VIRAL VACCINES AND METHODS OF FORMING THE SAME

Abstract

Provided herein is a method for producing an inactivated virus including a) heating the virus to a temperature sufficient to disrupt the virus membrane; b) exposing the virus of step (a) to a nucleic acid degrading enzyme; and c) cooling the virus to a temperature sufficient to reestablish the integrity of the virus membrane. Also provided herein is a vaccine produced using the instant method.

Claims

1. A method for producing an inactuated virus comprising: a) forming a denatured viral capsid through heating of the virus to a temperature of between 40° C. and about 44° C. for between 5 and 30 minutes; b) exposing viral genetic material within the denatured viral capsid of step (a) to a nucleic acid degrading enzyme; and c) following steps (a) and (b), reestablishing the integrity of the denatured viral capsid through cooling of the virus; wherein exposing the viral genetic material to the nucleic acid degrading enzyme in step (b) at least partially destroys the viral genetic material within the denatured viral capsid; and wherein steps (a)-(c) are performed without damaging the immunogenic capsid antigens of the virus.

2. The method of claim 1, wherein the virus is a pseudorabies virus.

3. The method of claim 1, wherein forming the denatured viral capsid comprises forming pores in the viral capsid.

4. The method of claim 1, wherein forming the denatured viral capsid comprises temporarily relaxing the viral capsid.

5. The method of claim 1, wherein the heating of step (a) is to a temperature of about 50° C. to about 60° C.

6. The method of claim 5, wherein the heating of step (a) is for at least 10 minutes.

7. The method of claim 1, wherein the exposing of step (b) is for at least 4 hours.

8. The method of claim 1, wherein the cooling of step (c) is to a temperature of 4° C.

9. The method of claim 1, wherein the cooling of step (c) is for at least 3 hours.

10. The method of claim 1, further comprising, after step (a) and before step (b), modifying a protein within the virus.

11. The method of claim 10, wherein the modifying relaxes a nucleic acid-to-protein association to expose the viral genetic material.

12. The method of claim 10, wherein the modifying includes acetylation.

13. The method of claim 1, wherein the exposing of step (b) destroys viral genetic material without damaging immunogenic capsid antigens of the virus.

14. A method for producing an inactuated virus comprising: a) heating the virus to a temperature of about 42° C. for at least 10 minutes to denature the viral capsid; b) exposing viral genetic material within the virus of step (a) to a nucleic acid degrading enzyme; and c) cooling the virus to a temperature of 4° C. or less for at least 3 hours to reestablish the integrity of the denatured viral capsid; wherein the exposing in step (b) at least partially destroys the viral genetic material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0027] FIGS. 1A-B show TEM images of a single untreated control PED virion (A) and a cluster of untreated control PED virions (B), illustrating the characteristic icosahedral shape and surface spikes.

[0028] FIGS. 2A-B show TEM images of a single gamma irradiated PED virion (A) and a cluster of gamma irradiated PED virions (B).

[0029] FIGS. 3A-B show TEM images of a single heat/RNase treated PED virion (A) and a cluster of heat/RNase treated PED virions (B).

[0030] FIGS. 4A-C show immunofluorescence images of inactuated PEDV cultures. Treated and untreated virus cultures were used to infect vero cell monolayers for 3 passages. Viral replication was assessed by staining the cell sheet with a PEDV-specific swine polyclonal antibody, followed by anti-swine FITC conjugate. (A) Untreated PEDV infected cells at passage 3, showing typical cytoplasmic, apple green fluorescence and the formation of syncytia. (B) Irradiated PEDV culture at passage 3, showing no viral growth. (C) Heat and RNase treated PEDV culture at passage 3, showing minimal replication.

[0031] FIG. 5 shows PCR detection of inactuated viral particles in infected cells. By column, the PCR shows (1) Ladder; (2) PEDV untreated control—5 Kb primers; (3) PEDV Heat/RNase treatment—5 Kb primers; (4) PEDV Irradiated treatment—5 Kb primers; (5) PEDV untreated control—3.5 Kb primers; (6) PEDV Heat/RNase treatment—3.5 Kb primers; and (7) PEDV Irradiated treatment—3.5 kb primers.

[0032] FIG. 6 shows a schematic representation of a swine vaccine challenge study according to an embodiment of the disclosure.

