbidity associated with the challenge virus.

This study also investigated the distribution and safety of the QIV vaccine. No edema or injection site reactions were detected in any of the studies. Biodistribution studies with QIV (measured RNA from TMV NtK carriers) showed that outside the injection site muscle, QIV was measured in the spleen and lymph nodes at all time points tested, and TMV viral RNA was detected in these cells. It was found to be relatively stable or slowly depleted in organs, indicating that this provides a potential mechanism of action and antigen presentation to the immune system. In repeated-dose toxicity studies, the only finding was a reversible increase in fibrinogen levels from the clinical chemistry profile in the treatment group, usually two days after dosing, an expected finding for an immunological agent. Treatment-related or toxicologically significant effects on body weight, body weight change, food consumption, body temperature, ophthalmology, clinical chemistry, hematology, organ weights, gross and microscopic pathology No other effects were observed.

In conclusion, the data support that advancement of TMV NtK conjugates into human clinical studies offers clear advantages over currently licensed influenza vaccines. As no toxicologically significant findings were observed with the QIV vaccine, no such events are observed for any other antigens purified according to the TMV NtK carrier conjugated antigen platform described herein. High probability. Therefore, other antigens conjugated to inactive TMV NtK are expected to have similar biodistribution and toxicity profiles and are therefore suitable for use in humans.

Example 15(a)-(c) - Coronavirus vaccine candidate: RBD-Fc121 (SARS-2) conjugated to TMV
A Covid-19 vaccine will be developed by expressing a recombinant version of the RBD SARS-2 spike protein fused to the Fc domain of human IgG1 in Nicotiana benthemiana (Nb) plants to form the antigen (non as a limited example) was generated. Prior to conjugation, the formed antigen is purified according to multiple embodiments and alternatives according to the antigen purification platform described herein, and the TMV virus particles are purified according to multiple embodiments and alternatives herein. Purification and inactivation were performed according to the virus purification platform described in the literature. Purified recombinant RBD-Fc antigen was then purified and conjugated to inactivated TMV virus particles 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 is a SARS-2 spike fused to a human IgG1 Fc domain via chemical conjugation to TMV viral particles. Glycoprotein RBD is presented. As discussed in more detail below, presentation of the RBD-Fc fusion in this embodiment was demonstrated to enhance Th1 and Th2 responses in all mammalian disease models tested to date.

In this example, we chose to target the RBD domain of the SARS-2 spike glycoprotein as the antigen for the Covid-19 vaccine, because it serves as the binding site for the human ACE-2 receptor and the binding site overlap with characterized neutralizing antibodies. The SARS-2 spike glycoprotein is found in the S1 subunit at approximately amino acids numbered 320-520. 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 an RBD domain fused to a human 171 allotypic IgG1 Fc domain expressed and purified from plants, according to multiple embodiments and alternatives.

Several considerations were made in selecting the SARS-2 RBD as a fusion partner for developing the RBD-Fc antigens described in this and subsequent examples. 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 a domain of the SARS-2 RBD domain. Binding of CR3022 can neutralize both SARS-1 and SARS-2 CoV. SARS-2 RBD also presents an ACE-2 (angiotensin II) binding domain.

By the methods of this example, a multigene construct was designed and constructed to contain the genes encoding the proteins required to synthesize the Covid-19 antigen targeting 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 acid numbers 331-632 of the SARS-2 virus spike:

３．「（配列番号３）」Ｆｃヒンジ：ＶＥＰＫＳＣＤＫＴＨＴＣＰＰＣＰ
４．「（配列番号４）］ＩｇＧ１ １７１アロタイプＦｃ：

3. "(SEQ ID NO:3)" Fc Hinge: VEPKSCDKTHTCPPCP
4. "(SEQ ID NO:4)] IgG1 171 allotype Fc:

Figure 52 illustrates, but is not limited to, TRBO expression plasmids constructed for SARS-CoV2 RBD-Fc fusion peptides (ie, antigens) for production in Nb plants. Following appropriate infiltration and plant incubation, antigen extraction and purification as described herein was performed. As exemplified and further discussed herein, a first receptor binding domain (RBD) of a pathogen (specifically a coronavirus, and namely the SARS-CoV-2 spike (S) glycoprotein RBD), comprises a pathogen receptor binding domain (RBD). 1 peptide, a second peptide comprising a fragment crystallizable (Fc) region of an antibody (sequence of amino acids set forth in SEQ ID NO: 4, comprising a human IgG1 Fc domain), the first peptide and a second peptide and a hinge portion (comprising the sequence of amino acids shown in SEQ ID NO: 3) connecting the , was fused to form a fusion peptide. In Example 15, the RBD contained the amino acid sequence set forth in SEQ ID NO:2, and in Example 17, the RBD contained the amino acid sequence set forth in SEQ ID NO:8. Note that references to codons in the plasmid of Figure 52 include 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. Antigen donor plasmids and TRBO expression plasmids were digested with appropriate restriction enzymes. The antigen was ligated into the TRBO expression plasmid and colonies were subsequently screened to confirm clones. The successfully ligated expression plasmid (Fig. 52) was transformed into E. It was amplified in E. coli and the DNA was purified. Clones that confirmed the presence of the antigenic vector DNA in the TRBO expression plasmid were used to transform Agrobacterium tumefaciens in preparations for penetration into Nb plants. Individual colonies were picked aseptically, incubated, and 20% glycerol stocks were made from the cultures and stored as an antigen master cell bank. As further shown in Figure 52, an exemplary construct contains the cauliflower mosaic virus (CaMV) 35S promoter, a DNA-dependent RNA promoter set for transcribing TMV expression vectors with correct 5' ends. there were. In addition, TMV replicase is composed of replication-associated proteins of 126 kDa and 183 kDa. In Figure 52, "30k" refers to a translocation protein produced from a subgenomic promoter at the 3' end of the 183kDa protein coding region. In an 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 contains a ribozyme to ensure termination of transcription near the true 3' end. have. In addition, there are E. coli origins for replication, border regions for TI plasmids and other elements for efficient replication in Agrobacterium and insertion into plant cells.

Thus, plasmids providing constructs for virus-conjugable antigens can be produced according to multiple embodiments and alternatives herein. Such constructs may comprise first and second coding regions that encode a fusion peptide. In a non-limiting manner, the first coding region comprises a nucleic acid sequence that encodes the amino acid sequence shown in SEQ ID NO: 2 above or, as a further example, SEQ ID NO: 8 below. The first peptide may comprise or substantially comprise a receptor binding domain of a pathogen such as a virus, non-limiting examples of which include coronaviruses and influenza viruses, discussed further herein. Also, in a similar non-limiting manner, the second coding region comprises a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:4 above. The second peptide may 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. A third coding region may comprise 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 the Fc region. In some embodiments, one or more of the nucleic acid sequences identified herein are part of a heterologous expression system.

