Patent Application
20220265808
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy of the Sequence Listing, which was created on Jul. 27, 2020, is named NO137-576WO.txt and is 13.8 kilobytes in size.
The presently-disclosed subject matter generally relates to a vaccine for porcine circovirus type 2 (PCV2). In particular, certain embodiments of the presently-disclosed subject matter relate to altered PCV2 vaccines and methods for developing altered vaccines.
Porcine circoviruses (PCVs) consist of the non-pathogenic porcine circovirus strain 1 (PCV1) and the pathogenic porcine circovirus strain 2 (PCV2) types. Porcine circovirus type 2 is a small, single-stranded DNA virus, with a circular genome and relatively high plasticity. It is an economically important swine virus which causes post-weaning multi-systemic wasting syndrome (PMWS) and lymphadenopathy in weanling piglets, along with a range clinical signs such as jaundice, nephropathy, reproductive and respiratory disorders collectively known as porcine circovirus associated diseases (PCVAD).
The approximately 1700 bp PCV2 genome encodes just two major proteins; the replicase (ORF1) and capsid (ORF2) proteins. The capsid protein is considered to be both necessary and sufficient for the prevention of PCV2, as subunit vaccination with the capsid protein alone is effective at preventing clinical signs. Hence, while the cell mediated immune response to PCV2 is not well studied, neutralizing antibody responses targeted to the capsid protein are considered to be critical for protection against PCV2. Strong binding Ab responses to PCV2 can be detected as early as 7 days post infection in naturally or experimentally infected pigs. However, neutralizing responses, whose appearance correlates with a reduction in viremia, are not detected until later in infection.
Infections characterized by delayed virus neutralizing Ab responses commonly present decoy epitopes, which are characterized by sequence variability, hydrophilicity, structural flexibility and proximity to conserved, functionally important regions such as receptor binding sites. Decoy epitopes are usually immunodominant and divert the Ab responses away from neutralizing epitopes. Immuno-dominance is the phenomenon by which the immune system preferentially mounts responses to selected antigens, or epitopes within antigens, and is an effective immuno-subversion mechanism for pathogens and a well-established confounding factor in the development of effective vaccines. While several studies on epitope mapping of the PCV2 capsid protein have identified four major immunodominant regions containing over-lapping linear and conformational epitopes, fewer studies have characterized the functionality of the identified epitopes. Of those regions which have been characterized, conformational neutralizing epitopes have been mapped to residues 47-58, 165-200, and 230-233. However, only one decoy epitope spanning residues 169-180 has been identified so far.
Despite the remaining need for a more complete picture of possible immuno-subversive strategies, several commercial vaccines against PCV2 are available and commonly deployed in pork production units. Most of the commercial vaccines continue to target the first discovered PCV2 subtype, PCV2a (SEQ ID NO: 1), either as whole inactivated virus, inactivated chimeric PCV1-2a virus preparations, or subunits of the capsid protein. Although existing vaccines are effective at preventing clinical signs of PCV2 and in reducing economic damage due to the virus, they do not prevent transmission or shedding of PCV2. As such, vaccinated animals continue to be viremic, transmitting the virus both horizontally and vertically. Additionally, since the introduction of commercial vaccines, the initially predominating PCV2a subtype was replaced by a 2nd subtype, PCV2b (SEQ ID NO: 2), and more recently by PCV2d (SEQ ID NO: 41). Therefore, it is possible that selection pressure induced by commercial vaccines could be driving viral evolution in the field.
Together, the delayed production of neutralizing Ab responses, coupled with the periodical emergence of new PCV2 subtypes following vaccination, suggests that antibody based immunodominance plays an important role in PCV2 pathogenesis and vaccine mediated protection. Thus, there remains a need for both a more complete picture of possible immune-subversive strategies as well as vaccines which enable differentiation of vaccinated and infected animals (DIVA) to facilitate possible eradication of PCV2 in the long term, and to ensure vaccine compliance during routine production.
Further, live attenuated vaccines against PCV2 may be more effective than current inactivated or subunit vaccines. However, attenuated PCV2 vaccines are not used in the field due to the need for a high safety margin to prevent reversion to virulence.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.
In some embodiments, the presently-disclosed subject matter includes a PCV2 vaccine including a PCV2 infectious clone with a re-engineered PCV2 capsid in the backbone thereof, wherein the re-engineered PCV2 capsid includes a modified immunogenic region. In some embodiments, the PCV2 infectious clone is selected from the group consisting of PCV2a (SEQ ID NO: 1), PCV2b (SEQ ID NO: 2), and PCV2d (SEQ ID NO: 41). In some embodiments, the modified immunogenic region includes at least one modification as compared to a region selected from the group consisting of wild type region 1, wild type region 2, wild type region 3, wild type region 4, and combinations thereof.