[0033] FIGS. 7A-C show graphs illustrating antibody responses in vaccinated pigs. (A) Mean reactivity against the PEDV neucleoprotein (NP) measured as ELISA OD values. (B) Mean reactivity against the PEDV spike protein measured as ELISA OD values. (C) Mean virus neutralization titers depicted as the average titer for each group (Y axis). DPV is days post vaccination. The virus neutralization assay was conducted by neutralizing 10.sup.3 TCID.sub.50 of virus with an equivalent volume of serum, serially diluted in doubling dilutions. Wells with no virus growth after incubation for 3 days are indicative of high levels of virus neutralizing antibodies and vice versa.

[0034] FIGS. 8A-B show graphs illustrating mean Ct values of qPCR analysis of fecal matter from individual pigs from each group, collected at day 3 (A) and day 7 (B) post-challenge.

[0035] FIGS. 9A-C show images of representative intestinal sections from study animals. (A) H & E section of healthy jejunum of an unvaccinated pig sacrificed prior to challenge showing intact villi and crypt architecture (10×). (B) H & E section of ileum of an unvaccinated pig and challenged pig showing mild villus atrophy and crypt hyperplasia (100×). (C) Immunohistochemistry (IHC) section of the jejunum of an unvaccinated pig and challenged pig. Yellow arrows indicate PEDV immune-positive enterocytes (400×).

[0036] FIGS. 10A-B show schematic representations of the PEDV and influenza virus. (A) PEDV virus and its genome (reproduced from (http://www.swinecast.com/) showing the linear, single-stranded RNA genome. (B) Influenza virus and its genome (reproduced from https://www.urmc.rochester.edu/labs/) showing the segmented RNA genome associated with viral proteins.

[0037] FIGS. 11A-I show images of representative images of cells infected with Heat/DNase treated virus. Depicted cell sheets are stained with a PRV-specific fluorescent antibody to verify viral replication. Cell sheets were stained with a PRV specific fluorescent antibody, with apple green fluorescence indicating detection of virus. By row, the top row, including images (A), (B), (C), shows uninfected cells; the middle row, including images (D), (E), (F), shows cells subjected to Heat/DNase treatment at 58° C. for 5 hrs; the bottom row, including images (G), (H), (I), shows cells subjected to Heat/DNase treatment at 58° C. for 6 hrs. By column, the left column, including images (A), (D), (G), shows a Brightfield images of cells; the middle column, including images (B), (E), (H), shows stained cells; the left column, including images (C), (F), (I), shows DAPI overlay of stained cells with blue nuclei.

[0038] FIG. 12 shows an image and a schematic representation of Pseudorabies Virus (PRV), which is a large enveloped virus with a size of approximately 180 nm containing dsDNA. Specifically, the PRV virus is composed of three (3) enveloping layers (outer envelope, tegument, capsid) with a linear double-stranded DNA genome of approximately 120-150 kilobase-pairs.

[0039] FIG. 13 shows a representation of a polyoma virus (polyomaviridae), which is a non-enveloped virus containing an icosahedral dsDNA genome with a diameter size of approximately 45-55 nm.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0040] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0041] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0043] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0044] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0045] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0046] The present application can “comprise” (open ended) or “consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0047] When open-ended terms such as “including” or ‘including, but not limited to” are used, there may be other non-enumerated members of a list that would be suitable for the making, using or sale of any embodiment thereof.

[0048] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0049] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0050] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0051] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0052] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0053] The presently-disclosed subject matter includes viral vaccines and methods of producing viral vaccines. Provided herein, in some embodiments, is a method for producing an inactivated and/or attenuated virus, including in some embodiments an inactuated virus. In one embodiment, the method includes a) heating the virus to a temperature sufficient to disrupt the virus membrane; b) exposing the virus of step (a) to a nucleic acid denaturing and/or degrading enzyme; and c) cooling the virus to a temperature sufficient to reestablish the integrity of the virus membrane. Also provided herein, in some embodiments, is a vaccine produced according to one or more of the instant embodiments.

[0054] As noted above, in one embodiment, the method includes disrupting the virus membrane through heating of the virus. In another embodiment, disrupting the virus membrane includes forming pores or temporarily denaturing the membrane. The temperatures and timing for disrupting the virus membrane will depend upon the specific virus. For example, for porcine pseudorabies virus (PRV), a herpes virus, suitable temperatures include, but are not limited to, about 50° C.-60° C.

[0055] As another example, for porcine epidemic diarrhea virus (PEDV), suitable temperatures include, but are not limited to, up to about 42° C., about 42° C., at least about 40° C., at least about 42° C., at least about 44° C., between about 40° C. and about 44° C., or any combination, sub-combination, range, or sub-range thereof. Suitable timing for PEDV includes, but is not limited to, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, up to 1 hour, up to 4 hours, up to 8 hours, up to 12 hours, between 1 minute and 12 hours, or any combination, sub-combination, range, or sub-range thereof. Suitable temperatures for other viruses may be lower than, the same as, or higher than the aforementioned temperatures for PEDV. Similarly, suitable timing for other viruses may be less than, the same as, or higher than the aforementioned timing for PEDV.