Example 15(a) - Plant Expression, Purification, and Characterization In this example, the RBD-FC121 fusion peptide (hereinafter referred to as "RBD-Fc121 antigen" in this example) was expressed in tobacco plants and harvested. and refined. Plant growth and incubation are carried out in a housed and controlled indoor environment, and an expression vector for protein replication of the RBD-Fc121 antigen (as a non-limiting example, Figure 2) according to multiple embodiments and alternatives herein. Expression was performed in naïve wild-type Nb plants infected with a vector such as that shown in 52). Purification of the RBD Fc-121 antigen was performed according to the antigen purification platform described herein (eg, as shown in Table 2 and FIG. 8), and in this particular example multimodal ceramic hydroxyapatite (CHT) The chromatography column step was omitted. Thus, for this example, FIG. 51(B) shows an RBD domain fused to a human 171 allotypic IgG1 Fc domain expressed and purified from plants, according to multiple embodiments and alternatives. Identification of such an exemplary pre-conjugation, SARS-COV2 RBD-Fc purified antigen intermediate can be confirmed by MALDI-TOF mass spectrometry. In some embodiments, the physicochemical properties of the antigenic intermediate include pH of about 7.0±0.4, osmolarity of about 250-350 mOsm/kg H ₂ O, bioburden of less than 10 CFU/mL and 100 ng /mg of residual host cell protein. The content of the inactivated TMV NtK intermediate and the RBD-Fc intermediate are incorporated into the conjugation reaction.

As shown in Figures 54-55, the antigen purification platform according to several embodiments and alternatives successfully purified the RBD-Fc121 antigen, in a manner compliant with GLP regulations, pure and stable as RBD-Fc monomer. This resulted in high yields (>400 mg of RBD-Fc protein) using a wide range of antigens. FIG. 53 contains an SDS page gel showing the purity of the RBD-Fc121 antigen resulting from the conclusion of the purification platform applied to this antigen, lane 1 shows markers and lane 2 purified RBD-Fc121 protein. indicate. The RBD-Fc121 antigen product is highly pure, as indicated by the distinct visible band in lane 2 of FIG. Moreover, the protein migration shown in FIG. 53 is consistent with the SEC-HPLC report of free purified RBD-Fc121 shown in FIG. In Figure 54, the SEC-HPLC report of free RBD-Fc121 antigen generated signal data detailed in Table 64 below.

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. Therefore, the RBD-Fc121 antigen was well and fully purified according to GLP.

The stability of the RBD-Fc121 antigen is reflected in Figure 55 and Table 65 (below). Figure 55 contains 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 after 5 weeks of purification. The distinct 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 ELISA results.

Example 15(a) - Conjugation and Preparation Purified RBD-Fc121 antigen was then conjugated to a 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 (eg, as shown in Table 1 and Figure 1). According to embodiments and alternatives herein, after purification, TMV NtK is subjected to micron filtration (e.g., 0.45) and UV-inactivated (as discussed in Example 8 herein). processed with

The purified inactive TMV NtK is then chemically conjugated to the RBD-Fc121 antigen to induce Covid-19 according to multiple embodiments and alternatives herein (eg, as shown in Table 3). produced a vaccine. As shown in FIG. 56, an exemplary conjugation procedure can include at least the following steps:
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.

Purified RBD-Fc121 antigen was reacted with EDC and Sulfo-NHS chemistry with 1 hour of mixing and conjugated to purified TMV NtK particles.

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.

Returning now to this example, the RBD-Fc121 antigen and TMV NtK were subjected to a conjugation reaction performed in EDC (4 mM) and Sulfo NHS (5 mM) at a pH of approximately 6.0. In this regard, the pH of the conjugation reaction and the pH of the purification step need not be the same. Conjugation reactions were quenched with free amines (Tris base), optionally using a 30 kDa UF membrane to remove residual EDC, Sulfo NHS and Tris base.

Therefore, the conjugated drug substance was diafiltered and formulated. All steps downstream of TMV NtK UV treatment and antigen 0.2 micron filtration were kept sterile since the conjugate exceeded 0.2 micron.

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

SV was measured using AUC to determine the percent conjugation of TMV NtK with RBD-Fc121 antigen. As described above, the fraction between 1-40S indicates the percentage of RBD-Fc monomer/trimer, and the fraction between 40-2000S, according to several embodiments and alternatives, TMV Percentage of NtK:RBD-Fc conjugates is shown.

In this example, Figure 57 shows the normalized sedimentation coefficient of sample 4 (28-fold dilution of TMV:RBD-Fc conjugate), and Figure 58 shows the normalized sedimentation coefficient of another sample (10-fold dilution of TMV:RBD-Fc conjugate). The normalized sedimentation coefficient of Figure 57 shows 100% total virus-related material (ie, virus-antigen conjugates) and Figure 58 shows 99.7% of total virus-related material (ie, virus-antigen conjugates). The results shown in Figures 57 and 58 demonstrate substantially complete engagement of the RBD-Fc product in the TMV conjugation event.

Successful conjugation between TMV NtK and RBD-Fc121 antigen was confirmed by SDS-Page analysis. FIG. 59 contains SDS-PAGE gels 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 minutes, lane 3 - conjugate mixture 5 minutes after conjugation, lane 4 - 15 minutes after conjugation. Conjugate mixture, Lane 5—Conjugate mixture 30 minutes post conjugation, Lane 6—Conjugate mixture 45 minutes post conjugation, Lane 7—Conjugate mixture 60 minutes post conjugation, Lane 8—Post conjugation. Final conjugate mixture, lane 9 - TMV:HA conjugate as control, lane 10 - conjugate mixture in 0 minutes, lane 11 - conjugate mixture 5 minutes post conjugation, lane 12 - conjugate mixture 15 minutes post conjugation. Gate mixture, lane 13—conjugate mixture 30 minutes after conjugation, lane 14—conjugate mixture 45 minutes after conjugation, lane 15—conjugate mixture 60 minutes after conjugation, lane 16—final conjugate after mixture. Gating mixture, 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.

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

Example 15(b) - In Vitro Studies To Determine the Efficacy of Conjugates of TMV NtK to RBD-Fc121 as Covid-19 Vaccine Candidates, Antigen Binding to CR3022 and ACE-2 Receptors, and in Mice Immune responses 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, early immune responses stimulated after one vaccination with TMV NtK:RBD-Fc conjugates supported this observation. Similarly, the RBD-Fc121 antigen was conjugated to the TMV NtK SARS-2 RBD-specific human neutralizing monoclonal antibody (mAb), CR3022.