In some embodiments, the modified immunogenic region includes at least one modification to a decoy epitope sequence contained therein. In some embodiments, the decoy epitope sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 26, and combinations thereof. In some embodiments, the decoy epitope sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 17, SEQ ID NO: 18, and combinations thereof. In some embodiments, the decoy epitope sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 20, and combinations thereof. In some embodiments, the modified immunogenic region includes at least one modification to each of SEQ ID NO: 5 and SEQ ID NO: 20. In some embodiments, the modified immunogenic region includes at least two modifications to each of SEQ ID NO: 5 and SEQ ID NO: 20. In some embodiments, the decoy epitope sequence is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and a combination thereof. In some embodiments, the modified immunogenic region includes a modified decoy epitope sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, and a combination thereof.
In some embodiments, the re-engineered PCV2 capsid further comprises at least one modified serine or modified leucine codon, wherein the modified serine codon include at least one mutation selected from the group consisting of UCA to UAA, UCA to UGA, and UCG to UAG, and wherein the modified leucine codon include at least one mutation selected from the group consisting of UUA to UAA, UUA to UGA, and UUG to UAG. In some embodiments, each serine and leucine codon is modified. In some embodiments, the mutation converts the at least one modified serine or modified leucine to a stop codon.
In some embodiments, the vaccine further comprises a marker for differentiating infected and vaccinated animals (DIVA). In some embodiments, the DIVA marker includes a peptide that is foreign to swine. In some embodiments, the DIVA marker includes SEQ ID NO: 27.
Also provided herein, in some embodiments, is a method of vaccinating against PCV2, the method including administering the vaccine according to one or more embodiments disclosed herein to a subject in need thereof. In some embodiments, after administration of the PCV2 infectious clone with the re-engineered PCV2 capsid in the backbone thereof refocus the immune response in the subject towards more protective regions on the capsid protein. In some embodiments, the method further comprises determining whether the subject is infected using the DIVA marker and removing infected subject from the herd.
Further features and advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.
The presently-disclosed subject matter will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, including the methods and materials are described below.
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 cells, and so forth.
The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
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.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, or the like is meant to encompass variations of in some embodiments ±50%, in some embodiments ±40%, in some embodiments ±30%, 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.
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.
All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. However, 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. As such, it should be understood that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims. Instead, 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.
The presently-disclosed subject matter includes articles and methods for vaccinating against porcine circovirus type 2 (PCV2). In some embodiments, the articles include a PCV2 vaccine including a reengineered PCV2 capsid in the backbone thereof. In some embodiments, the reengineered PCV2 capsid includes modifications (e.g., mutations) to linear decoy epitopes that are conserved or substantially conserved between PCV2 subtypes. As used herein, the phrase “substantially conserved between PCV2 subtypes” means that the corresponding linear decoy epitope(s) include no more than 2 mismatched amino acids between subtypes. For example, the decoy epitopes spanning amino acids 124-141 (SEQ ID NO: 5) and 166-180 (SEQ ID NO: 20) of PCV2a are conserved in PCV2b (i.e., they are identical), and are substantially conserved in PCV2d, with each containing a single amino acid mismatch as shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
As will be appreciated by those skilled in the art, since the linear decoy epitopes being modified are conserved or substantially conserved between subtypes, any PCV2 subtype may serve as the backbone for the PCV2 vaccine. For example, in one embodiment, the PCV2 vaccine includes a PCV2a infectious clone with a reengineered PCV2 capsid in the backbone thereof. In another embodiment, the PCV2 vaccine includes a PCV2b infectious clone with a reengineered PCV2 capsid in the backbone thereof. In a further embodiment, the PCV2 vaccine includes a PCV2d infectious clone with a reengineered PCV2 capsid in the backbone thereof.