[0056] As mentioned previously, suitable times and temperatures for disrupting the virus membrane may vary depending on the specific virus. As an additional example, for pseudorabies virus (PRV) suitable temperatures include, but are not limited to, up to about 42° C., about 42° C., at least about 40° C., at least about 42° C., at least about 44° C., between about 40° C. and about 44° C., or any combination, sub-combination, range, or sub-range thereof. Suitable timing for PRV includes, but is not limited to, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, up to 20 minutes, up to 1 hour, up to 4 hours, up to 8 hours, up to 12 hours, between 1 minute and 12 hours, or any combination, sub-combination, range, or sub-range thereof.

[0057] The disruption of the virus membrane according to step (a) permits small molecules, such as enzymes, to enter the virus. In some embodiments, this disruption permits the nucleic acid denaturing and/or degrading enzyme of step (b) to pass through the virus membrane or permit the nucleic acids to be extruded from the relaxed membrane. In one embodiment, step (b) includes exposing the virus to a solution that contains the nucleic acid denaturing and/or degrading enzyme in a sufficient concentration to allow the nucleic acid denaturing and/or degrading enzyme to pass through the denatured virus membrane resulting from step (a). In another embodiment, the virus is exposed to the solution of nucleic acid denaturing and/or degrading enzyme in step (b) for a sufficient time to allow an equilibrium of the concentration of nucleic acid denaturing and/or degrading enzyme inside and outside the virus. Suitable nucleic acid denaturing and/or degrading enzymes include, but are not limited to, nucleases or any other enzyme that has the ability to denature or degrade nucleic acid. As will be appreciated by those skilled in the art, the nucleic acid genetic material may alternatively be DNA or RNA with a suitable degrading enzyme, such as DNase, and/or Benzonase nuclease, or alternatively RNase, respectively. In one embodiment, step (b) includes exposing the virus to a solution that contains the RNA denaturing and/or degrading enzyme in a sufficient concentration to allow the RNA enzyme to pass through the denatured virus membrane resulting from step (a). In another embodiment, the virus is exposed to the solution of RNA enzyme in step (b) for a sufficient time to allow an equilibrium of the concentration of RNA enzyme inside and outside the virus. Suitable RNA denaturing and/or degrading enzymes include, but are not limited to, RNase or any other enzyme that has the ability to denature or degrade RNA. As will be appreciated by those skilled in the art, steps (a) and (b) may be performed in any order so long as the denatured virus membrane is exposed to the nucleic acid denaturing and/or degrading enzyme at some point. For example, the virus may be heated to denature the virus membrane first and then exposed to the nucleic acid degrading enzyme while the virus membrane is still denatured, or the virus may be exposed to the nucleic acid degrading enzyme and then heated to denature the virus membrane while remaining exposed to the nucleic acid degrading enzyme.

[0058] Additionally or alternatively, in certain embodiments, the virus includes one or more proteins closely associated with the virus genome thereof. For example, the influenza NS1 protein is closely associated with the viral genome in influenza, mimicking the histone:DNA association in human genomes. Accordingly, in some embodiments, the method includes disrupting an nucleic acid-to-protein interaction in the virus prior to exposing the virus to the nucleic acid degrading enzyme. In one embodiment, disrupting the nucleic acid-to-protein interaction includes chemically modifying the protein associated with the nucleic acid. In another embodiment, the chemical modification of the protein includes methylation and/or acetylation. In a further embodiment, the protein includes histone, a histone-like protein (e.g., influenza NS1 protein, polyoma), or any other protein that closely associates with the nucleic acid of the virus. The chemical modification of the protein, such as, for example, the chemical modification of NS1 with a histone acetylase enzyme, relaxes the RNA:protein association and exposes the genetic material. By exposing the genetic material, the disruption of the RNA:protein interaction permits the RNA denaturing and/or degrading enzyme to digest the viral RNA. As an additional example, the chemical modification of the histone-like proteins (e.g., H2A, H2B, H3, and H4), or any other protein that closely associates with viral DNA, with a histone acetylase enzyme, relaxes the DNA:protein association and exposes the viral genetic material.