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

ACE-2 functions as a cell surface receptor for the spike protein of SARS-CoV2 during invasion of respiratory epithelial cells. Two-color confocal microscopy and co-localization and competitive binding on Vero e6 cells to analyze the ability of the RBD-Fc121 antigen to bind the ACE-2 receptor and elicit a protective immune response against SARS CoV2 infection ACE-2 binding was quantitatively and functionally performed using a method-based analysis to determine ligand co-localization with ACE-2, and relative RBD-antibody complexes to ACE2 compared to angiotensin II. Both affinities were evaluated.

Vero cells are derived from the kidney of African green monkeys and are commonly used in cell assays. Vero e6 cells are subclones of Vero76 and exhibit a range of viral susceptibility. Here, by analyzing the concentration-dependent ability to block the binding of an ACE-2-specific antibody (ab15348, a homologue of angiotensin II) to its receptor on living Vero e6 cells, the natural agonist angiotensin II was compared to RBD-Fc binding. Plant-produced recombinant H7 strain influenza HA protein was used as a control when performing these studies. 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 the natural ligand of ACE-2, Angiotensin II, for their ability to bind to the receptor. . FIG. 62(A) is a graph showing ligand co-localization events in which FAM-Angiotensin II bound as expected to Vero e6 cells with an average of 452 particles per field and a non-specific H7 control. 2.79-fold increase over (n=10 analyses). (Considering Figures 61, 62(A) and 62(B), angiotensin II refers to ab15348.) As shown in the figure, the RBD-Fc121 antigen is 2.58-fold more potent at a concentration of 10 μg/ml. Increasingly, it bound to Vero e6 cells in a concentration-dependent manner (analysis with n=10). In contrast, in the same figure, binding of recombinant influenza HA-H7 remained at an average of 122.5 particles per field with no significant change at any concentration. FIG. 61 visually shows ligand co-localization and competitive binding between angiotensin II and RBD-Fc121 antigen in Vero e6 cells, as discussed in FIG. 62(A). The image in FIG. 61 was confocal with 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). Obtained using a microscope. In FIG. 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 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). , recombinant influenza HA-H7 did not correlate with a direct decrease with increasing concentration.

FIG. 62(B) shows that adding colocalization controls reduces overall specific binding, showing that ACE-2 antibody binding in the presence of both angiotensin II and RBD-Fc121 antigens Demonstrate decline. Note that binding of H7 HA did not affect detection of ACE-2 by monoclonal antibody at any concentration. These figures also demonstrate 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 to angiotensin II. indicates that the

Figures 60-62 thus demonstrate that the RBD-Fc121 antigen maintains functional conformation and activity through the binding of CR3022 and ACE-2. Binding to ACE-2 showed similar affinity and specificity to the natural agonist angiotensin II, and the conformation of the spike protein RBD in the recombinant RBD-Fc121 fusion peptide complex was similar to that of the natural SARS-2 spike. suggesting that it is comparable to that observed for proteins. This data indicates that the RBD-Fc121 antigen presents the correct structure required to elicit the production of neutralizing antibodies against the RBD of the Covid-19 spike protein.

Further evaluate the ability of the RBD-Fc121 antigen (both free and TMV conjugated) to not only stimulate immune responses, but also to neutralize viral infection by blocking cell entry at the ACE-2 receptor. To this end, pooled serum samples were analyzed using the SARS-CoV2 plaque neutralization assay before immunization, 12 days, 28 days, and 42 days after boost. Neutralization titers were calculated by determining the dilution of serum that reduced 50% of plaques. A standard 100 PFU amount of SARS CoV-2 was incubated with 2-fold serial dilutions of serum samples for 1 hour. The virus-serum mixture was then used to inoculate Vero e6 cells for 60 minutes, cells were overlaid with EMEM agar + 1.25% Avicel, incubated for 2 days, and plaques were stained with 1% crystal violet in formalin after counted. As Figures 63(A)-(D) show, preimmune 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 were not statistically different from vehicle controls (Fig. 63(B)). By day 28 (14 days after the second vaccination), a statistically significant reduction in titers was observed for the 15 μg neat (non-vaccinated) and 15 μg + CpG groups at a 1/20 dilution. However, the 45 μg neat group and the 45 μg + CpG group were significant at the 1/320 dilution (Fig. 63(C)). By day 42 (28 days after second vaccination), the 15 μg neat group showed significant neutralization titers at 1/640 dilution and significant neutralization titers at 15 μg + CpG group at 1/320 dilution. Indicated. Neutralizing titers increased at 1/5120 dilution for 45 mcg neat and 1/1280 dilution for 45 μg+CpG (FIG. 63(D)).

Figures 64(A)-(B) further highlight the increased neutralization titers of the 15 and 45 µg groups. This study compared the neutralizing antibody titers induced by the TMV:RBD-Fc121 vaccine in two mouse preclinical studies as measured by the CPE neutralization assay and by geometric mean titers (GMT) using the PRNT50 method, Table 68 below. 64(A), showing a dose response and no additional induction of neutralizing titers by inclusion of CpG compared to the neat vaccine group shown in FIG. 64(A). As further summarized in Table 52, day 42 (28 days after vaccination) sera were tested using the standard cytopathic effect (CPE) neutralization assay tested with SARS-CoV2. The CPE neutralization assay begins by titrating dilutions of heat-inactivated serum samples with 100 TCID50 of SARS-CoV-2, mixed in duplicate, 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, cell monolayers were fixed and stained with crystal violet to assess the presence of virus-induced CPE. The virus neutralization titer for each sample is reported as the reciprocal of the highest dilution that prevented CPM in 50% of wells. The results obtained from the CPE neutralization assay of individual serum samples from all groups correlate well with the pooled serum results obtained in the SARS-CoV2 plaque assay. TAP COVID-19 45 μg neat (GMT = 2702), 45 μg + CpG (GMT = 1280), 15 μg (GMT = 676), 15 μg + CpG (338), RBD-Fc antigen alone ( GMT=294) and PBS vehicle (<64). The group correspondence is clear, indicating no measurable contribution of CpG to promoting dose-response and enhanced neutralization titers.

Example 15(c) - In Vivo Testing The TMV NtK:RBD-Fc121 Covid-19 vaccine described above was evaluated for immunogenicity in two parallel evaluations using female C57BL/6 mice. Collected sera were tested for total IgG anti-RBD reactivity by ELISA, and recombinant COV2 RBD-His was used as the capture antigen to compare immune responses between groups, as shown in Figures 65(A)-(D). bottom.

