Irradiation-inactivated poliovirus, compositions containing same, and methods of preparation

Abstract

Compositions useful as poliovirus immunogens are provided, along with methods and compositions for their preparation. Compositions containing poliovirus immunogens can elicit a host response, including virus-neutralizing antibodies, that can protect the host from infection and / or disease. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] [Priority Statement] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 650,406, filed March 30, 2018, the entire contents of which are incorporated herein by reference.

[0002] [Statement of Government Support] This invention was made with government support under Grant Nos. 2R44AI120260-02 and 1R43AI120260-01 awarded by the National Institutes of Health, and HDTRA1-17-C-0030 and HDTRA1-15-P-0034 awarded by the Department of Defense. The government has certain rights in this invention.

[0003] [Statement regarding electronic filing of sequence listings] A Sequence Listing in ASCII text format filed under 37 CFR §1.821, entitled 1472-2WO_ST25.txt, 893 bytes in size, created on March 27, 2019, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated herein by reference for its disclosure.

[0004] [Field] The present invention relates to compositions comprising poliovirus immunogens inactivated by irradiation, as well as methods and compositions for their preparation. [Background technology]

[0005] [background] Chemically inactivated and live-attenuated polio vaccines have been effective in stimulating protective immunity and eradicating polio from most countries worldwide. The first polio vaccine, inactivated polio vaccine (IPV), developed by Dr. Jonas Salk, consisted of formalin-inactivated poliovirus types 1, 2, and 3 and was first used in 1955. The second polio vaccine, oral polio vaccine (OPV), developed by Dr. Albert Sabin, consisted of pathogenically attenuated live poliovirus types 1, 2, and 3. OPV has become the primary vaccine because of its lower cost compared to IPV and is widely used in vaccination campaigns aimed at achieving global eradication.

[0006] Both OPV and IPV have weaknesses that ultimately render them obsolete. After inoculation with OPV, the virus replicates in the intestine and stimulates strong immunity. However, during this replication process, the three viral components may lose their attenuated phenotype, allowing neuropathogenic progeny viruses to be excreted. With the decline in polio infections caused by natural wild-type strains, the relative incidence of disease and infection caused by vaccine-derived polioviruses (VDPVs) has become more significant. Because wild poliovirus type 2 has now been eradicated, the World Health Organization (WHO) recommends the use of bivalent OPVs composed of attenuated types 1 and 3. While the number of infections caused by VDPVs is relatively low, the numbers are prohibitive for the completion of global eradication. For this reason, most countries are transitioning to the use of IPV instead of OPV.

[0007] IPV is produced by large-scale production of wild-type, neuropathogenic strains of PV1, PV2, and PV3. The purified virus is concentrated and inactivated by incubation with formalin for 2–4 weeks. IPV stimulates strong systemic immunity. After global eradication, the WHO and other global health authorities intend to continue vaccination for 10 years or more to ensure that immune defenses in the general population remain strong enough to counter potential exposure to residual poliovirus in the environment. At the end of this approximately 10-year post-eradication vaccination period, many countries are expected to phase out polio vaccination. As global immunity wanes after eradication, the continued use of large quantities of neuropathogenic strains creates an increasingly significant biohazard.

[0008] Attempts to replace neuropathogenic strains in IPV with attenuated (Sabin) strains have had mixed results. Most notably, formalin treatment inactivates a major neutralizing epitope within the VP1 capsid protein of type 1 Sabin virus. Some countries have licensed IPV vaccines consisting of formalin-inactivated Sabin strains, but the efficacy of the type 1 component is unknown.

[0009] Vaccination should be the most effective defense against poliovirus infection and disease. Summary of the Invention [Means for solving the problem]

[0010] The present invention relates, in part, to poliovirus antigens based on attenuated and / or neuropathogenic strains of poliovirus that have been inactivated by exposure to ionizing radiation (e.g., gamma and / or x-ray radiation) and / or ultraviolet radiation (e.g., ultraviolet C (UVC) radiation having a wavelength of about 100 to about 280 nm). The antigenic compositions of the invention may comprise one, two, three, or more poliovirus strains. The antigenic compositions may serve as improved vaccines for stimulating protective immunity when introduced into a subject by injection or other delivery systems, such as those known to those skilled in the art.

[0011] The methods of the present invention may use antioxidants and / or antioxidant compositions to protect antigenic epitopes on the surface of the virus while subjecting nucleic acid within the virus to damage and / or destruction by radiation (e.g., ionizing radiation and / or UV radiation).

[0012] The antioxidant compositions of the present invention may comprise a divalent cation, a peptide, and a buffer system. In some embodiments, the antioxidant composition comprises manganese chloride (MnCl), a decapeptide (e.g., DEHGTAVMLK [SEQ ID NO: 1]), and a phosphate buffer (e.g., potassium phosphate buffer). Other buffers, such as TRIS and MES, can be substituted for the phosphate buffer.

[0013] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated into a different embodiment even if not specifically described therein. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. Applicant reserves the right to modify any originally filed claims and / or file any new claims as appropriate, including the right to amend any originally filed claims to depend on and / or incorporate any feature of any other claim(s), even if not originally claimed in that manner. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Additional features, advantages, and details of the invention will be apparent to those skilled in the art from a reading of the following drawings and detailed description of the preferred embodiments, such description being merely illustrative of the invention. [Brief explanation of the drawings]

[0014] [Figure 1] Graphs showing the reduction in infectivity of gamma-irradiated poliovirus (PV). PV2 Sabin (16 micrograms) combined with manganese-decapeptide-phosphate (MDP) complex was exposed to increasing doses of gamma irradiation and tested for infectivity using the CCID50 assay. The larger graph reports Log-10 values, while the inset graph reports non-log titers, showing no infectivity at 45 and 50 kilograys (kGy) of ionizing radiation. [Figures 2A-2C]Images of different analyses of irradiated PVs are shown. In Figures 2A and 2B, PV2S was γ-irradiated with and without decapeptide (Dp) or MnCl2 (Mn2+) as indicated. Aliquots were analyzed by Western blot, showing that the addition of Dp and Mn2+ protects the VP2 capsid protein from damage (Figure 2A). Similar results were obtained in blots where VP1 and VP3 were visualized. Analysis of intact viral RNA by reverse transcriptase polymerase chain reaction (RT-PCR) shows that the MDP complex does not protect the RNA from damage (Figure 2B). The blot shows that the addition of Dp and Mn2+ protects VP2 from damage. The PCR assay shows that the addition of Dp or Mn2+ does not protect the RNA from damage. Figure 2C shows electron micrographs of PV1S before irradiation (top), γ-irradiated without Dp or Mn2+ (middle), and γ-irradiated with Dp and Mn2+ (bottom). 91.8% of the unirradiated particles appear dense. After γ-irradiation, 90.0 and 2.0% were dense in samples with and without MDP, respectively (determined by counting >700 particles). [Figure 3] Log-2 anti-PV2 neutralization titers are shown for rats immunized with 1x, 0.5x, or reduced human doses of IPOL (circles), VeroPol (squares), or Ir-PV2S (triangles). Ir-IPVS irradiated without MDP conjugates was included as a control (star). Rats were immunized on days 1 and 21 and bled for test sera on days 35 (panels A, C, and E) and 49 (panels B, D, and F). Panels A and B show serum from a 1x human dose containing 8 DU of PV2 antigen. Panels C and D show serum from a 0.5x human dose containing 4 DU of PV2 antigen. Panels E and F show serum from reduced doses of PV2 antigen containing 1 / 8, 1 / 16, 1 / 32, or 1 / 64 of the human dose (containing 1, 0.5, 0.25, or 0.125 DU of PV2 antigen, respectively). [Figure 4]Graphs of anti-PV1 neutralization titers from rats immunized with irradiated 1x (panels A, B) or 0.5x (panels C, D) human doses of Ir-PV1 and the licensed IPV vaccines, IPOL (Sanofi) and VeroPol (Staten Serum Institute). Neutralization data are presented as Log-2 values ​​from immunization with IPOL (circles), IPV (squares), Ir-PV1 with MDP (triangles), or irradiated Ir-PV1 without MDP (stars). Immunizations were performed on days 1 and 21, and test bleeds for neutralization assays were performed on days 35 and 49, as indicated. Horizontal lines indicate the mean neutralization value for each group. BD = below the limit of detection (<1). [Figure 5] Anti-PV1 neutralization titers from rats immunized with UVC-inactivated PV1 Sabin are shown. PV1 Sabin virus was formulated with (upper panel) or without (lower panel) MDP conjugates and irradiated for 60 minutes with UVC light emitting 0.7 mW / cm2. Two inactivated virus preparations were analyzed for D antigen concentration, and an amount equivalent to one human dose was used to immunize rats. Rats were immunized on days 0 and 21, and sera were collected on day 49. Neutralization titers for each rat serum are shown, along with the Log-2 titer and group mean. Sera marked with an asterisk showed no neutralization at a 1:2 dilution. [Figure 6]Figure 5 shows the anti-PV1 neutralizing titers of sera from rats immunized with UVC-inactivated PV1 Sabin. Log-2 titers for each serum sample shown in Figure 5 are graphed, with the mean value shown as a horizontal line and error bars included. PV1 Sabin was formulated with or without MDP and inactivated for infectivity by exposure to UVC light emitting 0.7 mW / cm2 for 60 minutes. Rats were immunized on days 0 and 21 and test bled on day 49 for serum containing 40 D antigenic doses of inactivated virus (1x human dose). Serum was analyzed for neutralizing antibody activity in a standard TCID50 assay. Log-2 neutralizing titers are shown for each rat serum in two groups: with (solid symbols) or without (open symbols) MDP. [Figure 7] Analysis of UVC-irradiated PV3 Sabin virus is shown. The left panel shows infectivity. PV3 Sabin, complexed with MDP (squares) or not (triangles), was exposed to UVC light emitting 0.7 mW / cm2 for 0, 3, 10, or 30 minutes. Samples were analyzed in an infectivity assay using MRC5 cells, and Log-10 TCID50 titer values ​​were graphed. The right panel shows D antigen analysis of the same samples. Values ​​for the amount of D antigen per milliliter volume are shown, along with % residual values. [Figure 8] Anti-PV3 neutralizing titers from rats immunized with UVC-inactivated PV3 Sabin on days 0 and 21 and bled for serum on day 49 are shown. PV3 Sabin virus was formulated with (upper panel) or without (lower panel) MDP and irradiated for 60 minutes with UVC light emitting 0.7 mW / cm2. The two inactivated virus preparations were analyzed for D antigen concentration, and rats were immunized with 2x, 1x, 1 / 2x, and 1 / 8x the human dose equivalent (64, 32, 16, and 8 D antigen doses). Rats were boosted 21 days after the initial immunization, and serum samples were collected 28 days later. The neutralizing titers for each rat serum are shown, along with the Log-2 titer and group mean. [Figure 9]Neutralizing titers of sera from rats immunized with UVC-inactivated PV3 Sabin are shown. PV3 Sabin was formulated with or without MDP and inactivated by 60-minute exposure to UVC light emitting 0.7 mW / cm². Rats were immunized on day 0 and boosted on day 21 with 64, 32, 16, or 4D antigen doses (corresponding to 2x (circles), 1x (squares), 0.5x (triangles), and 0.125x (diamonds) doses, respectively). Log-2 values ​​of anti-PV3 neutralizing titers from sera for each animal within a group are shown. Data from animals immunized with virus formulated with MDP before UVC inactivation are shown as solid symbols, while sera from animals immunized with virus formulated without MDP are shown as open symbols. The data suggest that the inclusion of MDP reduces damage to neutralizing epitopes on the virus, an effect that is more pronounced at lower antigen doses. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0016] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0017] Unless otherwise specified, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflicts in terminology, the present specification controls.