[0059] Following the heating and exposing steps, step (c) of the method disclosed herein includes cooling the virus to a temperature and for a time sufficient to cause the virus membrane to reform. For example, in some embodiments, cooling the virus to a temperature of about 4° C. causes the virus membrane to renature and reform into the form it was before the first step of the method. Although the example above discusses a temperature of about 4° C., as will be appreciated by those skilled in the art, the disclosure is not so limited and may include any other suitable temperature for reforming the virus membrane. Such suitable temperatures will depend upon the specific virus, and may be lower than, the same as, or higher than 4° C.

[0060] Without wishing to be bound by theory, it is believed that partially or completely destroying the viral genetic material by degrading the virus membrane and exposing the virus to nucleic acid degrading enzymes, without damaging the immunogenic capsid antigens, produces a vaccine candidate which will enter host cells and can undergo abortive to no replication while still inducing effective immunity due to antigen presentation that resembles natural infection or vaccination with an attenuated vaccine. This process is interchangeably referred to herein as inactuation. In addition, it is believed that the vaccines produced according to one or more of the embodiments disclosed herein provide vaccination without causing any disease or other adverse effect. Accordingly, in some embodiments, the vaccines disclosed herein combine the safety and efficacy advantages of inactivated and attenuated vaccines respectively, to generate rapid, first-response vaccines.

[0061] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently-disclosed subject matter.

EXAMPLES

Example 1

[0062] Influenza A virus (Sandbulte, Spickler, Zaabel, & Roth) can be regarded as a prototype virus for the “one-health” concept as it can cross species boundaries with ease; affecting swine, humans, birds, horses as well as pet animals. Despite the concerted effort and investment in influenza vaccine research, the efficacy of influenza vaccines is limited by the accurate prediction of circulating strains for each season. Immunity is short-lived and annual vaccinations are recommended. With changing travel and trade practices, influenza pandemics occur roughly once in every ten years now, compared to once in 30-50 years in the 1800's. The 2009 pandemic caused by the Influenza/A/H1N1 virus (pH1N1) was believed to be of swine origin. The virus was hence dubbed the “swine flu” (swine influenza virus—SIV). It caused widespread panic and affected the export and trade of pork products, while health care systems, worldwide, were overwhelmed.

[0063] Avian influenza (AIV) is ranked among the highest emerging infectious disease risks for both animals and humans due to the very high rates (over 50%) of mortality in humans with the highly pathogenic strains (HPAI). While human to human transmission of the avian influenza strains is not the norm historically, the emergence of a strain which is transmitted between humans is possible. Should this occur, it would likely lead to a public health nightmare. The 2014-15 AIV outbreak affected 21 states in the U.S, including Minnesota, and cost the industry $3.3 billion (McKenna, 2015). Hence, the availability of technology to develop rapid-response vaccines against influenza viruses is a significant need for both human and veterinary medicine.

[0064] This example describes a method of producing a vaccine according to the instant disclosure. More specifically, this example describes an experiment testing the efficacy of the inactuated pH1N1 vaccine in a swine challenge model.

[0065] All animal experimentation was carried out at the biosafety level 3 (BSL3) swine experimentation facilities at SDSU. Two groups of twelve, 3-4-week-old pigs were randomly assigned to the treatment and negative control groups and housed separately. Pigs were vaccinated with 10.sup.4 TCID.sub.50 of the inactuated IAV (2 ml intra-nasally and 2 ml intra-muscularly) and boosted by the same route two weeks later. Serum (to assess antibody responses) and nasal swabs (to assess viral shedding) were collected every week. To assess cell-mediated immunity, whole blood samples were collected prior to immunization and challenge with the virulent virus. Antibody responses were assessed by a standard hemagglutination inhibition (USDA) test, every week post vaccination. A third booster was administered if the HI titers were less than 1:32 at week 4. To assess vaccine safety, 4 animals from each group were euthanized prior to challenge with the live virus. It was expected that vaccination would not produce any adverse effects at the vaccination site or cause clinical signs or lung lesions characteristic of IAV.

[0066] Two approaches were used to achieve inactuation of PEDV. In the first approach, virus cultures were exposed to gamma irradiation based on the hypothesis that the viral genetic material would be destroyed while the antigenic structures would remain intact. However, it was found that the irradiation approach may destroy viral antigenic structure and interfere with immune responses. The second approach was to expose the virus to heat to relax the capsid (shell), followed by RNase treatment genome and then treatment at 4° C. to refold the capsid. RNase is an enzyme which can selectively destroy the RNA genome of the virus.