For the initial evaluation, 10 animals were used per group and 5 animals per group were sacrificed on day 14 to have sufficient serum for extensive testing. The remaining 5 animals per group were boosted on day 14 and sacrificed on day 28. As used herein, the terms "boost" or "booster" or their similar and other synonyms 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 received prime and boost vaccines on days 0 and 14, respectively; Blood was collected on day 28, day 28 and final day. In both assessments, study endpoints included measurement of antigen-specific geometric mean antibody titers induced in mice, SARS-2 neutralization titers and antibody isotype analysis. The studies are outlined in Table 66 below and include various doses of each vaccine (including purified TMV NtK only, RBD-Fc only, TMV:RBD-Fc conjugate) and CpG oligodeoxynucleotides as adjuvants ( The same dose was compared both with and without CpG). CpG was added to the vaccine formulation such that the concentration of antigen was the same for 100 mcL injections as the neat (non-adjuvanted) vaccine formulation. In some embodiments, monophosphoryl lipid A (MPLA) and/or SE-M were utilized as adjuvants to enhance a 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.

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

Baseline serum antibody levels were very low, less than 100 ng/mL for the RBD-His and RBD-121-Fc antigens. Furthermore, no significant responses were measured for PBS vehicle control animals, unconjugated RBD-Fc antigen produced limited antibody responses, and conjugated to TMV NtK carriers produced strong immune responses. It has been demonstrated that it is necessary for the

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

As shown in Figure 65(D), the relative immune response to SARS-2RBD was tested as well as the human Fc portion of the antigen on the immune response. Day 42 sera were analyzed by ELISA using either SARS-2RBD-His or RBD-121-Fc proteins as capture agents. ELISA data were measured after a 1:100 dilution of the PBS/RBD-121-Fc antigen immunized group and a 1:1000 dilution of the TMV:RBD-Fc conjugate group. The titer recognizing RBD was significantly lower than that for RBD-Fc antigen, and the proportion of anti-RBD varied from 12% to 35% depending on the TMV:RBD-Fc group (Fig. 65(D)). Immune response titers were not significantly different between the TMV:RBD-Fc conjugate groups, regardless of the captured antigen.

A favorable balance of Th1/Th2 cytokines produced promotes safe and effective immune responses by balancing pro-inflammatory and anti-inflammatory responses, and given 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 FIG. FIG. 66(A) shows Th1 responses (IgG2a and IgG2C), FIG. 66(B) shows Th2 responses (IgG1), and FIG. 66(C) shows stack plot comparison of relative IgG2 to IgG1 antibody by ELISA. is. In Figure 66, " ^* " indicates p<0.05 compared to other groups.

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 significantly improved IgG2 titers with dose and adjuvant addition. 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 ratios were predominantly IgG1 isotype titers in the RBD-121-Fc group, with IgG1:G2 ratios greater than 1 in the non-adjuvanted TMV:RBD-Fc conjugate group, and TMV:RBD- The Fc conjugate + CpG adjuvant group varied between groups, with an IgG1:G2 ratio of less than 0.1. In summary, the CpG adjuvant strongly skewed the isotypic response to the TMV:RBD-Fc conjugate vaccine against the Th1 type, while the non-adjuvanted TMV:RBD-Fc conjugate showed a more balanced mix of Th1/Th2 responses. Unconjugated protein almost completely stimulated Th2 responses.

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

The data in Table 67 show measurable neutralization titers induced in mice following prime vaccination of adjuvanted and neat (non-adjuvanted) TMV:RBD-Fc121 vaccines. Figure 69 is a graph showing titer-based serum neutralizing activity after vaccination of mice against SARS CoV-2. The data shown in Figure 69 represent the log(2) transformed endpoint serum dilution that inhibited CPE by 50%. Dotted and dashed lines represent starting dilutions used for post-prime and post-boost assays, respectively. (*p<0.05, two-way ANOVA, Tukey's multiple comparison, GraphPad Prism 8.4.2). The data in FIG. 69 demonstrate that exemplary TMV:RBD-Fc vaccines (including but not limited to TMV:RBD-F121 vaccines), with or without adjuvant, produced measurable neutralization titers after a single vaccine. Further support that it can be induced. After the second vaccine boost these titers increase significantly at both the 15 and 45 μg concentrations. Referring to Figures 71 (A)-(C), an exemplary TMV:RBD-Fc vaccine confers protection to the organism following exposure to SARS-CoV2. Group 5 subjects received either control (PBS) or exemplary vaccines (CpG adjuvant only, 15 μg no adjuvant or 45 μg no adjuvant) at various levels of RBD-Fc concentrations or no immunization. Survival (Fig. 71 (A)), clinical symptoms (Fig. 71 (B)), post-challenge weight (Fig. 71 (C)) were evaluated and graphed. Clinical symptom data are based on temperature and physical appearance, the latter scored on a scale of 0 to 3: 0 = normal; 1 = mild, lack of grooming; 2 = moderate, coarse hair, nose. / eye discharge; 3 = severe, dyspnea, lack of response to stimulation, euthanized by human. Even without adjuvant, subjects receiving concentrations of 5 μg and 45 μg were the best for survival, the latter showing 100% survival after 12 days post-infection.

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

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

Further analysis of mouse sera showed a balanced Th1/Th2 immune response induced by the TMV:RBD-Fc121 vaccine, with immunogenicity to the vaccine largely dependent on conjugation to the TMV NtK carrier. In one in vitro model to evaluate the potential of a TMV:RBD-Fc vaccine to induce antibody-enhanced disease (ADE), sera containing neutralizing antibody titers ≧2700 were associated with SARS-CoV-2 to macrophages. It showed no evidence of enhanced invasion. This indicates that the vaccine strategy stimulates Nab titers without promoting ADE.

Additionally, Th1 immune responses were explored through cell stimulation assays. In this study, groups of 5 mice were treated on days 1 and 14 with antigen doses of either 15 μg or 45 μg, with or without CpG, in a total volume of 50 mcL (PBS with buffer). Immunization was given by subcutaneous administration. Sera were collected on days 0, 12, 18 and 42 and the extent of antibody response to the vaccine antigen was assessed as total titers to the target antigen and compared to PBS immunized or preimmune sera. The unconjugated protein RBD-Fc was used as a target for IgG evaluation and RBD-HIS to assess antigen responses to total antigen and RBD (spike S1 domain) components. Approximately 6 weeks after the first immunization, vaccinated mice were euthanized, terminal sera were collected, and spleens were harvested for IFNγ ELISpot analysis. A summary of the studies is shown in Table 69.

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

As shown in Figure 70, only cells recovered from mice vaccinated with a TMV:RBD-Fc vaccine containing a CpG adjuvant were statistically superior to IFNγ-secreting cells at day 42 (4 weeks after the second immunization). and similar numbers in both the 15 and 45 μg dose groups (Fig. 70(C)). This data supports IgG isotype analysis showing that TMV:RBD-Fc vaccine with CpG adjuvant co-delivery stimulates Th1 responses in mice.