[0018] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0019] Unless the context dictates otherwise, it is expressly intended that the various features of the invention described herein may be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. By way of example, if the specification states that a composite comprises components A, B, and C, it is expressly intended that any of A, B, or C, or any combination thereof, may be omitted and rejected.

[0020] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be understood to encompass the recited materials or steps "and those which do not materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising."

[0021] The term "about" as used herein when referring to a measurable value, such as an amount or concentration, means to encompass a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the particular value. For example, "about X," where X is a measurable value, means to encompass X and a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0022] As used herein, "pharmaceutically acceptable" means that the compound, anion, cation, or composition is suitable for administration to a subject to accomplish the treatment described herein without undue adverse side effects, given the severity of the disease and the need for treatment.

[0023] As used herein, the terms "increase," "increases," "increased," "increasing," "improve," "enhance," and similar terms refer to at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more increase in a specified parameter.

[0024] As used herein, the terms "reduce," "reduces," "reduced," "reduction," "inhibit," and similar terms refer to at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% reduction in a specified parameter.

[0025] As used herein, the term "sequence identity" has its standard meaning in the art. As is known in the art, several different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the BestFit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using default settings, or by searching

[0026] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151 (1989).

[0027] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program obtained by Altschul et al., Meth. Enzymol., 266:460 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are preferably set to default values. The parameters are dynamic and are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is being searched; however, values ​​can be adjusted to increase sensitivity.

[0028] Another useful algorithm is Gapped BLAST, as reported by Altschul et al., Nucleic Acids Res. 25:3389 (1997).

[0029] Percentage amino acid sequence identity values ​​are determined by dividing the number of matching identical residues by the total number of residues in the "longer" sequence within the aligned region, the "longer" sequence being the one having the most actual residues within the aligned region (ignoring gaps introduced by WU-Blast-2 to maximize the alignment score).

[0030] Alignment may include the introduction of gaps in the sequences being aligned. Furthermore, with respect to sequences that contain more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity is determined based on the number of identical nucleotides relative to the total number of nucleotides. Thus, for example, the sequence identity of a sequence shorter than the sequences specifically disclosed herein is, in one embodiment, determined using the number of nucleotides of the shorter sequence. In calculating the percentage identity, relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0031] In one embodiment, only identities are scored positively (+1) and all forms of sequence variation, including gaps, are assigned a value of "0," thereby eliminating the need for weighted scales or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one with the most actual residues in the aligned region.

[0032] "Immunogen" and "antigen" are used interchangeably herein to refer to a molecule that elicits a specific humoral and / or cell-mediated immune response, e.g., an immune response in which antibodies are stimulated to bind to the molecule or virus. The binding site for an antibody on an antigen and / or immunogen can be referred to as an epitope (e.g., an antigenic epitope).

[0033] As used herein, "D antigen content" refers to the combined activity of multiple immunostimulatory poliovirus epitopes. Early studies of poliovirus pathogenesis described two distinct antigenic forms of poliovirus, classified as noninfectious empty particles and / or low-infectivity viruses containing "C" antigens, and infectious viruses containing "D" antigens (which can be inactivated and converted to C antigens by heating at 56°C). D antigen assays, e.g., ELISA assays involving antibodies reactive to D antigen content (e.g., infectious virus epitope(s)), are a measure of antigenicity and have been used to predict the immunogenicity of vaccine formulations, where higher D antigen content correlates with higher immunogenicity.

[0034] A vaccine is an immunogen used to generate an immunoprotective response, e.g., by priming the immune system, such that upon further exposure to an antigen (e.g., an immunogen and / or antigen of an infectious agent, e.g., an infectious virus), the immune response is more protective for a host (e.g., a vaccine recipient, e.g., a subject) than the immune response upon exposure to the antigen without prior vaccination. For example, induced antibodies are provided by the vaccine that reduce the negative effects in the host of immunogens found on infectious viruses or agents expressing them. Vaccine dosages can be obtained, estimated, and / or determined by preclinical and clinical trials, as known to those skilled in the art. Multiple doses of the vaccine can be administered as known in the art and / or as needed to ensure long-term protection and / or a memory state (e.g., a primed state). In some embodiments, a successful endpoint of a vaccine's usefulness for purposes of the present invention is the occurrence of an induced immune response (e.g., humoral and / or cell-mediated) that results, for example, in the production of a host-produced serum antibody(ies) that recognizes the intended antigen. Such antibodies can be measured by various assays, as known in the art, such as, for example, antiviral neutralization assays of serum sampled from animals or humans immunized with the vaccine and / or immunogen.

[0035] As used herein, the terms "antigen sparing" and / or "dose sparing" refer to the effect that the amount of immunogen / antigen used in a vaccine and / or the amount of vaccine provided can be reduced without negatively affecting the protective nature of the immunoprotective response induced in the vaccine recipient, e.g., without affecting the efficacy, quality of the immunoprotection induced in the vaccine recipient, and / or protection from disease.

[0036] Poliovirus is a non-enveloped virus containing positive-sense genomic RNA and exists as three antigenically distinct serotypes: types 1, 2, and 3 (i.e., PV1, PV2, and PV3). Poliovirus is a member of the Enterovirus genus in the family Picornaviridae and replicates within the gastrointestinal tract. At relatively low frequency, the virus infects neurological tissue, where it can cause temporary paralysis, permanent paralysis, or death. Because poliovirus is less hydrated than enveloped viruses such as influenza and Venezuelan equine encephalitis virus (VEEV), higher doses of ionizing radiation are typically required to inactivate infectivity.