[0067] Validation of the inactuation process by electron microscopy showed intact viral structures following both treatments. An assessment of the integrity of the viral RNA genome by PCR showed that both processes resulted in the fragmentation of the genome, while detection of viral RNA within cells showed that the inactuated virus was entering host cells in a manner similar to the live virus. Vaccination of the pigs with the vaccine candidates induced strong virus neutralizing antibody responses in the group immunized with the heat+RNase treated virus, while the responses were not as strong with the irradiated vaccine. Examination of virus shedding and intestinal pathology following challenge with the virulent virus showed similar patterns, with the heat+RNase treated vaccine inducing excellent protection and no detection of virus shedding or intestinal lesions while the irradiated vaccine was less protective. The safety margin of both process with very high, with no detection of vaccine virus shedding in fecal matter at any time of the experiment and no lesions being produced in vital organs or injection sites. In conclusion, the approach of using heat+RNase treatment for the development of first response vaccines against PEDV shows strong potential for commercialization and application to other viral pathogens.

[0068] Results

[0069] Inactuation of PEDV Cultures.

[0070] Two approaches were followed to achieve optimal inactuation. Viral cultures of PEDV were re-suspended in DMEM to 10.sup.5 TCID.sub.50 viral particles/ml and subjected to either a) gamma-irradiation for 24 hours or an equivalent of 10.85 KGy, or b) 42° C. for 10 mins, followed by 2 mg/ml of RNase A and 10,000 units/ml of RNase H and incubated at 42° C. for 4 hours. The inactuated culture was then placed at 4° C. for a minimum of 3 hours for refolding. Untreated virus cultures were used as controls in both cases.

[0071] The Inactuation Process Maintains Viral Structural Integrity.

[0072] Assessment of the inactuated viral cultures by transmission electron microscopy (TEM) showed that the viral capsid structures remained intact in both the irradiation and heat/RNase treatments (FIGS. 1A-3C). An untreated viral culture was used as a control. As expected, the processes used did not damage the antigenic structures which are important for vaccine-induced immunity.

[0073] Inactuation Diminishes Viral Replication.

[0074] To test the hypothesis that the inactuation process would abrogate or diminish viral replication significantly, Vero cell monolayers were infected with the inactuated viruses and passaged serially three times. This “amplification” test is commonly used to determine whether the virus can replicate after the treatment, as the number of viral particles will increase in each passage to detectable levels. All three passages and an untreated control virus inoculum were examined by immunofluorescent microscopy to determine the extent of viral replication. No viral replication was detected in any of the three passages of the irradiated virus culture, while very slight replication (in comparison to the untreated control) was detected only in the third passage of the heat+RNase treatment (FIGS. 4A-C). Since the post-treatment viral replication was not robust, the likelihood of the vaccine virus being safe in the host was expected to be high.

[0075] Antigen Presentation of Inactuated Viral Particles is Similar to that of Natural Infection.

[0076] To ensure that the inactuation treatments did not affect the ability of the viral particle to enter cells, but damaged the integrity of the nucleic acids, the inactuated viral cultures were used to infect Vero cells. After thorough washing to remove the inocula, the cell monolayers were subject to PCR analysis to detect the presence of the virus (FIG. 5). As expected, viral cDNA bands of approximately 5 Kb and 3.5 Kb were detected by PCR, indicating the inactuated viral particles had entered the cells. Several bands of smaller sizes were also detected in the cells infected with the inactuated viruses, indicating that the treatments caused fragmentation of the parent RNA strands, as expected.

[0077] Assessment of Immune Responses and Vaccine Efficacy in Pigs.

[0078] Three groups of 8 pigs each, housed at the South Dakota State University (SDSU) swine experimentation facility, were immunized with A) Sham inoculation B) Irradiated PEDV vaccine C) heat+RNase treated PEDV vaccine. The pigs were boosted twice with the same dose at two week intervals (FIG. 6).

[0079] Antibody Responses.

[0080] Antibody responses were assessed by a commercial ELISA and virus neutralization assays at the SDSU veterinary diagnostic laboratory. Post-challenge viral loads in fecal matter were assessed by qPCR, using a commercial kit at the NDSU veterinary diagnostic lab. Intestinal tissues were examined for pathology by hematoxylin and eosin sections as well as immunohistochemistry using a PEDV specific antibody.

[0081] No significant binding antibody responses to the nucleoprotein were detected by ELISA, except at post-challenge for the Vac-I group (FIG. 7A). However, significant responses against the protective spike (S) protein were noted in the Vac-II group and post-challenge for Vac-I (FIG. 7B). Significant virus neutralization titers were detected on DPI 28, following the booster, vaccination for Vac-II, while only low level responses were detected following the second booster and prior to challenge for Vac-I. Following the challenge on DPV 43, significant incremental responses were noted for both vaccine groups (FIG. 7C). The unvaccinated animals showed no responses as expected, except for minimal responses post-challenge (FIG. 7C). The S protein pattern correlated with the virus neutralization data, indicating that Vac-II was stimulating robust antibody responses against the S protein, which in turn were able to neutralize the virus challenge effectively.