In addition, VaxArray® SeroAssay analysis of mouse sera in the presence or absence of adjuvant, immunized with various TMV:RBD-Fc vaccine doses, was performed to assess its association with binding to heterologous coronavirus antigens. In FIG. 72, nCoV antigen (i) is the intact SARS-CoV2 spike protein, (ii) is the S1 (RBD) domain, SARS (i) is the intact SARS-1 spike, MERS (i) and HKU1 are similar, each produced in a mammalian system. Relative titers are expressed as fold enhancement of signal over control as reported. Strong reactivity was observed with both SARS-2 antigen and apparent cross-reactivity with SARS-1 antigen. As shown in Figure 72, no binding to the derivative MERS or HKU1 spike proteins was observed. Thus, the results in Figure 72 show that antisera from mice with neutralizing titers recognized xenogeneic (mammalian cell) SARS-2 spike protein, RBD and SARS-1 spike protein (i.e., cross-reactive ).

Specificity and relative titers of sera from the final bleed (day 42) were analyzed by VaxArray®. Nine different capture antigens were used, including eight heterologous antigens against SARS-CoV2 vaccine components: SARS-CoV2 spike (produced in insect cells), SARS-CoV2 spike (produced in mammalian cells), RBD (S1 ) - human Fc plant produced (homologous antigen), SARS-CoV2 RBD (S1) (produced in mammalian cells), SARS-CoV2 S2 domain (produced in insect cells), SARS-CoV2 S1 - ovine Fc (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 100, 350 and 700 ms exposures. Reactivity to each antigen by each serogroup is shown in Figures 73(A) and 73(B). FIG. 73(A) shows the reactivity measured against each captured antigen. FIG. 73(B) shows a comparison of the reactivity of each test group to the specific capture antigen. As a result of the 100ms exposure, reactivity of vaccine sera against all captured antigens except SARS-CoV2 S2 domain, MERS and HKU1 spike protein was observed. However, a significant response was observed for the SARS-COV1 S1 domain. Overall different reactivities were observed with intact and S1 capture antigens from SARS-CoV2 sources. With respect to overall reactivity, strong reactivity was observed from 45 μg + CpG, 45 μg neat, 15 μg neat, 15 μg + CpG and RBD alone in descending order, whereas sera from PBS-treated animals showed no reactivity. In addition, FIG. 74 shows further correspondence when individual animal responses measured by CPE found in Table 52 are correlated with plaque group microneutralization titers measured by VaxArray®. In FIG. 74, titers lower than those 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, indicated by absolute counts and inability to be reduced by dilution, indicate higher neutralization values. The overall correlation between inter-group titers and group neutralization titers corresponds to absolute numbers of individual titers, GMT and pooled serum values.

Further data on survival are shown in FIGS. 79(A)-(B), respectively. The former shows information from the mouse challenge study on survival and the latter shows information from the ferret challenge study on survival. In a 14-day mouse study (FIG. 79(A)), subjects (other than the placebo group) received the HA7 pandemic influenza vaccine (i.e., TMV:H7 antigen with or without CpG adjuvant) at or near the time of infection. vaccine) were inoculated at a ratio of 8:1 or 1:1. Subjects receiving adjuvant had a 100% survival rate by day 14. FIG. 79(B) shows information about the results of the ferret challenge test. Again, this was done with TMV-H7 conjugates in the presence or absence of CpG adjuvant. Based on viral titers collected from nasal turbinates, results showed that the vaccine improved recovery rate with lower titers in the adjuvanted group compared to the non-adjuvanted group.

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

Mouse studies compared the ability of specific antibodies, including those generated by the subject TMV:RBD-Fc vaccine, to enhance antibody-dependent 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 antibody. Test antibodies (or pooled sera) were co-incubated for 1 hour to facilitate viral inactivation by neutralizing antibodies prior to incubation with macrophages. After this incubation, medium containing both antibody and SARS-CoV2 was added to the macrophages and incubated for 48 hours, at which point viability was assessed through the addition of Cell Titer Glo (Promega) and luminescence output. evaluated based on

As further shown in Figure 75 (A-B), non-neutralizing antibodies to the nucleocapsid and spike proteins of SARS-CoV2 induced mouse macrophages, i. enhanced viral entry into certain cell types. Viability of virus-exposed macrophages in the absence of antibody averaged 91.5% of cell viability controls at 48 hours post-infection (n=10). Figure 75(A), Figure 75(B) first and fifth bars. In the presence of anti-nucleocapsid antibody raised against 6.25 μg/ml total spike protein, viability decreased to an average of 55.69%, with a maximal concentration-dependent reduction of >1 μg/ml. FIG. 75(A). In the presence of polyclonal antibody raised against 6.25 ug/ml of total spike protein, survival decreased to an average of 59.78%. FIG. 75(A). In contrast, the pooled mouse sera from the study of Example 15(b) against the 6.25 μg/ml TAP COVID-19 vaccine is shown by an average cell viability of 125.56% compared to the control. showed high neutralization titers without observable antibody-dependent enhancement. One-way ANOVA with Tukey's P-value was used to measure statistical differences and the results are shown in FIG. 75(C). Thus, non-neutralizing antibodies (against the nucleocapsid or spike protein) promoted viral entry into macrophages, whereas antibodies generated from the TMV:RBD-Fc vaccine did not show this effect.

Thus, the in vivo studies show that the TMV:RBD-Fc121 vaccine induces measurable and statistically significant SARS-2 neutralizing antibody titers over controls. 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. IgM levels can be assessed by ELISA and neutralizing antibody titers in vaccinated subjects. In general, IgM antibody levels are typically expected to rise for a period of about several weeks, eg, about two weeks, after vaccination. Therefore, testing by ELISA and/or neutralizing antibody titers on days 0, 7, 14 and 28, although other time periods can be used, is a Covid-19 It can be done after administration of the vaccine. Such studies after such administration are expected to confirm the production of IgM and its increased levels between days 0 and 14, which serves as an indicator of immunogenic response to vaccination. Thereafter, IgM levels are expected to decline by day 28 as IgG antibody titers increase. Also, the entire vaccine manufacturing process from initial sequencing of the Covid-19 antigen to final production of the cGMP vaccine for drug product sterility and release can be accomplished within a period of eight weeks, which is typically Note that this is a significant advantage over conventional vaccine production methods, which are typically over 6 months.

To assess potential adverse reactions to vaccination, the exemplary TMV:RBD-Fc121 vaccines described herein were tested for effects 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 body weight of each animal was recorded weekly after vaccination. The data graphed in FIG. 80 represent animals receiving Vaccine 1 (Day 0) and Vaccine 2 (Day 14), with the study ending on Day 28. As shown in Figure 80, the weight of all subjects increased.

In another challenge study, New Zealand White rabbits were administered an exemplary vaccine at different rates, with or without adjuvant, and compared to the control group in terms of post-challenge weight (FIG. 81A) and survival (FIG. 81(B)). evaluated. As shown in these figures, the vaccine group recorded significantly better results than placebo over 12 days in both categories.