Any suitable conserved or substantially conserved linear decoy epitope in the PCV2 subtype may be modified to form the reengineered PCV2 capsid backbone. In some embodiments, the vaccine includes at least one modification to the PCV2a (SEQ ID NO: 1), PCV2b (SEQ ID NO: 2), PCV2d (SEQ ID NO: 41), or other PCV2 capsid protein. In some embodiments, the vaccine includes at least two modifications to the PCV2a (SEQ ID NO: 1), PCV2b (SEQ ID NO: 2), PCV2d (SEQ ID NO: 41), or other PCV2 capsid protein. In some embodiments, the modifications are to an immunogenic region of the PCV2 capsid. For example, in one embodiment, the vaccine includes at least one modification to region 1, 2, 3, and/or 4 (TABLE 1). In another embodiment, the at least one modification is to a major immunogenic region having a sequence according to SEQ ID NO: 7, 8, 9, and/or 10. In a further embodiment, the at least one modification is to an immunodominant decoy epitope having a sequence according to SEQ ID NO: 3, 4, 5, and/or 6. In one embodiment, the vaccine includes at least two modifications to region 1, 2, 3, and/or 4 (TABLE 1). In another embodiment, the at least two modifications are to a major immunogenic region having a sequence according to SEQ ID NO: 7, 8, 9, and/or 10. In a further embodiment, the at least two modifications are to an immunodominant decoy epitope having a sequence according to SEQ ID NO: 3, 4, 5, and/or 6. As will be appreciated by those skilled in the art, the immunodominant decoy epitope sequences according to SEQ ID NOS: 3-6 are within the major immunogenic regions according to SEQ ID NOS; 7-9, and thus any modification to an immunodominant decoy epitope will also be considered a modification to the overlapping immunogenic region.
In some embodiments, the modifications are to decoy epitope sequences such as, but not limited to, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 25, and/or SEQ ID NO: 26. For example, in one embodiment, the reengineered PCV2 capsid includes at least one modification to YTVKATTVRTPSWAVDMM (SEQ ID NO: 3), WPCSPITQG (SEQ ID NO: 17), and/or KATALTYDPY (SEQ ID NO: 18). Additionally or alternatively, in one embodiment, the reengineered PCV2 capsid includes at least one modification to SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 20. In another embodiment, the reengineered PCV2 capsid includes two modification to each of SEQ ID NO: 5 and SEQ ID NO: 20. In one embodiment, the reengineered PCV2 capsid includes at least one modification to SEQ ID NO: 25 and/or SEQ ID NO: 26. In some embodiments, the reengineered PCV2 capsid includes SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments, the reengineered PCV2 capsid includes SEQ ID NO: 23 and SEQ ID NO: 24.
In some embodiments, the PCV2 capsid is also mutated such that the vaccine virus undergoes suicidal replication in the host. This eliminates the possibility of vaccine-induced disease or recombination with field strains to produce new variants. Serine and leucine amino acids are encoded by 6 redundant codons each. Of these 6 codons, two codons for each amino acid (UUA, UUG for Leucine and UCA, UCG for Serine) require just one single mutation to be converted to a stop codon. To increase the chances of a stop codon occurring during viral replication in the pigs, all the serine and leucine amino acids of the capsid protein of the vaccine virus were redesigned as in Table 2.
Additionally or alternatively, in some embodiments, the PCV2a infectious clone with the reengineered PCV2 capsid also includes a marker for differentiating infected and vaccinated animals (DIVA). Suitable DIVA markers include, but are not limited to, peptides which are “foreign” to swine. For example, in one embodiment, the marker includes a highly immunogenic, 18 amino acid long segment from the surface antigen-1 related sequence 2 (SRS2) protein (AAD04844.1) of N. caninum. In another embodiment, the marker includes Amino acids 324 QSSEKRDGEQVNKGKPP 348 (SEQ ID NO: 27) of the SRS2 protein. In some embodiments, the marker has an antigenicity index score sufficient to ensure that it will not cross react serologically with other swine related proteins. In some embodiments, the marker is inserted into the 5′ end of the capsid gene of the PCV2 vaccine disclosed herein.
Also provided herein, in some embodiments, is a method of vaccinating swine against PCV2. In some embodiments, the method includes administering one or more of the articles disclosed herein to a swine. In one embodiment, after administration the modifications to the one or more immunodominant decoy epitopes refocus the immune response in the swine towards more protective regions on the capsid protein, as compared to PCV2 capsids without the immunodominant decoy epitope modifications. In some embodiments, administration of the articles disclosed herein provides a lower total IgG Ab response against the capsid protein as compared to existing commercial vaccines (e.g., vaccines without one or more modified immunodominant decoy epitopes), while providing a clear anamnestic response. In some embodiments, the administration of the articles disclosed herein may be used to vaccinate against any PCV2 strain, such as, but not limited to, PCV2a, PCV2b, and/or PCV2d. Additionally or alternatively, in some embodiments, the method includes determining whether the swine is infected using the DIVA marker, and removing infected swine from the herd.
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.