[0082] Viral Shedding.

[0083] No virus was detected by qPCR at any of the time points prior to challenge in all three groups. At 3 days post challenge all the animals in the heat+RNase treatment showed no viremia, while all animals in the control group were shedding virus in their fecal matter. Four of six animals in the irradiated vaccine group showed shedding (FIG. 8A). At 7 days, post-challenge, the trend continued, with viral shedding increasing in both the unvaccinated controls as well as animals administered the irradiated vaccine, with all 6 animals in the vac-I group turning positive (FIG. 8B). These results indicate that the heat+RNase treated vaccine induced very high levels of protection while the irradiated vaccine did not.

[0084] Vaccine-Induced Protection Against Intestinal Lesions.

[0085] Two slides with three intestinal sections (Ileum, Jejunum and Duodenum) were prepared for each pig for the blinded assessment of microscopic lesions as well as immunohistochemistry (IHC) staining to detect the accumulation of viral antigen in the intestinal cells (FIGS. 9A-C). Scores were assigned as described in Table 1. Very severe lesions were not encountered in any of the groups, including the unvaccinated animals, as the pigs were well over 3 months of age at necropsy. In agreement with the other assessed parameters, the pigs vaccinated with the RNase and heat treated vaccine showed no lesions, and only one out of six pigs had semi-formed stools at necropsy, indicating complete protection by vaccination. Compared to the unvaccinated controls, the pigs administered the irradiated vaccine had higher microscopic scores and comparable IHC scores, but very low fecal scores, indicating that while viral replication occurred on challenge, the high viral titers did not result in clinical signs.

TABLE-US-00001 TABLE 1 Lesion scores to assess vaccine-mediated protection against disease. Microscopic Immuno- Lesions* histochemistry** (No. of positive (No. of positive animals/Total animals/Total Fecal Group animals) animals) Score*** Unvaccinated 4 7 2.5 (4/6) (4/6) (5/6) Irradiated 12  8 0.5 Vaccine (4/6) (4/6) (1/6) RNase + Heat 0 0 0.5 Vaccine (0/6) (0/6) (1/6) *Mean microscopic lesion scores calculated as the number of positive sections (Ileum, jejunum, duodenum), multiplied by the atrophic enteritis score, where 0 = negative, 1 = mild, 2 = moderate, 3 = severe. **Mean immunohistochemistry (IHC) score calculated as the number of positive sections (Ileum, jejunum, duodenum), multiplied by the IHC score, where 0 = negative, 1 = positive, ≤10%, 2 = positive, 11-50%, 3 = positive, >50%. ***Fecal score at necropsy-Formed Feces = 0, Semi-formed feces = 3.

[0086] Vaccine Safety.

[0087] Two pigs from each group were sacrificed prior to challenge to examine whether the vaccines caused any side effects or lead to shedding of the vaccine virus in feces. No virus was detected in the fecal matter at necropsy or after the primary vaccination and boosters, indicating that both vaccines viruses were not amplified or shed in vaccinated animals. Similarly, no microscopic or IHC lesions were detected in these pigs in intestinal, heart, spleen and lung tissues. Therefore, both vaccine development approaches were safe and induced no side effects.

[0088] Conclusions.

[0089] Of the two approaches used to develop rapid-response first generation vaccines for emerging RNA viruses, the vaccine developed using heat+RNase treatment induced complete protection against challenge with a virulent virus, indicating that this was an excellent and safe process. The pigs vaccinated with the irradiated virus appeared to experience viral replication and developed lesions which were comparable to the unvaccinated controls. However, they did not show clinical signs, indicating that the mechanisms of protection by the two vaccines were likely to be different. The heat+RNase treatment approach was very effective for the inactuation of PEDV and the development of the first response vaccine.

Example 2

[0090] Other RNA Viruses.

[0091] Although the PEDV virus used in EXAMPLE 1 and IAV are both RNA viruses, the RNA genome organization in PEDV and IAV is very different. The PEDV genome consists of a 12-15 Kb long, positive sense, single strand of RNA. On the other hand, the influenza viral genome consists of 8 short segments of negative-sense RNA, wound around viral non-structural proteins (FIGS. 10A-B).