Furthermore, as described in Example 14, studies with QIV vaccines have shown very high potential in laboratory animals for H1, H3, H5 and H7 influenza models, with no toxicologically significant findings observed. it wasn't. This data, and the role of inactivated TMV as a carrier, suggest similar potential for the Covid-19 vaccines disclosed herein due to similar structural components and development platform. As discussed in Example 16 below, the Covid-19 vaccines disclosed herein exhibit high stability at room temperature and under room temperature conditions for at least 6 months.

Thus, through practice of certain embodiments herein, conjugable antigens suitable for conjugation with a carrier can be produced. The teachings herein contemplate or cover compositions of antigens, viral particles, vaccines, constructs and other materials that can be conjugated to viral particles, and all methods used in their production. . Such antigens can be recombinant antigens, which generally comprise a first peptide comprising the receptor binding domain of a pathogen (coronavirus in a non-limiting example) and a fragment of an antibody capable of binding to an Fc receptor. A fusion peptide with a second peptide that includes a crystallizable (Fc) region. In some embodiments, the first peptide and the second peptide are linked by a hinge portion. Optionally, this hinge portion may be part of the Fc region. In some embodiments, the hinge portion comprises the amino acid sequence set forth in SEQ ID NO:3. Exemplary coronaviruses from which receptor binding domains can be obtained include, but are not limited to, SARS-CoV-1, SARS-CoV-2 and MERS.

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

Consistent with the teachings herein, in some embodiments a vaccine comprising at least one conjugable antigen and a carrier comprising a viral particle as described according to embodiments and alternatives of the present disclosure is provided. manufactured. In some embodiments, such viral particles are viruses, such as TMV. The first peptide of the conjugable antigen may comprise the amino acid sequence set forth in SEQ ID NO:2, or alternatively the amino acid sequence may be the amino acid sequence set forth in SEQ ID NO:8. The first peptide may comprise a contact residue located in the range of about position 289 to about position 662 of the S-1 subunit of the coronavirus spike protein, which allows the antigen to be released from the viral particle carrier after administration. contact with ACE-2 receptors on cells of mammalian subjects to which such vaccines are administered. For the RBD-FC121 antigen, contact residues can range from about position 301 to about position 662 of the S-1 subunit. For the conjugable antigen referred to herein as the 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 lacks this sequence was found to contain In both cases, when the conjugateable antigens of the vaccine are released in a mammalian subject having cells containing one or more ACE-2 receptors, at least one conjugateable antigen is one or more binds to the ACE-2 receptor of In some embodiments, the viral particles used to produce such vaccines have surface lysine residues that chemically associate with the fusion peptide, more particularly the first peptide, resulting from the conjugation reaction. do.

Consistent with the teachings herein, in some embodiments, vaccines according to the teachings herein are multivalent and contain at least one conjugable antigen having an influenza A virus receptor binding domain. and at least one conjugable antigen having a coronavirus receptor binding domain. Other possible combinations are within the scope of the present embodiments, and the combinations provided herein are exemplary and non-limiting. In some embodiments, vaccines according to the teachings herein are multivalent and comprise at least one conjugable antigen having an influenza B virus receptor binding domain and at least one having a coronavirus receptor binding domain. Contains one conjugable antigen. Alternatively, a vaccine in accordance with the teachings herein is multivalent, comprising at least one conjugable antigen having an influenza A virus receptor binding domain, at least one conjugate having an influenza B virus receptor binding domain, and It comprises a gating antigen and at least one conjugable antigen having a coronavirus receptor binding domain. Various ratios can be selected and expressed as the virus particle:antigen mass ratio (ie, the mass ratio of virus particles to at least one bindable 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 vaccines according to the teachings herein as an adjuvant to enhance the mammalian subject's immune response to the vaccine.

Example 16 Stability of Coronavirus Vaccine Candidates Under Refrigerated and Room Temperature Conditions: TMV:RBD-Fc121 Vaccine Similar to QIV vaccine stability discussed in Example 13, RBD-Fc121 antigen and TMV:RBD-Fc121 All vaccines showed consistency and stability under refrigerated and room temperature conditions for at least 6 months. In this example, the studies discussed in Example 13 were used to measure the stability of the RBD-Fc121 antigen, as well as the stability of the TMV:RBD-Fc121 vaccine.

As previously described, the 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 table below shows the purified RBD-Fc121 antigen measured at release and at various times after filling into vials and storage at refrigerated conditions (2° C.-8° C.) and room temperature (22° C.-28° C.). stability data and preservative efficacy of In these tables, "HMW" is an abbreviation for high molecular weight.

Tables 70 and 71 show that the purified RBD-Fc121 antigen is highly stable and potent as a result of following the production and purification methods according to several embodiments and alternatives.

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

The table below shows the TMV:RBD-Fc121 vaccine (TMV to antigen ratio 8:1) at the time of release and filled into bags under refrigerated (2°C to 8°C) and room temperature (22°C to 28°C) conditions. Show the stability data at various times after storage with :

Tables 72 and 73 show that the TMV:RBD-Fc121 vaccine showed robust stability measurements for at least 6 months under refrigerated conditions. Similarly, Table 74 shows that TMV:RBD-Fc121 vaccines produced according to several embodiments and alternatives show robust stability measurements for at least 6 months at room temperature storage (22°C-28°C). indicates that This, in most situations, requires storage under ultra-low freezer (e.g., -20°C to -70°C) or refrigerated conditions, or has a stability of no more than a few hours at room temperature, compared to conventional SARS- 2 vaccine. Moreover, the purification and formulation process according to multiple embodiments and alternatives stabilizes the RBD-Fc121 antigen itself, well beyond the stability limits of conventional approaches.

Example 17 - Coronavirus Vaccine Candidate: Plant Expression, Purification, Characterization and Conjugation of RBD-Fc139 (SARS-2) to TMV In addition to the RBD-Fc121 antigen, any other Covid-19 antigen It is anticipated that viral antigens of this type can be purified, conjugated to inactivated TMV, and used as effective vaccine candidates according to multiple embodiments and alternatives. In some embodiments, the following antigen sequences (referred to herein as "RBD-Fc139 constructs") are used for Covid-19 antigen synthesis:
1. (SEQ ID NO: 1) Signal peptide: MGKMASLFATFLVVLVSLSLASESSA
2. (SEQ ID NO: 5) SARS-2 virus spike amino acid numbers 319-330: RVQPTESIVRFP
3. (SEQ ID NO: 6) SARS-2 virus spike amino acid numbers 331-591:

４．（配列番号７）タバコエッチウイルスＮＩＡ切断配列：ｅｎｌｙｆｑｇ
５．（配列番号３）Ｆｃヒンジ：ＶＥＰＫＳＣＤＫＴＨＴＣＰＰＣＰ
６．（配列番号４）ＩｇＧ１ １７１ アロタイプＦｃ：

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:

In one embodiment of the RBD-FC139 construct, the following antigen sequences are used for Covid-19 antigen synthesis:
(SEQ ID NO:8) SARS-2 virus spike amino acid numbers 319-591:

A polygenic construct was designed and constructed to contain the genes encoding the proteins necessary to synthesize the RBD-Fc139 antigen, which also included the RBD domain of SARS-2 in a manner similar to RBD-Fc121. target. According to several embodiments and alternatives, the RBD-Fc139 construct was ligated into a TRBO vector (as a non-limiting example) and subsequent colonies screened to confirm clones. A constructed expression plasmid containing the RBD-Fc139 construct similar to the plasmid shown in FIG. 52 was amplified and then purified for use in generating a master cell bank.