Porcine circovirus type 2 (PCV2) is an economically important swine virus which causes post-weaning multisystemic wasting syndrome (PMWS) in weanling piglets. Commercial vaccines against PCV2 are highly effective. Yet, a recurring emergence of new subtypes in vaccinated herds necessitates a better understanding of protective immunity. As such, this Example is directed to identifying previously unrecognized decoy epitopes in the PCV2 capsid protein and demonstrating that early antibody responses map to potential decoy epitopes and vice versa. Since virus neutralizing Ab responses are not detected until later in PCV2 infection, the premise that the earliest detected immunodominant Ab responses in PCV2 infected animals would correspond to potential decoy epitopes is also discussed herein. Further discussed herein is the identification of immune-subversive regions of the capsid protein which dominate the early Ab response in PCV2 infection.
Using a peptide library spanning the PCV2a capsid protein (SEQ ID NO: 1) and weekly sera collections from PCV2a infected animals, three major immunodominant regions mapping to the early responses were identified. Regions with potential decoy activity were further narrowed down using peptide blocking fluorescent focus inhibition assays to residues 55 YTVKATTVRTPSWAVDMM 72 (SEQ ID NO: 3), 106 WPCSPITQGDRGVGSTAV 123 (SEQ ID NO: 4), and 124 ILDDNFVTKATALTYDPY 141 (SEQ ID NO: 5). Post-vaccination responses also largely recognized the three identified regions, which appeared to dominate the antibody responses to PCV2 in both infection and vaccination.
Material and Methods
Peptides, antibodies, and viruses: A peptide library spanning the entire 233 amino acids of the capsid protein (ORF2) of PCV2a strain 40895 (GenBank Accession AF264042) was commercially synthesized (Mimotopes, Victoria, Australia) as overlapping 12mer biotinylated peptides with a 3 aa overlap (total 75 peptides). Serum was collected weekly from 3-week-old, PCV2 negative piglets which were experimentally infected with PCV2a strain 40895 as previously described. Sera from 12 pigs collected on days post infection (DPI) 0, 7, 14, 21 and 28 were pooled for the assessment of binding antibody responses to the peptides. Similarly, sera collected at 35 days post-vaccination (DPV) from 8 pigs each, which were vaccinated with either a commercial inactivated or subunit PCV2 vaccine were used to assess post-vaccination Ab responses to the pep-set. All sera used in the study were previously tested with PCV2 capsid protein specific ELISAs. To prepare pure cultures of the virus for the virus neutralization assays, an infectious clone of PCV2a strain 40895 was used to rescue recombinant virus cultures as described previously.
Detection of antibody responses to the peptide library: An indirect ELISA format was used for the detection and differentiation of the early and mature PCV2 Ab responses to biotinylated peptide library spanning the PCV2 capsid protein. The same protocol was used to test post-vaccination Ab responses to the individual peptides. To coat the ELISA plates (Maxisorp, Nalge Nunc, Rochester, N.Y.), 100 μl of a 10 μg/ml solution of streptavidin in sterile distilled water was added to the wells and allowed to dry overnight. After washing 5 times with phosphate buffered saline with Tween20 (PBST) containing 2% BSA, the plates were the incubated with 100 μl of a 10 μg/ml solution of each biotinylated peptide at 37° C. for 1 hr. Plates were then blocked with 2% BSA, 2% skimmed milk powder and 2% normal goat serum in PBST for 2 hrs at 37° C. Test samples were prepared by pooling equal volumes of sera from twelve PCV2 infected pigs collected at DPI 0, 7, 14, 21 and 28 each or DPV 35 sera from 8 pigs each administered a commercial inactivated or subunit vaccine. Each pool was then was diluted to 1:50 in PBST containing 2% BSA and added in 100 μl volumes to the peptide coated plates and incubated for 1 hr at 37° C. After washing 5 times with PBST, anti-swine IgG HRPO conjugate (KPL, Gaithersburg, Md.) at a 1:5000 dilution in blocking buffer was added plates incubated at 37° C. for 1 hr. Detection was achieved using the tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, Md.) and incubation in the dark for 15 mins at room temperature. Finally, 1M HCl was added to stop the reaction. Optical density (OD) readings were obtained at 450 nm using a microplate reader (BioTek Instruments, Winooski, Vt.). All samples were assessed in duplicate. The mean signal to negative [S/N] ratio for each peptide was calculated as the OD value for each peptide divided by the corresponding value of the day 0 sample. Values above an S/N ratio of 1 were considered positive (Table 1).