[0037] The two common types of poliovirus vaccines used since their approval in the 1950s are inactivated polio vaccine (IPV) and oral attenuated (non-inactivated) live polio vaccine (OPV). IPV, originally developed by Jonas Salk and colleagues, is produced by growing and purifying neurovirulent strains of types 1, 2, and 3 polioviruses, followed by chemical inactivation of infectivity with formaldehyde and subsequent purification. IPV is typically administered intramuscularly without an adjuvant to stimulate systemic immunity and protect the host from neuropathogenic disease. OPV is produced by growing and purifying strains of three serotypes that are attenuated for pathogenesis. The attenuated strains were originally developed by Albert Sabin and colleagues and are referred to as "Sabin" strains. As an orally administered attenuated live virus vaccine, OPV replicates in the intestine to stimulate both gastrointestinal and systemic immunity against infection and / or disease. Both IPV and OPV are used to control poliovirus infection and the incidence of polio, and the vaccines have contributed to the eradication of polio in all but a few countries.

[0038] Between 2005 and 2015, it is estimated that over 10 billion doses of OPV were administered to more than 2.5 billion children, preventing over 10 million cases of polio. In some cases, the OPV vaccine loses its disease-attenuating phenotype during replication in the gastrointestinal tract, resulting in excretion of neuropathogenic virus in feces. Progeny virus can infect inadequately vaccinated bystanders exposed to infected feces. Furthermore, improperly treated sewage can result in contamination of water bodies, which can also lead to infection of inadequately vaccinated individuals. During past years, when thousands, if not hundreds of thousands, of individuals developed polio disease through contact with wild poliovirus strains, the relatively low rate of OPV atavism posed a less significant population health hazard than the risk of natural infection. In contrast, once eradication is complete or nearly complete in defined geographic regions and ultimately in the global population, disease outbreaks due to vaccine revertants will become more significant and problematic. In 2017, 22 cases of polio were reported, down from approximately 350,000 in 1988. Because there are approximately 200 asymptomatic cases for every case of paralytic polio, the incidence of virus circulating in the population is higher than reported. In countries where the three polio serotypes have been eradicated for many years, OPV has been replaced by IPV to prevent cases of vaccine-associated infection and disease.

[0039] IPV also has drawbacks. Vaccines are produced using neuropathogenic strains. The production and handling of large quantities of neuropathogenic strains requires a high level of biological containment to prevent accidental release of the virus from production facilities. Published reports have documented such accidental release of the virus into the surrounding environment at more than one production facility. The World Health Organization has documented that PV2 has been eradicated, with no cases of infection or disease caused by wild-type PV2 virus reported in recent years. No cases of PV3 infection have been reported since 2012, and the World Health Organization expects that PV1 will be eradicated in the near future. Upon eradication, the use of large quantities of neuropathogenic strains will increasingly pose a biohazard risk. After eradication is achieved and mass vaccination against poliovirus begins to cease in many countries, the accidental or intentional release of neuropathogenic poliovirus could undermine the enormous efforts being made to eradicate the three viruses. For this reason, replacing neuropathogenic strains with attenuated Sabin strains reduces the biohazards associated with IPV production. Published reports demonstrate that a major epitope within the VP1 capsid protein of PV1-Sabin is damaged by formaldehyde treatment. Although some countries have licensed the use of formaldehyde-inactivated Sabin strains as IPV vaccines, concerns remain regarding their ability to induce long-lasting protective immunity.

[0040] The efficacy of poliovirus vaccines or immunogens can be measured by analyzing neutralizing antibody activity in immunized humans or selected test animals, where higher quantification of neutralizing antibody (e.g., IgA, IgM, and / or IgG, e.g., IgG1, IgG2, IgG3, and / or IgG4) activity in immunized subjects (e.g., humans and / or test animals) correlates with improved immune protective responses upon subsequent challenge with wild-type poliovirus. In neutralization assays, diluted serum samples are mixed with standardized amounts of PV1, -2, and / or -3 to allow for the formation of antibody-virus complexes. The mixture is then applied to a monolayer of mammalian cells, such as HeLa, Vero, and / or MRC5 cell lines, allowing infectious virus to attach to the cells. Typically, assays are performed in tissue culture plates with multiple wells (48, 96, or 386 wells per plate), with multiple well replicates for each serum-virus test. The inoculum is washed with standard buffer, growth medium is added to the cells, and the culture plate is incubated at approximately 35°C to approximately 37°C to allow for viral growth and multiple replication cycles. Wells are scored as infected or uninfected after 3–6 days to determine whether the serum inhibited viral infection of the monolayer (e.g., via neutralizing antibodies in the serum). The virus neutralization titer is expressed as the reciprocal of the serum dilution that results in a 50% reduction in infected wells among replicate wells. For example, dilutions of 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12 that result in a 50% reduction in infected wells correspond to virus neutralization titers of 2, 4, 6, 8, 10, and 12, respectively, with higher neutralization titers being more effective in providing protection. A virus neutralization titer of 8 corresponds to a 1:8 dilution of serum and is generally accepted as equivalent to protection in humans. A titer of 8 is equivalent to a Log-2 titer of 3.Thus, a vaccine containing a reduced mass of antigen is considered "antigen-sparing" if it stimulates equivalent or higher neutralization titers compared to a reference vaccine (e.g., a current commercially available (e.g., formalin / formaldehyde) inactivated poliovirus immunogen, e.g., IPV).

[0041] Immunization of Wistar rats is a common test animal model for predicting vaccine efficacy. Vaccine antigens that stimulate neutralizing titers of 8 in the vast majority of immunized rats are equivalent to protection in humans.

[0042] According to an embodiment of the present invention, an irradiation-inactivated poliovirus immunogen is provided. The irradiation-inactivated antigen of the present invention can stimulate neutralizing antibodies in a standard Wistar rat model, which closely correlates with protective immunity in immunized humans. The irradiation-inactivated poliovirus immunogen of the present invention can stimulate protective immunity in humans (e.g., humans immunized with the immunogen).

[0043] Compositions of the invention may comprise one or more different, irradiation-inactivated poliovirus immunogens, such as one or more (e.g., 1, 2, 3, 4, 5, or more) attenuated and / or neuropathogenic Sabin strain(s). In some embodiments, compositions of the invention may comprise one or more attenuated S19 strain(s). Compositions of the invention may comprise a pharmaceutically acceptable carrier. In some embodiments, compositions of the invention are vaccines. In some embodiments, inactivated trivalent whole virus compositions are provided that can replace currently licensed OPV and IPV vaccines.

[0044] The irradiation-inactivated poliovirus immunogens of the present invention can stimulate neutralizing antibodies in a subject (e.g., an immunized human) and can have a virus neutralization titer of about 8 or more (e.g., about 8, 10, 25, 50, 80, 100, 300, 500, 800, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 14,000, 16,000, 20,000 or more, and / or any value or range therein). For example, in some embodiments, the irradiation-inactivated poliovirus immunogens of the present invention can stimulate neutralizing antibodies in a subject with a virus neutralizing titer of about 8 to about 20,000; about 100 to about 15,000; about 1,000 to about 10,000, or about 15,000 to about 20,000.

[0045] According to some embodiments of the present invention, methods for producing inactivated poliovirus vaccines are provided, which may include one or more (e.g., 1, 2, 3, 4, 5, or more) attenuated and / or neuropathogenic Sabin strain(s). Instead of using formaldehyde, which can damage neutralizing epitopes and require lengthy incubation periods of two weeks or more, the methods of the present invention may include inactivating viral infectivity using ionizing radiation (e.g., γ) and / or ultraviolet (e.g., UVC) irradiation. In some embodiments, the ionizing radiation irradiation may be γ irradiation. In some embodiments, the ultraviolet irradiation may be ultraviolet C (UVC) irradiation.

[0046] In some embodiments, the methods of the invention include providing an antioxidant composition (e.g., a peptide-containing composition, e.g., a manganese-decapeptide-phosphate (MDP) composition) that includes a complex capable of protecting poliovirus epitopes during irradiation (e.g., supralethal irradiation). The antioxidant compositions of the invention include a divalent cation (e.g., Mn 2+), a peptide, and a buffer system. In some embodiments, the antioxidant composition comprises manganese chloride (MnCl), a decapeptide, and a phosphate buffer. In some embodiments, the antioxidant composition comprises manganese chloride (MnCl), a decapeptide, and a Tris buffer. In some embodiments, the antioxidant composition comprises manganese chloride (MnCl), a decapeptide, and an MES buffer. In some embodiments, the antioxidant composition of the present invention comprises a manganese-decapeptide-phosphate (MDP) complex.

[0047] The methods of the present invention may use antioxidants and / or antioxidant compositions to protect antigenic epitopes on the surface of the virus while subjecting nucleic acid within the virus to damage and / or destruction by ionizing radiation (e.g., gamma irradiation) and / or UV radiation (e.g., UVC radiation).