[0092] Mammalian genomes are closely associated with proteins called histones. The histone:DNA association shields the genetic material of the mammalian genome. It was recently found that the influenza NS1 protein, which is closely associated with the viral genome, mimics human histones. Therefore, since very little of the IAV RNA is exposed to digestion by enzymes, it was expected that the inactuation protocol, as used for PEDV, would not be successful with the IAV's. However, similar to the histone:DNA association which can be relaxed through chemical modifications such as acetylation to expose the genetic material, the histone-like motifs found in NS1 can be methylated and acetylated. Accordingly, to inactuate the influenza virus, following heat treatment the virus is first exposed to the histone acetylase enzyme to disrupt the RNA:protein interaction, and then the RNase treatment.

[0093] Briefly, the pH1N1 virus was cultured in MDCK cells by standard methods. The virus culture was suspended to 10.sup.4 TCID.sub.50/ml. The inactuation treatment was optimized using a series of time points ranging from 1 hour to 12 hours, after exposure to heat at 44° C. for 10 minutes. Assessment of inactuation was carried out as described in EXAMPLE 1, by electron microscopy, PCR and serial passaging of the inactuated virus culture. As an alternative to the heat+enzyme treatment, the gamma-irradiation method may be used to inactuate the virus. Without wishing to be bound by theory, it is expected that the irradiated vaccine will stimulate stronger cell-mediate immunity than antibody-based immunity. Additionally, to improve responses to both arms of the immune response, an adjuvant, such as the Montanide Seppic adjuvant, may be used to improve antibody mediated immunity.

Example 3

[0094] The porcine pseudorabies virus (PRV), a herpes virus, is used as a model for large DNA viruses. The general approach is to expose the DNA virus to heat to relax the capsid, followed by DNA degrading enzyme treatment genome and then treatment at 4° C. to refold the capsid. DNase and Benzonase are both enzymes which can selectively destroy the DNA genome of the virus—collectively, DNA degrading enzymes. Large DNA viruses have several layers of lipid and lipoprotein that envelop and enclose the core nucleocapsid protein. Due to the additional layers of large DNA viruses, the duration of DNA degrading enzyme treatment is optimized by a time point assay. For example, the heat/DNase treatment may include exposure to the DNase treatment of approximately 8-15 hours at a temperature of at least 37° C.

[0095] Following partial degradation of the viral DNA genome by the heat+DNA degrading enzyme treatment, the treated DNA viruses are used to infect susceptible mammalian cells in culture and passaged until replication of the “rescued” virus is detectable. These treated and inactuated cultures are subjected to plaque purification techniques that isolate individual viral clones; the individual viral clones are then submitted for next-generation sequencing. Mutations acquired due to DNA damage repair occurring during replication of the treated virus in cells result in an attenuated phenotype.

[0096] This example describes a method of producing a vaccine according to the instant disclosure. More specifically, this example describes an experiment testing the efficacy of the attenuated PRV in a swine challenge model.

[0097] Results

[0098] Inactuation of PRV Cultures.

[0099] Porcine pseudorabies virus (PRV) cultures were reconstituted to 10.sup.5TCID.sub.50. The PRV culture was incubated at 54° C. and 58° C. for 20 mins to effect reversible denaturation. Subsequently, digestion of DNA was carried out with either 40 units of DNase I or 50 units of Benzonase nuclease by incubating the culture with the enzymes at 42° C. for 5 or 6 hours. The heat and enzyme treated cultures were refolded at 25° C. for 1 hour and then at 4° C. for 2 hours and layered on porcine kidney (PK-15) cells. Untreated virus cultures were used as controls in both cases.

[0100] Inactuation Diminishes Viral Replication.