Virus neutralization assay: A conventional virus neutralization (V/N) assay format was used to obtain the V/N titers for the pooled samples as described before, with some modifications. Each of the pooled sera, prepared as described above, was serially diluted two-fold from 1:2 to 1:1024 dilutions in PBS, in sterile U bottom plates. The PCV2a strain 40895 virus culture was diluted to 103.5TCID50/ml, and equal volumes added to the diluted sera. The U bottom plates were incubated for 1 hr at 37° C. The mixture was then layered on pre-formed PK-15 cells at 60% confluence in 96 well tissue culture plates. Virus replication was visualized after 36 hrs by staining with a PCV2-specific monoclonal antibody as previously described. The virus neutralization titer was determined as the log2 serum dilution at which 80% or higher reduction in the number of fluorescent foci was noted, when compared to the virus only control.
Virus neutralizing activity of peptides: To localize virus neutralizing activity within the immunodominant regions identified by the pep-scan ELISA, a peptide-blocked fluorescent focus neutralization [FFN] assay was performed essentially as described before, with some modification. Blocking of virus neutralizing Abs by a peptide was expected to increase virus replication and hence, the number of fluorescent foci detected, and vice versa. To block the activity of Abs specific to the peptides, a pool of 5-6 peptides [20-23 aa total] spanning the length of each identified immunogenic region was first tested.
To prepare the pool, equal volumes of a 1 μg/ml solution of each peptide was mixed well. Each pool (10 μl) was incubated for 60 mins at 37° C. with 50 μl heat inactivated, pooled DPI 28 PCV2a anti-serum or pooled DPV35 serum at a 1:4 dilution in PBS. A non-specific swine-influenza virus-specific peptide [EALMEWLKTRPI] (SEQ ID NO: 11) and DPI 0 serum were used as controls. The PCV2a culture was adjusted to 100-150 fluorescent focus units/well and 50 μl was mixed with the peptide blocked antisera, followed by incubation at 37° C. for 60 mins. The serum/peptide/virus mixtures were incubated for 36 hrs on preformed PK-15 monolayers at 60% confluence, in 8 well chamber slides.
Virus replication was visualized by a PCV2-specific immunofluorescence assay, as previously described. The number of fluorescent foci in each well was counted in a blinded fashion by two individuals, in two independent experiments, with 3 replicates for each peptide pool (total 12 values). Activity was assessed as the mean percentage change in the number of fluorescent foci in the sample blocked with peptides, when compared to the unblocked DPI 28 PCV2 antiserum. To further narrow down the residues involved, smaller pools of 2-3 peptides spanning the regions identified to have potential decoy activity in the first screen were tested next. Each peptide pool was tested in 4 replicates and 2 independent experiments (total 8 values). All other procedures were similar to the initial screen (Table 1).
Pairwise statistical differences at p<0.05 between the blocked and unblocked serum for each peptide pool was assessed by the Mann Whitney U test. To determine location and surface exposure of the aa identified as having potential decoy activity, the residues were visualized on the crystal structure of the monomeric unit of the PCV2 capsid protein (PDB ID 3R0R) using the EzMol molecular visualization tool (
Results and Discussion
Early antibody responses map to three major immunogenic regions. To test the premise that the early Ab responses in PCV2 infected animals would be directed towards non-protective regions of the PCV2 capsid protein, the differential antibody responses between early and late infection were characterized using the pep scan ELISA and post-infection sera collected at weekly intervals. In agreement with our previous findings, PCV2-specific Ab responses were detected as early as DPI 7, although the magnitude of the responses was low. Early Ab responses mapped to peptides 20-24 (58 KATTVRTPSWAVDMMRFNIDDFVP 81) (SEQ ID NO: 12), 33-46 (97 RIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPY 141) (SEQ ID NO: 8) and 56-61 (166 SGSGVLDSTIDYFQPN NKRNQLWMRLQTSRN 192) (SEQ ID NO: 9) (
The Ab responses to the three regions persisted and increased in strength at DPI 14. In addition, weak responses to peptides 74-75 containing the N terminal amino acids (226 LKDPPLKP 233) (SEQ ID NO: 10) were observed, suggesting the residues could participate in the formation of a neutralizing epitope. The V/N titer at DPI 14 was 1:8. Between DPI 21 and 28, responses to all four regions increased in magnitude (
Mapping of virus neutralizing activity: To determine which peptides would be able to block Abs with virus neutralizing activity, pools of 5-6 peptides spanning the identified immunodominant regions were reacted with the DPI 28 serum pool. The extent of blocking was visualized as an increase or decrease in viral replication in a fluorescent focus neutralization assay. Overall, potential decoy activity appeared to localize to residues 58-160 (peptides 19-43), while protective activity was detected between residues 160-233 (peptides 51-75). Possible decoy activity were detected for peptide pools 19-24, 33-38 and 39-43 with values for peptides 33-39 being statistically significant. When 2-3 peptides were used instead of 5-6 peptides in the 2nd screen to narrow down the regions responsible for the identified activity, peptides 19-21 [55 YTVKATTVRTPSWAVDMM 72] (SEQ ID NO: 3) showed potential decoy activity, while peptides 22-24 did not. The aa sequence 59 KATTVR 64 (SEQ ID NO: 14) was previously identified as immunodominant in studies where linear or conformational epitopes were mapped, with residues 59 and 60 being critical for subtype or strain specific reactivity. These residues were previously found to map to Abs with neutralizing activity. However, interestingly, when residues 59 and 60 were mutated neutralizing activity was significantly improved in vitro, indicating that the identified epitope could actually be a decoy epitope, as identified in this study.