[0048] The peptides of the present invention may comprise two or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more), where the peptide may comprise two or more amino acid residues from the sequence DEHGTAVMLK (SEQ ID NO: 1) in any order and / or length. The exact sequence and / or length of the peptide may vary, and the peptide may contribute to antioxidant activity and / or function as an antioxidant in the compositions of the present invention. For example, in some embodiments, the peptide may be a quadripeptide (4-mer), pentapeptide (5-mer), hexapeptide (6-mer), heptapeptide (7-mer), octapeptide (8-mer), nonapeptide (9-mer), and / or decapeptide (10-mer). In some embodiments, a peptide (e.g., a decapeptide) of the invention may comprise the amino acids DEHGTAVMLK (SEQ ID NO: 1) in any order and / or length, for example, the peptide may comprise the sequence of amino acids HMLK (SEQ ID NO: 2), a scrambled sequence of amino acids HMLK (SEQ ID NO: 2), the sequence of amino acids HMHMHM (SEQ ID NO: 3), a scrambled sequence of amino acids HMHMHM (SEQ ID NO: 3), the sequence of amino acids DEHGTAVMLK (SEQ ID NO: 1), and / or a scrambled sequence of amino acids DEHGTAVMLK (SEQ ID NO: 1). In some embodiments, the peptide may comprise an amino acid sequence having at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence DEHGTAVMLK (SEQ ID NO: 1). In some embodiments, the peptide may comprise an amino acid sequence having at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence HMLK (SEQ ID NO: 2).In some embodiments, the peptide may comprise an amino acid sequence having at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence HMHMHM (SEQ ID NO: 3).

[0049] In some embodiments, the antioxidant compositions of the present invention may contain a peptide at a concentration of about 0.5 mM to about 10 mM, e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mM, or any value or range therein. For example, in some embodiments, the antioxidant compositions of the present invention may contain a peptide in an amount of about 2.5 mM to about 5 mM, about 0.7 to about 3.7 mM, or about 2 mM to about 8 mM. In some embodiments, the antioxidant compositions of the present invention may contain a peptide in an amount of about 3 mM, e.g., about 3 mM of a decapeptide.

[0050] In some embodiments, the antioxidant compositions of the present invention contain, for example, manganese, Mn 2+ In some embodiments, the divalent cation may be provided as a salt, such as MnCl. In some embodiments, the antioxidant compositions of the present invention may contain a divalent cation (e.g., Mn) at a concentration of about 1 mM to about 10 mM, e.g., about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mM, or any value or range therein. 2+ For example, in some embodiments, the antioxidant compositions of the present invention may contain Mn in an amount of about 1.4 mM to about 5.3 mM, about 2 mM to about 7 mM, or about 1 mM to about 9.8 mM. 2+ In some embodiments, the antioxidant compositions of the present invention may comprise about 3 mM Mn 2+ In some embodiments, the antioxidant compositions of the present invention may include about 3 mM MnCl.

[0051] In some embodiments, the antioxidant compositions of the present invention may include a buffer, such as phosphate buffer, Tris buffer, MES buffer, HEPES buffer, and / or the like. In some embodiments, the buffer may be at a concentration of about 10 mM to about 500 mM, e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 495, 496, 497, 498, 499, or 500 mM, or any value or range therein. For example, in some embodiments, antioxidant compositions of the present invention may comprise about 20 mM to about 100 mM phosphate buffer, about 10 mM to about 200 mM phosphate buffer, or about 40 mM to about 120 mM phosphate buffer. In some embodiments, antioxidant compositions of the present invention may comprise about 50 mM phosphate buffer. In some embodiments, the buffer and / or antioxidant composition may have a pH of about 5 to about 9, or any value or range therein, e.g., about 6.8 or about 7.4. In some embodiments, antioxidant compositions of the present invention and / or methods of use thereof may include compositions and / or methods described in PCT / US2008 / 073479; PCT / US2011 / 034484; and / or PCT / US2012 / 062998, the disclosures of which are incorporated herein by reference.

[0052] In some embodiments, the antioxidant compositions of the present invention comprise MnCl2 at a concentration of about 1 mM to about 10 mM, a decapeptide (e.g., DEHGTAVMLK [SEQ ID NO: 1]) at a concentration of about 0.5 mM to about 10 mM, and a phosphate buffer at a concentration of about 10 mM to about 500 mM. In some embodiments, the antioxidant compositions of the present invention comprise about 3 mM MnCl2, about 3 mM decapeptide (e.g., DEHGTAVMLK [SEQ ID NO: 1]), and about 200 mM phosphate buffer. However, the concentrations of the components in the antioxidant composition may vary as long as there is only a small degradation in efficacy. The antioxidant compositions may further comprise one or more excipient(s), such as sorbitol, trehalose, and / or one or more peptide(s), such as HMHMHM (SEQ ID NO: 3), HMLK (SEQ ID NO: 2), and / or the like.

[0053] The methods of the invention may expose a virus present in an antioxidant composition to radiation (e.g., ionizing radiation (e.g., gamma) radiation and / or ultraviolet radiation (e.g., UVC) radiation), which may result in protection of one or more epitopes (e.g., surface protein epitope(s), e.g., VP1, VP2, VP3, and / or VP4 epitope(s)), while leaving the viral genome exposed to radiation damage and / or destruction. In some embodiments, the virus is exposed to an amount of ionizing radiation (e.g., gamma radiation and / or X-rays) of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 kGy. In some embodiments, the virus is exposed to an amount of ionizing radiation (e.g., gamma radiation) of about 30, 35, or 40 kGy to about 45 or 50 kGy. In some embodiments, the virus has a fluence of about 0.01, 0.5, or 0.1 kJ / m 2 ~approximately 5, 10, or 15 kJ / m 2(e.g., about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 kJ / m 2 ) or an equivalent derived exposure time, surface area and / or light source wavelength and / or wattage. In some embodiments, the poliovirus is exposed to about 0.7 mW / cm of UV (e.g., UVC) radiation. 2 and / or the poliovirus is exposed for about 60 minutes (e.g., in a thick-walled plastic tube) to a UVC light source emitting about 5 mW / cm for about 1, 5, 10, or 30 seconds to about 1, 1.5, or 2 minutes. 2 In some embodiments, the poliovirus is exposed to a UV source (e.g., a UVC light source) that emits UV light, where the poliovirus may be contained within a UV-transparent (e.g., thin-walled) tube or pipe. In some embodiments, the poliovirus is exposed to a UV source (e.g., a UVC light source) that is of sufficient intensity and / or for a sufficient period of time to inactivate infectivity.

[0054] Due to their lower water and lipid concentrations, non-enveloped viruses typically require higher doses of radiation for inactivation than, for example, influenza and VEEV, thereby increasing damage to protein antigens. In some embodiments, the compositions and / or methods of the invention have and / or provide improved protection of one or more surface epitope(s).

[0055] In some embodiments, the methods of the present invention replace air and / or dioxygen contacting the compositions of the present invention with argon. For example, the air in a tube containing a virus and an antioxidant composition can be at least partially replaced with argon. In some embodiments, the methods of the present invention reduce and / or remove the concentration of metals, such as iron, from compositions containing a virus and / or an antioxidant composition. For example, trace amounts of iron contamination in phosphate buffers and other reagents can lead to increased oxidative damage to protein epitopes. Thus, in some embodiments, iron and / or other metals can be removed from buffers and water using methods known to those of skill in the art, such as by passage through a chelating chromatography column (Chelex column, BioRad). In some embodiments, iron and / or other metals can be present at a concentration of less than about 100 mM.

[0056] Some embodiments of the present invention provide increased protection of poliovirus epitopes (e.g., surface protein epitopes) from damage during irradiation treatment (e.g., ionizing radiation and / or ultraviolet radiation) compared to currently commercially available (e.g., formalin / formaldehyde) inactivated poliovirus immunogens (e.g., but not limited to, IPV). In some embodiments, the currently commercially available inactivated poliovirus immunogen(s) include, but are not limited to, IPV (e.g., IPOL and / or VeroPol). Increased protection of poliovirus epitopes can be increased protection of epitopes compared to a control, e.g., during gamma and / or UVC radiation inactivation compared to formalin / formaldehyde inactivation. Increased protection can be achieved by at least partially replacing ambient air in a container (e.g., a tube) containing the virus with a non-reactive gas (e.g., argon) and / or by removing and / or reducing the amount of iron in a composition containing the pre-inactivated virus. In some embodiments, the air may be at least partially replaced with a non-reactive gas (e.g., argon) such that the oxygen content is reduced by about 50% or more, e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, compared to the oxygen content in the atmosphere and / or the oxygen content prior to the at least partial replacement.