[0101] To test the hypothesis that the inactuation process would abrogate or diminish viral replication significantly, cell monolayers were infected with the inactuated viruses and passaged serially three times. This “amplification” test is commonly used to determine whether the virus can replicate after the treatment, as the number of viral particles will increase in each passage to detectable levels. Cell monolayers were observed for the presence of viral replication, manifested as cytopathic effects consisting of rounded and bunched up cells (CPE). The infected cells were sub-cultured three (3) times to rescue viral particles which had undergone DNA damage and to repair replication within cells. Cell sheets were stained with a PRV-specific fluorescent antibody to verify CPE and characteristic cytoplasmic viral replication. No viral replication was detected in the first passage for cell cultures unfolded at 54° C. and 58° C. and exposed to DNase for 5 or 6 hours. CPE was detected in both treatments in the second (2.sup.nd) passage. Thus, time of exposure was increased to six (6) hours to increase stringency of DNase. When exposure time of DNase was increased to six (6) hours, no viral replication via CPE was detected; therefore, an inactuated virus may be produced between exposure time to DNase between five (5) and six (6) hours. Cultures treated with Benzonase nuclease exhibited detectable CPE at temperatures of both 54° C. and 58° C. for exposure times of five (5) hours. For exposure times of six (6) hours, Benzonase nuclease treatment exhibited viral replication via CPE for the second (2.sup.nd) passage. It is contemplated that, based upon the results of passages 1-3 for both the Heat/DNase and Heat/Benzonase treatment, that inactuation via exposure to the Heat/DNase treatment will continue to evidence significantly reduced viral replication and that inactuation via exposure to heat/Benzonase treatment will yield reduced viral replication. Thus, inactuation via exposure to DNA degrading enzyme Benzonase nuclease requires exposure time greater than six (6) hours and/or heat treatment greater than 58° C. CPE viral replication was also recorded as described in Table 1.

TABLE-US-00002 Treatment Heat/DNase Heat/Benzonase Reversible 54° C. 58° C. 54° C. 58° C. 54° C. 58° C. 54° C. 58° C. Denatu- (5h) (5h) (6h) (6h) (5h) (5h) (6h) (6h) ration CPE CPE CPE CPE CPE CPE CPE CPE Passage 1 — — — — +++ +++ — — Passage 2 +++ +++ — — Abandoned Abandoned +++ +++ Passage 3 Abandoned Abandoned — — Abandoned Abandoned Passage 4 Under Under test test

[0102] In addition, FIGS. 11A-11I show the effectiveness of the heat+DNase treatment, especially the reduction in viral replication when the treatment includes exposure to temperature of 58° C. for six (6) hours. With reference to the specific gridded representative images of cells infected with heat+DNase treated virus, (A) shows a Brightfield image of PRV uninfected cells as the control, (B) shows stained uninfected cells as the control, and (C) shows a DAPI overlay of stained uninfected cells as the control with blue nuclei. FIG. 11D shows a Brightfield image of PRV cells after Heat/DNase treatment at 58° C. for five (5) hours with the groupings of rounded and bunched up cells constituting cytopathic effects (CPE) as a result of viral replication. Grid panel (E) further shows CPE with the characteristic green staining of PRV cells after Heat/DNase treatment at 58° C. for five (5) hours, and panel (F) shows the same cells with DAPI overlay that indicates cytoplasmic green viral florescence—cytoplasmic viral replication in the treated cells. Lastly, FIG. 11G shows a Brightfield image of PRV cells after Heat/DNase treatment at 58° C. for six (6) hours without any groupings of rounded and bunched up cells. The results of the Heat/DNase treatment at 58° C. for six (6) hours are further evidence in panels (H) and (F) that show reduced green fluorescence and grouping of cells—low viral replication—and no green fluorescence in the DAPI overlay—indicating no cytoplasmic viral replication.

[0103] Conclusion.

[0104] The vaccine developed using heat+DNase treatment indicated protection against challenge with a virulent virus, indicating inactuated of PRV and development of a first response vaccine.

Example 4

[0105] Other DNA Viruses.

[0106] Although the PRV virus used in EXAMPLE 3 is similar in many respects to other DNA viruses, the DNA genomic organization may vary among different viruses. For example, the PRV virus is a large enveloped virus containing a linear double-stranded DNA genome approximately 120-150 kpb in length (FIG. 12) (Díaz C, Celer V, Frébort I. The Main DNA Viruses Significantly Affecting Pig Livestock. J Vet Res. 2020; 65(1):15-25. Published 2020 Dec. 29. doi:10.2478/jvetres-2021-0001). As has been found, some large DNA viruses utilize DNA-packing proteins, such as histones, to wrap the viral genetic material and that may affect infectivity.

[0107] Mammalian genomes are closely associated with proteins called histones. The histone:DNA association shields the genetic material of the mammalian genome. As introduced above, some DNA viruses, such as pathogenic polyomaviruses, wind their viral DNA around proteins, called histones, to form nucleosomic structures (FIG. 13). Similar to the histone:DNA association which can be relaxed through chemical modifications such as acetylation to expose the genetic material, the histone-like motifs found in large DNA viruses can be methylated and/or acetylated. Accordingly, to inactuate some DNA viruses with specifically-packed viral DNA, following heat treatment, the virus is exposed to a histone acetylase enzyme to disrupt the DNA:viral protein interaction, and then the DNase or Benzonase treatment.

[0108] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:

REFERENCES

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[0125] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.