According to embodiments and alternatives, the RBD-Fc139 antigen is expressed in plants and purified using the antigen purification platform described herein. As shown in FIGS. 76-77, according to multiple embodiments and alternatives, the antigen purification platform successfully extracts pure and stable antigen and RBD-Fc139 as RBD-Fc monomers in a GLP-compliant manner. Purify well in high yield. Figure 76 from the conclusion of the antigen purification platform includes SDS-page gels in both reducing and non-reducing conditions demonstrating the purity and successful purification of the RBD-Fc139 antigen. In Figure 76, the lanes are:
Lane 1 - 11.8 ug purified antigen in reducing conditions, lane 2 - blank, lanes 3-5 - 1.5 ug purified antigen in reducing conditions, lane 6 - blank, lane 7 - marker, lane 8 - blank, Lanes 9-11—1.5 ug of purified antigen in non-reducing conditions, lane 12—blank and lane 13—11.8 ug of purified antigen in non-reducing conditions. A clear visible band indicates that the RBD-Fc139 antigen product is highly pure.

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

Thus, the SDS-page gel and SEC-HPLC report 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.

In parallel with Covid-19 antigen production, 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 inactivated. Using the conjugation of recombinant antigen embodiments described herein, the RBD-Fc139 antigen is then conjugated to inactivated TMV via surface-exposed lysine residues.

Also, TMV:RBD-Fc139 conjugates according to the present embodiments herein are currently under investigation as vaccine candidates for Covid-19 disease. However, based on the success of the tetravalent vaccine and the strong immune response by the first TMV:RBF-Fc121 conjugate, the TMV:RBD-Fc139 conjugate is also expected to be a promising Covid-19 vaccine.

It will be readily appreciated that the teachings herein are consistent with embodiments having a wide variety of alternative ways to implement the embodiments. Thus, without limitation, the fusion peptide in the antigen-directed embodiment, referred to herein as embodiment A, is a first peptide comprising a pathogen receptor binding domain and binding to an Fc receptor. is formed from a second peptide that contains the crystallizable (Fc) region of the antibody capable of The antigen in an embodiment within embodiment A, referred to herein as embodiment B, further comprises a hinge portion connecting the first and second peptides. A 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. The pathogen in embodiments falling within embodiment A and referred to herein as embodiment D is a coronavirus having a receptor binding domain, and more specifically, embodiments herein The coronavirus designated E is selected from the group consisting of SARS-CoV-1 and SARS-CoV-2. In an embodiment within embodiment E and referred to herein as embodiment F, the coronavirus receptor binding domain is about contact residues located in the range of position 289 to about position 662, wherein the contact residues contact an ACE-2 receptor on a cell of a mammalian subject, alternatively referred to herein as embodiment G The contact residues in certain embodiments are located in a range from about position 301 to about position 662 of the S-1 subunit. The coronavirus receptor binding domain in an embodiment falling within embodiment F and referred to herein as embodiment H lacks the amino acid sequence shown in SEQ ID NO:5.

The coronavirus in the embodiment falling within embodiment D and referred to herein as embodiment I is Middle East respiratory syndrome coronavirus. In an embodiment within embodiment A, referred to herein as embodiment J, the second peptide is the Fc domain of an IgG1 antibody, more specifically, , wherein 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 performed (ie, made, formed, designed, used, etc.) according to embodiment A as more fully described herein. Optionally, in practicing the embodiments described herein, the antigen described herein is any one of embodiments B, C, D, E, F, G, H, I, or J. It may be implemented by incorporating into Embodiment A above, which may be directed to compounds, methods and genetic constructs in practicing any one or more of these alternative embodiments. Similarly, embodiments may include any of the antigens, methods of forming antigens, or methods useful for forming antigens described in Embodiments A, B, C, D, E, F, G, H, I, or J. A vaccine comprising the genetic construct may be directed, in combination with any of the foregoing embodiments, further comprising a carrier comprising a viral particle, which in some embodiments is a virus, more particularly tobacco mosaic virus and the fusion peptide is chemically associated with lysine residues on the surface of the carrier.

Further, in a non-limiting embodiment, referred to herein as embodiment K, the vaccine comprises a carrier comprising influenza hemagglutinin antigen (HA) and viral particles having surface lysine residues, wherein HA is surface lysine Chemically associate with the residue. The viral particle in an embodiment falling within embodiment K and referred to herein as embodiment L is a viral particle that targets at least one antigen in a mammalian subject having cells containing one or more ACE-2 receptors. released, the at least one antigen binds to one or more ACE-2 receptors. The viral particle in the embodiment within embodiment K and referred to herein as embodiment M is a virus, and more specifically in the embodiment referred to herein as embodiment N, the virus is tobacco mosaic virus. The vaccine in the embodiment within any of embodiments K, L, M, or N and referred to herein as embodiment O is multivalent and the HA consists of HA type A and HA type B. selected from the group, wherein 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 surface lysine residues . In an embodiment within embodiment O, referred to herein as embodiment P, the HA comprises two or more hemagglutinin antigen (HA) type A and two or more HA type B include. Also, for embodiments within the scope of embodiment O and referred to herein as embodiment Q, embodiments A, B, C, D, E, F, G, H, I, or J are incorporated into the coronavirus element of the vaccine. Further, in the embodiment referred to herein as embodiment R, the ratio (by weight) of viral particles to at least one antigen in the vaccine within embodiment Q is from 1:1 to 8:1. Within the range, and more specifically in the embodiment designated as embodiment S, the range is 8:1.