[0057] UV light may be used to inactivate poliovirus with minimal to no damage to epitopes that stimulate neutralizing antibodies (e.g., antibodies against viral surface protein epitopes). UV light can be divided into categories based on wavelength: UVA is 315-400 nm, UVB is 280-315 nm, and UVC is 100-280 nm. The infectivity of poliovirus is approximately 0.7 mW / cm. 2For approximately 60 minutes (e.g., in a thick-walled plastic tube) exposed to a UVC (e.g., wavelengths of approximately 220 to approximately 280 nm) light source emitting approximately 5 mW / cm 2 Poliovirus can be completely inactivated by exposure to a UVC light source emitting UV-C for about 1, 5, 10, or 30 seconds to about 1, 1.5, or 2 minutes (if the poliovirus is contained within a UV-transparent (e.g., thin-walled) tube or tubing).

[0058] As described herein, the methods of the present invention can include exposing poliovirus to radiation (e.g., ionizing radiation and / or UV radiation), and the poliovirus can be present in a tube (e.g., a tube or container). One of skill in the art will understand that exposure conditions (e.g., radiation intensity and / or exposure time) can vary depending on the type and / or characteristics of the tube. Appropriate tube characteristics (e.g., thickness and transparency) for exposure to and / or penetration by radiation (e.g., UVC) can be selected based on the radiation conditions (e.g., radiation source and intensity), and / or exposure conditions can be modified based on the tube. For example, in some embodiments, the tube and / or tube can be clear and / or transparent, or opaque and / or matte. In some embodiments, the pipe or tube may have a thickness of about 1 mm or more (e.g., about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 mm or more) (e.g., a "thick-walled" pipe or tube). In some embodiments, the pipe or tube may have a thickness of less than about 1 mm (e.g., about 0.05, 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, or 0.95 mm) (e.g., a "thin-walled" pipe or tube). In some embodiments, the methods of the invention may include exposing the immunogens of the invention to radiation while they are flowing within and / or being transported through pipes and / or tubing (e.g., flow cells).

[0059] Thus, in some embodiments, the methods of the invention may expose poliovirus to ultraviolet light (e.g., UVC), optionally in a UV-transparent tube or conduit, in an amount sufficient to at least partially inactivate the infectivity of the poliovirus. In some embodiments, the amount sufficient to inactivate the poliovirus may be a wavelength of about 220 to about 280, e.g., about 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280, or any range or value therein. In some embodiments, the amount sufficient to inactivate the poliovirus is about 0.5 mW / cm. 2 ~about 10mW / cm 2 , for example, about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mW / cm 2 The UVC light source may emit a UVC light source emitting a UVC light source of about 10 seconds to about 75 minutes, e.g., about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, or 75 minutes, or any value or range therein. For example, in some embodiments, the methods of the present invention provide a UVC light source emitting a UVC light source of about 0.7 mW / cm at a dose sufficient to completely inactivate the poliovirus. 2 Approximately 60 minutes of exposure to a UVC light source with a wavelength of approximately 254 radiating approximately 5 mW / cm 2 The method may include exposing the poliovirus to UVC, e.g., in a UVC-transparent tube or conduit, for about 10 seconds to 5 minutes of exposure to a UVC light source emitting at a wavelength of about 254. In some embodiments, during UVC inactivation, complexing with the MDP complex protects poliovirus epitopes from damage as evidenced by stimulation of neutralizing antibodies.

[0060] In some embodiments, the methods of the invention include pre-treating a composition comprising a virus and optionally an MDP composition with ultraviolet (UV) light (e.g., UVC) followed by ionizing radiation.

[0061] The sterilizing effect of X-rays and / or gamma rays in vaccine production is the result of direct damage to proteins and nucleic acids by photons and (much) more significantly, indirect damage due to reactive oxygen species (ROS) produced by radiolysis of water molecules.

[0062] Some embodiments of the invention provide protection of all or at least a portion (e.g., 10% or more, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more) of the outer proteins that form the epitopes of the virus while facilitating destruction of the RNA genome.

[0063] The MDP complex was developed from a long-term study of radioresistant bacteria. Deinococcus radiodurans is exceptionally resistant to oxidation and can survive both desiccation and 12–16 kGy of gamma irradiation (1 kGy = 100,000 rads) (Daly et al., 2010; Daly et al., 2004). Radioresistance in Deinococcus radiodurans is due to the presence of intracytosolic Mn. +2The MDP complexes of the present invention can protect the structural integrity of antigens at doses of radiation that abolish infectivity. Studies have been published demonstrating the protective effect of MDP and its use in protecting antigenic proteins in VEEV, chikungunya virus, and Staphylococcus aureus (MRSA) (Dabral et al., 2014; Gaidamakova et al., 2012; Honnald et al., 2014). The inventors of the present invention have discovered that MDP compositions can be used to prepare attenuated and / or neuropathogenic Sabin strains of poliovirus (a picornavirus and therefore lacking an envelope) that have been inactivated by irradiation. The methods of the present invention are highly scalable. In some embodiments, the immunogens of the invention are not subject to epitope inactivation during formalin-inactivation, as is PV1-Sabin, as evidenced by D antigen content.

[0064] In some embodiments, the methods of the invention provide and / or the compositions of the invention comprise improved antigens compared to existing polio vaccines. Unlike OPV, the irradiation-inactivated vaccines of the invention (Ir-PV) do not replicate and are unable to genetically evolve into pathogenic viruses. Furthermore, unlike currently licensed IPV vaccines, Ir-PV is produced using attenuated and / or neuropathogenic Sabin strains of virus, resulting in reduced biohazards associated with producing and handling large quantities of pathogenic strains.

[0065] The irradiation-inactivated polio immunogens of the invention may be formulated as trivalent mixtures containing different amounts of three components: irradiated poliovirus type 1 Sabin (Ir-PV1S), irradiated poliovirus type 2 Sabin (Ir-PV2S), and irradiated poliovirus type 3 Sabin (Ir-PV3S). In some embodiments, the compositions of the invention contain about 2 to about 50 D antigenic amounts of Ir-PV1S (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, or 50 D antigenic amounts of Ir-PV1S, or any value or range therein), about 0.1 to about 10 D antigenic amounts of Ir-PV2S (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 D antigenic amounts of Ir-PV1S, or any value or range therein). The compositions may comprise 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 D antigenic amounts of Ir-PV2S, or any value or range therein, and / or about 5 to about 50 D antigenic amounts of Ir-PV3S (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, or 50 D antigenic amounts of Ir-PV3S, or any value or range therein). Alternatively, monovalent compositions containing a single serotype or bivalent compositions containing two serotypes may be formulated. In some embodiments, the D antigen content of the polio immunogen inactivated by irradiation of the present invention is reduced by less than about 50% (e.g., less than about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) compared to the D antigen content of the purified virus before inactivation. For example, in some embodiments, the D antigen content of the polio immunogen inactivated by irradiation is reduced by, e.g., less than about 40%, less than about 25%, or less than about 7% compared to the D antigen content of the purified virus before inactivation.

[0066] Irradiation-inactivated polio immunogens can be formulated in simple solutions, such as water, standard buffers, standard saline, and / or the like. In some embodiments, adjuvants may be included in the compositions of the invention, thereby even increasing the magnitude and / or duration of the immune response.

[0067] In some embodiments, the poliovirus immunogens of the invention can stimulate neutralizing antibodies (e.g., neutralizing antibody titers of about 8, 10, 25, 50, 80, 100, 300, 500, 800, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,000, 14,000, 16,000, 20,000, or higher, and / or any value or range therein), which may be determined in a standard Wistar rat model. In some embodiments, the poliovirus immunogens of the invention can stimulate substantially similar amounts (e.g., ±5%, 10%, 15%, or 20%) of neutralizing antibodies as current commercial polio vaccines (e.g., IPV and / or OPV), which may be determined in a standard Wistar rat model. In some embodiments, the poliovirus immunogens of the invention can stimulate an increased amount of neutralizing antibodies (e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, or, e.g., about 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, 10-log, or more) over current commercially available polio vaccines (e.g., IPV and / or OPV), as may be determined in a standard Wistar rat model. For example, in some embodiments, the poliovirus immunogens of the invention (e.g., irradiated poliovirus serotypes 1, 2, and / or 3) can stimulate an amount of neutralizing antibodies within ±20% or less of the amount of neutralizing antibodies provided by current commercially available polio vaccines (e.g., IPV and / or OPV). In some embodiments, the poliovirus immunogens of the invention (e.g., irradiated poliovirus serotypes 1, 2, and / or 3) stimulate neutralizing antibodies in an amount at least about 2-fold greater than the amount of neutralizing antibodies provided by current commercially available polio vaccines (e.g., IPV and / or OPV). In some embodiments, the poliovirus immunogens of the invention (e.g., irradiated poliovirus serotypes 1, 2, and / or 3) stimulate neutralizing antibodies in an amount at least about 2-log2 greater than the amount of neutralizing antibodies provided by current commercially available polio vaccines (e.g., IPV and / or OPV).