In the second immunodominant region spanned by peptides 33-44 (Table 1) peptides 33-35 (97 RIRKVKVEFWPCSPITQG 114) (SEQ ID NO: 15) and 39-41 (115 DRGVGSTAVILDDNFVTK 132) (SEQ ID NO: 16) either blocked neutralizing activity or had no activity in the 2nd screen using fewer peptides. Hence, it could be deduced that decoy activity was localized to 106 WPCSPITQG 114 (SEQ ID NO: 17) and 132 KATALTYDPY 141 (SEQ ID NO: 18), while neutralizing activity could be attributed to 97 RIRKVKVEF 105 (SEQ ID NO: 19). Indeed, a putative receptor binding site function has been proposed for residues RIRKVK. The location of neutralizing epitopes, adjacent to decoy epitopes resulting in steric interreference with Ab binding to the neutralizing epitope as a mechanism of immune evasion has been described before.
The third immuno-dominant region, 166 VLDSTIDYFQPNNKRNQLWMRLQTSRN 192 (SEQ ID NO: 9) spanning peptides 56-61 (Table 1), contained the decoy epitope (166 VLDSTIDYFQPNNKR 180) (SEQ ID NO: 20) identified by Trible et. al. This region showed very strong responses to the DPI 7 serum, which persisted for the duration of the study on the pep scan analysis. However, no significant decoy activity was detected in the first screen. When the peptides containing the core epitope (peptides 55 and 56) and key residues (173 YFQ 175, 179 K) alone were tested separately, the activity in the FFN assay was non-neutralizing. However, the values were not statistically significant. These findings are in agreement with Lekcharoensuk et. al., who found that residues 165-200 could interact with residues 58-63 to form conformational neutralizing epitopes. Hence, without wishing to be bound by theory, it is believed that overlapping linear and conformational epitopes are present in this location.
The 4th immunodominant region which was recognized later in infection spanned peptides 70-75 and contained a previously identified neutralizing epitope involving the last 3 aa “231 LKP 233.” The three N terminal residues, “231 LKP 233,” often vary between newly emerging subtypes, with several PCV2b strains having the sequence LNP, and the more recently emerged PCV2d having a single N terminal amino acid elongation to LKPK.
Mapping of the residues to the crystal structure using PDB structure ID 3R0R and the EzMol molecular visualization tool showed that of the three putative decoy epitopes identified in this study, residues 55 YTVKATTVRTPSWAVDMM 72 (SEQ ID NO: 3) [
Antibody responses in vaccinated pigs: When post-vaccination serum from pigs administered either an inactivated or subunit vaccine was tested on the pep scan to assess differential responses between infection and vaccination, the trends were similar in infected and vaccinated animals, with the higher magnitude responses being directed towards immunodominant regions 1 [residues 55 YTVKATTVRTPSWAVDMM 72] (SEQ ID NO: 3) and 3 [residues 166 VLDSTIDYFQPNNKRNQL 183] (SEQ ID NO: 6). Responses to region 2 were low with detectable responses to residues 106 WPCSPITQGDRGVGSTAV 123 (SEQ ID NO: 4) but not 124 ILDDNFVTKATALTYDPY 141 (SEQ ID NO: 5) (
Trible et al., found that vaccination with the monomeric form of the capsid protein induced high levels of Abs to the decoy epitope, 169 STIDYFQPNNKR 180 (SEQ ID NO: 22), while vaccination with the fully assembled VLP did not. The level of Abs to this epitope was found to correlate inversely with neutralizing Abs in vaccinated animals. However, significant differences in the pattern of responses between the inactivated and subunit vaccines were not found in this Example. Instead, the findings in this Example are in agreement with Worsfold et. al., who also found strong Ab responses to this epitope in vaccinated pigs, with the strength of the response increasing with the age of the pigs. While regions with neutralizing activity were not characterized in this study, only low magnitude responses to the C-terminal aa with known neutralizing activity were detected in vaccinated animals (
Thus, three new PCV2 capsid protein sequences, YTVKATTVRTPSWAVDMM (SEQ ID NO: 3), WPCSPITQG (SEQ ID NO: 17), and KATALTYDPY (SEQ ID NO: 18), with possible immuno-subversive activity were identified in this Example. The data described supports the belief that the earliest detectable Ab responses in PCV2 infected pigs will likely localize to decoy epitopes. It also supports the broader premise that the approaches used in this Example can be applied to other pathogens with a delayed virus neutralizing Ab response to characterize Ab responses at the linear epitope level. Hence, the data and approaches described in this Example contribute to further understanding PCV2 Ab mediated immunity.