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

In one embodiment, directed to a viral-antigen conjugate comprising a virus and at least one antigen, referred to herein as embodiment DD, wherein the at least one antigen comprises a first A peptide, and a fusion peptide comprising a second peptide, wherein the fusion peptide can be conjugated to a virus. In an embodiment within embodiment DD, referred to herein as embodiment EE, the conjugate is multivalent and comprises at least It further contains one influenza hemagglutinin antigen (HA). In an embodiment within embodiment DD or EE, referred to herein as embodiment FF, the conjugate is multivalent and the second peptide binds to an Fc receptor. is the crystallizable (Fc) region of an antibody, which is the Fc domain of an IgG1 antibody. In embodiments within any of embodiments DD, EE, or FF, and referred to herein as embodiment GG, the conjugate comprises a first peptide and a second peptide. Further comprising a linking hinge portion, 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. Within any of embodiments DD, EE, FF, or GG, and referred to herein as embodiment HH, the first peptide is either SEQ ID NO:2 or SEQ ID NO:8 contains the amino acid sequences described in In embodiments 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. be. Embodiments within any of embodiments DD, EE, FF, GG, HH, or II, wherein the pathogen in embodiments referred to herein as embodiment JJ is a coronavirus having a receptor binding domain and the receptor-binding domain of coronaviruses is located on the S-1 subunit of the coronavirus spike protein and includes contact residues located in the range from about position 289 to about position 662.

Further Embodiments and Uses of the Novel Subject Matter Herein In addition to the antigens described above, including the RBD-Fc antigen for treatment against SARS-CoV2, according to multiple embodiments and alternatives described herein, A myriad of other antigens can be formed. It is intended that the scope of the description and teachings herein be limited only according to the claims. For example, using strategies similar to Examples 14, 15 and 16, candidate vaccines are derived from other human-infecting coronaviruses, including acute respiratory syndrome coronavirus (SARS-1) and Middle East respiratory syndrome coronavirus (MERS). Obtainable. As shown in Figure 78, homology domains can be identified in functional regions of the spike S1 domain that correlate with receptor binding and other essential activities.

In this regard, Figure 78 shows the receptor binding domains in spike protein sequence alignments of SARS-CoV-2 and other related coronaviruses. Presented here is a sequence alignment of the interacting (ie binding) domains of SARS-CoV-2 (MN938384), Bat-CoV (MN996532 and MG772933) and SARS-CoV (NC004718). Key amino acids described for interaction with ACE2 and SARS-CoV2 are underlined. (Line (-) = same amino acid, dot (.) = deletion). FIG. 78 shows the results of 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. EXCLIJ. 2020;19:410-417, published 18 March 2020, from Doi: 10.17179/excli2020-1167.

As seen in the constructs and N-terminal readings of the RBD-Fc121 and RBD-Fc139 antigens, the functional core element was changed from the "RVQPT" motif for the RBD-Fc139 construct to the to the "CGPKK" domain for Looking more closely at the RBD-Fc121 construct, beyond the core domain there is a longer protein domain that facilitates proper folding. Indeed, this strategy is expected to extend to any type of coronovirus antigen through the following process:
1. 2. Protein homology analysis shown in FIG. In silico protein folding using existing coronavirus spike models3. 4. 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, represented by the RBD-Fc121 and 139 constructs. Expression in plants6. 7. Purification by protein A and other methods exemplified in this patent application. Conjugation to TMV according to multiple embodiments and alternatives described herein

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

Therefore, 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, multivalent vaccines comprising 2, 3, 4, 5 or more different antigens against various viruses and other pathogens according to the teachings herein is provided. At least one antigen is capable of neutralizing or stimulating an immune response to one type of virus (e.g. influenza), while at least one other antigen is capable of neutralizing or stimulating an immune response to another type of virus (e.g. coronavirus or The same can be done for pandemic viruses like HA7). Pre-determined antigens on the carrier may consist of a modeled fusion protein in which the receptor binding domain of a virus is followed by a fusion partner such as the Fc domain of an antibody derived from the tail region of the 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 combinable antigenic-viral conjugates contemplated herein often allows for broad-spectrum Facilitate the ability to manufacture vaccines on a large scale.

It is to be understood that the embodiments described herein are not limited in their application to the details of the teachings and description set forth or shown in the accompanying drawings. Rather, it is understood that the embodiments and alternatives described and claimed herein can be implemented or used in various ways. Also, it is to be understood that the words and phrases used herein are for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "for example," "containing," or "having" herein and variations of those words are enumerated thereafter. and their equivalents, as well as additional items.

Accordingly, the above description of several embodiments and alternatives is intended to be illustrative rather than acting as a limitation on the scope of what is disclosed herein. The description herein is not intended to be exhaustive, nor is it intended 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 (15)

a first antigen comprising influenza hemagglutinin antigen (HA);
A multivalent vaccine comprising: a second antigen; and a carrier comprising viral particles having surface lysine residues, wherein said first antigen and said second antigen are chemically associated with the surface lysine residues. 2. The multivalent vaccine of claim 1, wherein said second antigen is influenza HA other than said first antigen. 3. A multivalent vaccine according to claim 1 or 2, comprising two or more type A hemagglutinin antigens (HA) and two or more type B HAs. 2. The multivalent vaccine of claim 1, wherein said second antigen comprises a coronavirus receptor binding domain. 5. A multivalent vaccine according to claim 4, comprising two or more type A hemagglutinin antigens (HA) and two or more type B HAs. 2. The multivalent vaccine of claim 1, wherein said viral particles are tobacco mosaic virus. the integrity or concentration of the vaccine at the end of said predetermined period of time is at least 90% of the initial integrity or initial concentration of said vaccine when placed in an unrefrigerated environment at storage temperature for a predetermined period of time; 2. The multivalent vaccine of Claim 1, wherein said predetermined period of time is at least 42 days after the date of release of said vaccine. 8. A multivalent vaccine according to claim 7, wherein said storage temperature is at least 20<0>C. A virus-antigen conjugate comprising a virus and at least one antigen,
A conjugate wherein said at least one antigen comprises a fusion peptide comprising a first peptide comprising a receptor binding domain of a pathogen and a second peptide, said fusion peptide being capable of being conjugated to said virus. 10. The conjugate of claim 9, wherein said conjugate is multivalent and further comprises at least one influenza hemagglutinin antigen (HA) selected from the group consisting of HA type A and HA type B. 11. The claim 9 or 10, wherein said second peptide is a fragment crystallizable (Fc) region of an antibody capable of binding to an Fc receptor, said Fc region being the Fc domain of an IgG1 antibody. Conjugate. further comprising a hinge portion connecting the first peptide and the second peptide, wherein the hinge portion comprises the amino acid sequence set forth in SEQ ID NO: 3, and the pathogen has the receptor binding domain The conjugate of any one of claims 9-11, which is a coronavirus. The conjugate of any one of claims 9-12, wherein said first peptide comprises the amino acid sequence set forth in either SEQ ID NO:2 or SEQ ID NO:8. The conjugate of any one of claims 9-13, wherein said virus is tobacco mosaic virus comprising an N-terminal lysine residue. said pathogen is a coronavirus having said receptor binding domain, wherein said receptor binding domain of said coronavirus is located in the S-1 subunit of said coronavirus spike protein and is located at about position 289 to about 662; 10. The conjugate of claim 9, comprising contact residues located in the range of .

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