[0068] The use of irradiation-inactivated poliovirus of the present invention can provide and / or enable a reduction in the amount of antigen contained within a single immunization dose, thereby providing a dose-sparing effect, thereby allowing for the production of a greater number of doses per unit of purified starting virus in the manufacturing process. In some embodiments, the dose of poliovirus antigen of the present invention for one or more of the three serotypes can be reduced, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, as may be defined by the D antigen content for current commercially available IPV vaccines (e.g., IPOL and / or VeroPol). For example, in some embodiments, the dose of a poliovirus antigen of the invention for serotypes 1, 2, and / or 3 can be reduced to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, or less of the usual human dose currently defined by D antigen content and / or mass (e.g., micrograms). In some embodiments, a composition (e.g., a vaccine) of the invention comprises a radiation-inactivated PV1 serotype having a D antigen content of less than 40 D antigen amounts, a radiation-inactivated PV2 serotype having a D antigen content of less than 8 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of less than 32 D antigen amounts.For example, in some embodiments, the compositions (e.g., vaccines) of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 D antigen amounts. In some embodiments, the compositions of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of about 0.1, 1, 2, 3, 4, 5, 6, 7, or 8 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 D antigen amounts. In some embodiments, the compositions of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of about 40 D antigen amounts, a radiation-inactivated PV2 serotype having a D antigen content of about 8 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of about 32 D antigen amounts. In some embodiments, the compositions of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of 20 D antigen amounts, a radiation-inactivated PV2 serotype having a D antigen content of 8 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of 32 D antigen amounts. In some embodiments, the compositions of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of 40 D antigen amounts, a radiation-inactivated PV2 serotype having a D antigen content of 2 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of 32 D antigen amounts. In some embodiments, the compositions of the invention comprise a radiation-inactivated PV1 serotype having a D antigen content of 40 D antigen amounts, a radiation-inactivated PV2 serotype having a D antigen content of 8 D antigen amounts, and / or a radiation-inactivated PV3 serotype having a D antigen content of 18 D antigen amounts.In some embodiments, compositions (e.g., vaccines) of the invention comprise radiation-inactivated PV1, PV2, and / or PV3 serotypes with D antigen content that is reduced by about 2-fold, 4-fold, 8-fold, 16-fold, or 32-fold compared to a composition (e.g., a conventional 1× human dose) having a 40D antigen amount for PV1, an 8D antigen amount for PV2, and / or a 30D antigen amount for PV3.

[0069] In some embodiments, the immunogens and / or compositions (e.g., vaccines) of the invention can stimulate neutralization titers that are equivalent to or greater than acceptable neutralization titers (e.g., 8 and / or 3-Log2 titers) that correlate with protection from infection and / or disease for poliovirus when the antigenic mass (e.g., D antigen content) of the poliovirus immunogen is less (e.g., at least about 50%, 25%, 12.5%, or 6% less) than the antigenic mass (e.g., D antigen content) in a standard human dose for current commercially available poliovirus vaccines (e.g., oral poliovirus vaccines and / or inactivated poliovirus vaccines that are not inactivated using ionizing radiation (e.g., formaldehyde and / or formalin-inactivated polio vaccines)). In some embodiments, the immunogens and / or compositions of the invention may have a D antigen content by mass (e.g., D antigen amount per gram weight (e.g., per microgram or milligram)) that is equivalent to or increased / greater than the D antigen content by mass of current commercially available poliovirus vaccines (e.g., oral poliovirus vaccines and / or inactivated poliovirus vaccines that are not inactivated using ionizing radiation (e.g., formaldehyde and / or formalin-inactivated polio vaccines). For example, in some embodiments, the methods of the invention may be gentler and / or less damaging to epitopes than formalin inactivation. Thus, 1 mg of immunogen of the invention may have a higher D antigen content than formalin / formaldehyde-inactivated poliovirus and, therefore, may allow a greater number of doses to be produced per unit of virus than formalin / formaldehyde-inactivated poliovirus.In some embodiments, the immunogens and / or compositions (e.g., vaccines) of the invention may have at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more increased D antigen content by mass (e.g., amount of D antigen per gram weight) compared to current commercially available poliovirus vaccines (e.g., oral poliovirus vaccines and / or inactivated poliovirus vaccines that are not inactivated using ionizing radiation (e.g., formaldehyde and / or formalin-inactivated polio vaccines).

[0070] The immunogens and / or compositions of the invention may be provided and / or packaged in any suitable package and / or container. In some embodiments, the immunogens and / or compositions of the invention may be provided in packaging suitable for administering the immunogens and / or compositions to a subject. In some embodiments, the immunogens and / or compositions of the invention may be contained in glass vials, ampoules, or other containers known to those of skill in the art, which may be single or multiple doses.

[0071] The amount of immunogen administered to a subject and / or present in a composition of the invention is typically sufficient to induce a desired immune response in the target host. Generally, dosages used may be from about 0.1 micrograms to about 100 micrograms of protein per dose (e.g., about 0.1, 0.2, 0, 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms of protein per dose, or any value or range therein). In some embodiments, dosages may be calculated using D antigen concentrations. Doses may or may not correlate with the D antigen content of currently licensed IPV vaccines, which consist of 40, 8, and 32 D antigen amounts of PV1, -2, and -3. In some embodiments, compositions (e.g., vaccines) of the invention may include at least one antigen in an amount that is at least about 10% less than the amount in an IPV vaccine (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less than the amount in an IPV vaccine (e.g., a current commercially available IPV vaccine, e.g., IPOL and / or VeroPol)). For example, in some embodiments, the compositions of the invention may contain at least one PV1 antigen in an amount that is at least about 10% to about 55% less than the amount in an IPV vaccine, at least one PV2 antigen in an amount that is at least about 20% to about 60% less than the amount in an IPV vaccine, or at least one PV3 antigen in an amount that is at least about 15% to about 50% less than the amount in an IPV vaccine.

[0072] The irradiation-inactivated poliovirus immunogens of the present invention can be used to stimulate protective immunity in subjects (e.g., humans). The immunogens can be injected into animals and / or humans intramuscularly, intradermally, subcutaneously, and / or similarly, and may be administered using a standard syringe. In some embodiments, the immunogens of the present invention can be introduced into animals or humans using microneedles, patches designed to allow the immunogen to penetrate the skin surface, and / or other methods known to those skilled in the art.

[0073] In some embodiments, manufacturing processes for immunogens of the invention may include drying the immunogen (e.g., by freeze-drying, spray-drying, and / or similar methods). In some embodiments, drying may increase the heat resistance of the immunogen and / or immunogen-containing composition (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more) and / or may extend the measured shelf life of the immunogen and / or immunogen-containing composition (e.g., by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more), which may result from maintaining the immunogenic properties of the composition. The drying process may include combining the immunogen of the present invention with one or more stabilizing excipient(s) known to those of skill in the art, such as, but not limited to, sorbitol, trehalose, sucrose, polyethylene glycol, amino acids, and / or other additives. The drying procedure may utilize freeze-drying, e.g., lyophilization, spray drying, and / or other methods known in the art.

[0074] In some embodiments, an adjuvant may be present in the vaccine of the invention and may be capable of stimulating an improved immune response. Examples of adjuvants include, but are not limited to, alum, aluminum hydroxide, aluminum phosphate, monophosphoryl lipid A, saponin derivatives (e.g., QS-21), nucleic acids, including oligonucleotides such as CpG, lipopolysaccharides, oil-and-water emulsions, squalene, saponins, and / or other adjuvant substance(s) (e.g., flagellin).

[0075] The invention is described in more detail in the following non-limiting examples. [Example]

[0076] Example 1: Poliovirus serotype 2 Sabin strain (PV2S) The irradiation process involved exposing the virus preparation to radiation on wet ice. Poliovirus was mixed with an antioxidant composition containing 3 mM MnCl, 3 mM decapeptide DEHGTAVMLK (SEQ ID NO: 1), and 50 mM phosphate buffer (pH 7.2), placed in a polypropylene tube, purged with argon, and exposed to either X- or γ-irradiation. In this example, the virus was inactivated on ice using a radioactive cobalt-60 source with appropriate shielding to protect the human user from harmful radiation.

[0077] Once mixed with the MDP complex, the virus was exposed to 0–50 kGy of γ-irradiation. Figure 1 shows the effect of γ-irradiation on the viability of PV2-Sabin in a dose-dependent manner. In the left panel of Figure 1, viral infectivity is graphed in a semilogarithmic plot against the dose of γ-irradiation up to 35 kGy. In the insert of Figure 1, viral infectivity is graphed in a linear plot. These graphs show that 45 kGy of γ-irradiation is sufficient to destroy 100% of the infectivity of a virus preparation containing 16 micrograms of purified PV2S.