Despite the availability of commercial vaccines which can effectively prevent clinical signs, porcine circovirus type 2 (PCV2) continues to remain an economically important swine virus, as strain drift followed by displacement of new subtypes occurs periodically. Commercial vaccines against PCV2 were introduced in the U.S in 2006. They solely target the PCV2a subtype and are effective in preventing clinical signs. However, the recent viral evolution and emergence of new PCV2 strains suggest that the existing vaccines require updating or improvement in efficacy. While antibody responses to the PCV2 capsid protein are considered to be both necessary and sufficient for protection, as discussed in Example 1 above, a significant portion of the early antibody responses are non-functional, thus serving as a host immune-evasion mechanism. More specifically, the present inventors had previously determined that the early antibody responses to the PCV2 capsid protein in infected pigs map to immunodominant but non-protective, linear B cell epitopes of the PCV2 capsid protein.
With that in mind, the primary objective of this Example was to determine if the threshold of protection against PCV2 can be improved by further rationalization of current vaccine design. This included mapping the putative protective and non-protective regions of the PCV2 capsid protein and then reengineering the PCV2 capsid in the backbone of a PCV2b infectious clone, such that the immune response is refocused towards more protective regions on the capsid protein. Using sequential anti-sera from infected pigs and a panel of overlapping peptides spanning the PCV2 capsid protein, 3 new linear, immunodominant but non-protective regions of the PCV2 capsid protein were identified and the presence of a previously identified immuno-dominant decoy epitope was confirmed. It was also found that a majority of the Abs produced by vaccination mapped to the non-protective, immunodominant epitopes identified. Based upon these findings, the present inventors tested the hypothesis that abrogation of the immunodominance patterns induced by two of the previously identified, non-protective epitopes would raise the threshold of protection attained PCV2 by vaccination. More specifically, to further improve PCV2 vaccine efficacy, two of the previously identified immunodominant epitopes were mutated in the backbone of a PCV2b infectious clone to rationally restructure the immunogenic viral capsid protein. The rescued virus was used to immunize 3-week-old weanling piglets, followed by challenge with a virulent heterologous PCV2d strain.
Additionally, in veterinary medicine, the successful eradication of an infectious disease requires a vaccine that is both effective and has the capability of differentiating infected and vaccinated animals (DIVA). DIVA vaccines are usually accompanied by an immuno-assay which can help to differentiate infected and vaccinated animals. Infected animals which are removed from the herd eventually lead to a disease free population. However, none of the current PCV2 vaccines have DIVA capabilities, nor is a PCV2 DIVA immuno-assay available. As such, a secondary objective of this Example was to develop a marker vaccine against PCV2 by introducing an immunogenic foreign peptide in the vaccine construct, to enable detection (Absof antibodies) against the marker to distinguish between vaccinated and infected pigs (i.e., to serve as a DIVA marker). This construct was designated as modified live vaccine I (MLV-I) (
Vaccination of pigs with the restructured PCV2b vaccine (rPCV2-Vac) encoding a DIVA marker, which is also referred to herein as MLV-II, and challenge with the currently predominating heterologous PCV2d strain resulted in improved heterosubtypic virus neutralization responses, protection against tissue pathology, lack of viremia due to the challenge virus, improved weight gain, and Ab responses specific to the DIVA tag. More specifically, a loss of immunodominant antibody responses to the targeted epitopes and an overall reduction in the magnitude of the antibody responses was detected. The loss of immunodominance to the targeted epitopes correlated with a broadening of the virus neutralization responses and absence of tissue pathology in the lymphoid organs. Challenge viral replication was detected in only 1/7 pigs at day 21 post-challenge. Thus, as hypothesized, rational redesign of the PCV2 capsid antigen resulted an alteration of the immunodominance hierarchy and improved PCV2 vaccine performance. Accordingly, the strategy described in this Example provides insights into the mechanisms of vaccine mediated protection against PCV2 and has long term implications for improving the control and prevention of PCV2.