[0078] When mixed with the MDP complex, the viral surface capsid protein is protected from degradation. Figures 2A-2C show three analyses of irradiated PV2. In the studies, PV2-Sabin was exposed to 0, 5, 10, 20, 30, 40, and 45 kGy of irradiation. In Figures 2A and 2B, prior to irradiation, the virus was treated with decapeptide (Dp) and MnCl2 (Mn 2+ ) (lower panel), or Mn 2+ The irradiated virus samples were mixed with Dp alone (middle panel), Dp alone (top panel), or neither (top panel). For Fig. 2A, samples of irradiated virus were denatured with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), electrophoresed in polyacrylamide gels, transferred to membranes, and probed with an antibody against the VP1 capsid protein. The figure shows the irradiated virus samples mixed with Dp and Mn before irradiation. 2+The figure shows that the inclusion of both nucleotides results in protection of the protein components (lower panel). For Figure 2B, RNA was extracted from irradiated virus samples and reverse-transcribed using oligo(dT) to generate a cDNA library from the genomic poly(A) tract. The cDNA was used as a template in polymerase chain reaction (PCR) to assess genomic RNA fragmentation with increasing irradiation. PCR products were electrophoresed on a 2% agarose gel, and DNA fragments were visualized using ethidium bromide staining. The upper band was synthesized using a primer corresponding to nucleotides 6786–7403 (617 base pairs located near the cDNA priming site). The lower band was synthesized using a primer corresponding to nucleotides 5478–5941 (463 base pairs located further from the cDNA priming site). Figure 2B shows that the presence of the MDP complex during irradiation (lower panel) does not protect the RNA from damage. As expected, the signal from the lower band diminishes more rapidly than the signal from the upper band due to its greater distance from the cDNA synthesis priming site within the poly(A) tract. Thus, Figures 2A and 2B show that the MDP complex protects viral proteins but not the genome from fragmentation and inactivation. Figure 2C shows transmission electron micrographs of non-irradiated (top) and irradiated virus (middle and bottom). In the presence of MDP, viral particles appear less fragmented than when irradiated in the absence of MDP.

[0079] Prior to analysis of the immunogenicity of the irradiated virus, a commercially produced vaccine, IPOL R (Sanofi Pasteur) and VeroPol RInactivated virus preparations were standardized for D antigen concentration for comparison with the Staten Serum Institute (Denmark). The D antigen assay is a measure of antigenicity and has been used to predict the immunogenicity of vaccine preparations, with higher D antigen content correlating with higher immunogenicity. The assay is performed in the form of a solid-phase enzyme-linked antibody detection and is well standardized. Irradiated virus preparations were standardized to a 1x human dose containing 40 D antigen doses of PV1, 8 D antigen doses of PV2, and 32 D antigen doses of PV3.

[0080] Immunization of Wistar rats is a commonly used method for testing the immunogenicity of poliovirus vaccines. Animals are immunized by intramuscular injection of the vaccine or test antigen, and rat sera sampled 35 and 49 days after the initial immunization are analyzed for virus-neutralizing antibodies. A neutralizing titer of 1:8 is recognized as the level of antiviral antibody that confers protection from infection and / or disease.

[0081] In quantitative neutralization assays, a two-fold dilution series of antibody is incubated with 100 tissue culture infectious units (TCID50) of virus for 1 hour. The antibody-virus complexes are applied to replicate wells containing poliovirus-tropic indicator cells, such as MRC-5 cells, for 1 hour, and then unbound material is washed away with a standard phosphate-buffered saline (PBS) solution. Standard growth medium is added to the wells, and the plates are incubated at 37°C for 3–4 days to allow virus replication. Wells are scored microscopically as infected or uninfected. The neutralization titer is calculated using the Karber formula and is expressed as the reciprocal of the serum dilution that reduces the number of infected wells by 50%.

[0082] The immunogenicity of irradiation-inactivated type 2 Sabin strain (Ir-PV2S) was compared with IPOL and VeroPol in a Wistar rat immunization study. Immunization with 1× or 0.5× doses of Ir-PV2S stimulated anti-PV2 neutralizing antibodies with titers similar to those of IPOL (Fig. 3, panels A, B, C, and D).

[0083] The immunogenicity of Ir-PV2S and VeroPol was compared using decreasing doses of antigen. In addition to comparing the two antigens, a dose-sparing study was conducted to determine whether a reduced dose could stimulate protective levels of neutralizing antibodies. Panels E and F of Figure 3 show the anti-PV2 neutralizing titers for individual rat sera in groups immunized with decreasing amounts of immunogen. In the leftmost pair of data points, sera from rats immunized with 1 / 8 (one-eighth) dose of antigen (1 D antigen dose) show that Ir-PV2S sera (indicated by triangles) have higher neutralizing titers compared to anti-VeroPol sera. The figure shows that further reductions in the VeroPol dose stimulated barely detectable PV2 neutralizing antibodies. In contrast, as little as 1 / 64 (one-six-fourth) of the normal human dose, or 0.125 D antigen dose, of Ir-PV2S was sufficient to stimulate neutralizing antibodies.

[0084] Example 2: Poliovirus serotype 1 Sabin strain (PV1S) PV1S was inactivated using gamma irradiation. In this process, PV1S was mixed with an antioxidant composition containing 3 mM MnCl, 3 mM decapeptide DEHGTAVMLK (SEQ ID NO: 1), and 50 mM phosphate buffer (pH 7.2) and placed in a polypropylene tube. The ambient air was purged with argon to remove dioxygen, and the tube was exposed to gamma irradiation. In this example, the virus was inactivated on ice using a radioactive cobalt-60 source (approximately 12 kGy / hr) with appropriate shielding to protect human users from harmful radiation.

[0085] The immunogenicity of irradiation-inactivated poliovirus type 1, Sabin strain (Ir-PV1S), was compared with IPOL and VeroPol in a Wistar rat immunization model. Groups of eight rats were immunized with either 1x or 0.5x human dose equivalents (containing 40 or 20 D antigenic doses of PV1 antigen, respectively). A control group of rats was immunized with irradiated virus without the addition of MDP conjugates. Figure 4 shows the neutralization titers of serum samples from rats 35 and 49 days after the initial immunization with the four antigens. Serum from rats immunized with a 1x dose of IPOL is shown by circles, 1x dose of VeroPol by squares, 1x dose of Ir-PV1S with MDP by triangles, and 1x dose of Ir-PV1 without MDP by circles. Serum from rats immunized with half the dose (0.5x) is shown in the right panel. The horizontal line indicates the mean titer for the group of rats, expressed as Log-2 values. At a 1× dose of Ir-PV1S, 6 of 8 rats seroconverted to protective titer levels of 8 or 3-Log2. In contrast, none of the rats immunized with Ir-PV1S produced in the absence of MDP conjugate seroconverted. At half the dose (0.5×), Ir-PV1S stimulated titers nearly equivalent to those of IPOL administered at an equivalent dose (right panel).

[0086] Example 3: Inactivation of PV1 Sabin using UVC radiation Poliovirus can be inactivated by exposure to UVC light, but neutralizing epitopes are not significantly destroyed. PV1 Sabin was complexed with MDP containing 3 mM DP1 decapeptide, 3 mM MnCl2, and 50 mM potassium phosphate buffer (pH 7.2). The virus-MDP complex was placed in a tube transparent to UVC light. Samples were exposed to 0.7 mW / cm2 of UVC light. 2 Emits UVC light for 60 minutes or 5mW / cm 2 The inactivation process was evaluated using a standard infectivity assay. Irradiation inactivates poliovirus infectivity more rapidly when the virus is contained within a thin-walled UVC-transparent tube. In a follow-up study, polioviruses were exposed to UVC light emitting 5 mW / cm for 2 minutes.2 Irradiation with light emitting β-glucan for as little as 10 or 20 seconds inactivated all detectable infectivity (data not shown).

[0087] The immunogenicity of UVC-inactivated poliovirus can be assessed by analyzing sera from immunized rats. Wistar rats were immunized on days 0 and 21 by intramuscular injection with 40D antigen doses (1x human dose) of UVC-inactivated PV1 Sabin. No adjuvant was used. Four weeks later, on day 49, serum samples were collected from the rats for analysis of neutralizing antibodies. Figure 5 shows the linear and Log-2 titers of neutralizing activity from sera produced by rats immunized with UVC-inactivated PV1. The mean Log-2 titer of sera from PV1 Sabin with MDP was 9.7, corresponding to a linear titer of 1,960. The mean Log-2 titer of sera from PV1 Sabin without MDP was 4.4, corresponding to a titer of 1:229.3. The data from Figure 5 are graphed in Figure 6. Figure 6 shows a graph of the Log-2 titers of sera from rats immunized with UVC-irradiated PV1 Sabin with (filled circles) and without (open circles) MDP. The horizontal line indicates the mean Log-2 titer.

[0088] Example 4: Inactivation of PV3 Sabin using UVC light Not all viruses can be inactivated using γ-irradiation without damaging protective epitopes. In the case of PV3 Sabin, exposure to 40 kGy of γ-irradiation resulted in antigens that appeared intact by Western blot analysis, but D antigen concentrations were greatly reduced. In multiple experiments, the amount of D antigen was reduced from approximately 60,000 units / mL to undetectable levels (data not shown). As expected, rats immunized with PV3 Sabin-MDP complexes inactivated for infectivity using γ-irradiation did not mount acceptable anti-PV3 neutralizing titers (data not shown).

[0089] Inactivation of PV3 Sabin by exposure to UVC light was investigated. Similar to PV1 Sabin, virus samples were complexed with or without MDP (3 mM Dp1 decapeptide, 3 mM MnCl2, and 50 mM potassium phosphate buffer, pH 7.2). 0.7 mW / cm 2 Samples were titrated for infectivity using HeLa cells after 3, 10, and 30 minutes of exposure to UVC light emitting MDP. The left panel of Figure 7 shows the Log-10 infectivity titer of PV3 Sabin exposed to UVC with (circles) or without (triangles). There was little difference in the infectivity curves, indicating that the presence or absence of MDP conjugates has little effect on inactivation of infectivity. PV3 Sabin samples treated with UVC light in the presence of MDP conjugates were evaluated using a D antigen ELISA assay for protection of viral epitopes that stimulate neutralizing antibodies (Figure 7, right panel). High doses of UVC irradiation inactivate almost all infectivity but reduce D antigen concentrations by only 25%. A 60-minute exposure with the same UVC source inactivates 100% of infectivity while maintaining the immunogenic activity of the neutralizing epitopes.

[0090] To provide a more definitive measure of immunogenicity, rats were immunized with UVC-inactivated PV3 Sabin. In this example, PV3 Sabin samples with and without MDP were immunized at 0.7 mW / cm 2After 60 minutes of exposure to UVC light emitting D antigen, no detectable infectivity was found. Duplicate samples were analyzed for D antigen concentration, and samples equivalent to 2x, 1x, 1 / 2x, and 1 / 8x the human dose (64, 32, 16, and 4 D antigen doses, respectively) were formulated without adjuvant. Rats were immunized by intramuscular injection on days 0 and 21 and bled for serum on day 49. Figure 8 shows the linear and Log-2 titers of neutralizing activity by sera produced from rats immunized with UVC-inactivated PV3 Sabin, with the mean values ​​for each group shown. Figure 9 graphs the data shown in Figure 8. Figure 9 shows the Log-2 titers for individual rats immunized with PV3 Sabin with (closed symbols) or without (open symbols) MDP. The horizontal line indicates the mean group titer. The inclusion of MDP improves protection of neutralizing epitopes during UVC-inactivation. The effect of MDP on improving neutralization titers was more pronounced when rats were immunized with a lower antigen dose: at a dose equivalent to 1 / 8 the standard human dose, UVC-inactivated PV3 Sabin stimulated neutralization titers greater than the acceptable protective titer of 3-log2 when irradiated in the presence of MDP conjugates.

[0091] The foregoing is illustrative of the invention and is not to be construed as limiting thereof. The invention is defined by the following claims, including equivalents of the claims. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is set forth.

Claims

1. 1. A poliovirus immunogen comprising: the poliovirus immunogen is inactivated by UV radiation in the presence of an antioxidant composition comprising a divalent manganese cation, a peptide having the amino acid sequence of DEHGTAVMLK (SEQ ID NO: 1), and a buffer; wherein the poliovirus immunogen comprises an attenuated and / or neuropathogenic strain of poliovirus serotype 1, 2, and / or 3; The poliovirus immunogen stimulates neutralizing antibodies, as determined in a standard Wistar rat model; the poliovirus immunogen has a higher D antigen content than a poliovirus immunogen inactivated using UV radiation in the absence of the antioxidant composition and / or a poliovirus immunogen inactivated using formaldehyde and / or formalin. Poliovirus immunogen.

2. 2. The poliovirus immunogen of claim 1, the poliovirus immunogen stimulates neutralizing antibodies in amounts substantially similar to or greater than the amounts of neutralizing antibodies provided by current commercial polio vaccines; Poliovirus immunogen.

3. 3. The poliovirus immunogen of claim 1 or 2, the poliovirus immunogen stimulates neutralizing antibodies at a dose lower than a typical vaccine dose; may stimulate neutralizing antibodies at doses less than 1 / 32 or 1 / 64 of the normal human dose and / or at doses of 0.125D antigen amounts; Poliovirus immunogen.

4. 2. The poliovirus immunogen of claim 1, the poliovirus immunogen is further inactivated by ionizing radiation; Poliovirus immunogen.

5. The poliovirus immunogen according to any one of claims 1 to 4, the D antigen content of the poliovirus immunogen is reduced by less than about 50% compared to the D antigen content of the purified virus before UV inactivation; Poliovirus immunogen.

6. A poliovirus immunogen comprising the poliovirus immunogen of any one of claims 1 to 5. composition.

7. 1. An inactivated immunogenic composition comprising an attenuated and / or neuropathogenic strain of poliovirus serotype 1, 2, and / or 3, the viral infectivity is abolished by UV radiation in the presence of an antioxidant composition comprising a divalent manganese cation, a peptide having the amino acid sequence of DEHGTAVMLK (SEQ ID NO: 1), and a buffer, and the viral protein containing the epitope is protected from damage; The inactivated immunogenic composition stimulates neutralizing antibodies, as determined in a standard Wistar rat model; the immunogenic composition has a higher D antigen content per milligram of virus than a poliovirus immunogenic composition inactivated with UV radiation in the absence of the antioxidant composition and / or a poliovirus immunogenic composition inactivated with formaldehyde and / or formalin; Immunogenic composition.

8. 8. The immunogenic composition of claim 7, The attenuated and / or neuropathogenic strain of poliovirus is further inactivated by treatment with gamma irradiation. Immunogenic composition.

9. The immunogen according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 8, vaccine.

10. 1. A method for producing an inactivated poliovirus immunogen, comprising: The method comprises: exposing the poliovirus to an antioxidant composition comprising a divalent manganese cation, a peptide having the amino acid sequence of DEHGTAVMLK (SEQ ID NO: 1), and a buffer; and exposing the poliovirus in the presence of the antioxidant composition to ultraviolet radiation in an amount sufficient to inactivate the poliovirus; thereby providing said inactivated poliovirus immunogen; Here, the inactivated poliovirus immunogen produced stimulates neutralizing antibodies, as determined in a standard Wistar rat model. The method produces a greater number of doses per unit of poliovirus compared to polio vaccine production methods that use formaldehyde and / or formalin for inactivation. method.

11. 11. The method of claim 10, the poliovirus comprises an attenuated strain of poliovirus serotype 1, 2, and / or 3; method.

12. 12. The method of claim 10 or 11, The method produces an inactivated poliovirus vaccine having a higher D antigen content per mass than formaldehyde and / or formalin-inactivated poliovirus vaccines and / or inactivated poliovirus vaccines that have not been inactivated using ionizing radiation. method.

13. The method according to any one of claims 10 to 12, The step of exposing the poliovirus to the divalent Mn cation, a peptide having an amino acid sequence of DEHGTAVMLK (SEQ ID NO: 1), and a buffer causes the poliovirus to react with the divalent cation (Mn ２＋ ), a peptide having an amino acid sequence of DEHGTAVMLK (SEQ ID NO: 1), and a composition comprising a buffer to provide a combined composition; method.

14. 14. The method of claim 13, The composition contains MnCl at a concentration of about 1 mM to about 10 mM. ２ a peptide at a concentration of about 0.5 mM to about 10 mM, and a phosphate buffer at a concentration of about 10 mM to about 100 mM; method.

15. The method according to any one of claims 10 to 14, The method further comprises exposing the poliovirus to ionizing radiation. method.

16. The method according to any one of claims 10 to 15, further comprising at least partially replacing the air in contact with the poliovirus and / or the air in a container containing the poliovirus with a non-reactive gas prior to exposing the poliovirus to ultraviolet radiation. method.

17. The method according to any one of claims 10 to 16, At least a portion of the poliovirus epitope is intact and / or active in the inactivated poliovirus immunogen. method.

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