Material and Methods
Cells and viruses: The PCV1 free porcine kidney cell line, PK-15N (005-TDV, National Veterinary Services Laboratory, Ames, Iowa, USA), was used to culture all PCV2 strains. An infectious clone of PCV2b strain 41513 (GenBank accession number KR816332) was used as the backbone for the vaccine. An infectious clone of a heterologous PCV2d strain (GenBank accession number JX535296.1) was used to prepare the challenge virus. For virus neutralization assays, infectious clones of PCV2a (AF264042.1), PCV2b (EU340258.1), and PCV2d (JX535296.1) were used to generate virus stocks by transfection as described below.
Cloning of the vaccine construct: Using the infectious clone of PCV2b 41513 as the backbone, two previously identified linear immuno-dominant, but non-protective epitopes in the immunogenic PCV2 capsid protein were mutated. The capsid gene segment encoding the desired mutations was commercially synthesized, and cloned into the backbone of PCV2b 41513 by restriction digestion. To minimize the risk of producing a lethal mutation, selected amino acids in the linear decoy epitopes were replaced with other amino acids with a low penalty score on a point accepted mutation (PAM) matrix as follows; Epitope A—124 ILDDNFVTKATALTYDPY 141 (SEQ ID NO: 5) was modified to 124 ILDDNFVNKSTALTYDPY 141 (SEQ ID NO: 23), and Epitope B—166 VLDSTIDYFQPNNKR 180 (SEQ ID NO: 20) was modified to 166 VLDSTIDYFNPNNSR 180 (SEQ ID NO: 24) (Table 3,
STALTYDPY 141
Insertion of a marker to differentiate vaccinated and infected (DIVA) pigs: As a high percentage of production swine are naturally infected with PCV2, the vaccine construct was designed to include a positive marker to enable DIVA capabilities. Neospora caninum is an apicomplexan parasite which has not been detected in pigs. A highly immunogenic segment of 18 amino acid length selected from the surface antigen-1 related sequence 2 (SRS2) protein (AAD04844.1) of N. caninum was selected following the in silico prediction of antigenicity (Lasergene 11, Protean 13, DNASTAR, USA). The selected sequence was subjected to a protein blast to rule out possible serological cross reactivity with other swine related proteins. Amino acids 324 QSSEKRDGEQVNKGKPP 348 (SEQ ID NO: 27) of the SRS2 protein, with an antigenicity index score of 1.7 was inserted into 5′ end of the capsid gene of the rPCV2-Vac construct described above as a separate transcriptional unit (
Preparation of PCV2 virus cultures: The vaccine and challenge virus cultures, as well as the virus cultures required for the virus neutralization assay were prepared by transfection of PK-15 cells with some modifications. Briefly, the PCV2 genome was excised from the shuttle plasmid by restriction digestion and re-circularized with DNA ligase, unless dimerized infectious clones were available. For transfection, 12 μg of viral genomic DNA or plasmids containing the dimerized infectious clones were diluted in Opti-MEM, mixed with 36 μl of TransIT-2020 (Minis Bio, USA) and incubated at room temperature for 30 mins. After the incubation period, the mixture was overlaid on cell culture flasks (25 cm2, Corning, USA) containing 50% confluent monolayers of PK-15 cells and incubated at 37° C. in a CO2 incubator for 3 h, followed by addition of Dulbecco's Modified Eagle's Medium (DMEM) with 2% fetal bovine serum and 1× penicillin streptomycin. The flasks were frozen and thawed 3 times after 72 h of incubation. The rescued viruses were titrated by the TCID50 method. The stock cultures were stored at −80° C. until used.
Immunofluorescence assay: As PCV2 does not produce cytopathic effects, replication of the PCV2 strains was visualized by IFA as previously described. Briefly, 50% confluent PK-15 monolayers grown in 8 well chamber slides were either transfected as described above or infected with the virus cultures. After 72 hrs of incubation in a CO2 incubator, the cells were fixed with a 1:1 mixture of methanol: acetone. The fixed cell sheets were stained with a PCV2 specific monoclonal antibody (Rural Technologies, USA) or Neospora caninum specific mouse polyclonal antibody, followed by detection with a FITC-conjugated secondary antibody (KPL, USA), and counter-staining with DAPI (Life Technologies, USA). The stained cells were evaluated for apple green nuclear fluorescence indicative of PCV2 replication or expression of the SRS2 DIVA tag (
In vitro vaccine stability: The rPCV2-Vac cultures rescued by transfection of PK-15 cells were serially passaged three times in PK-15 cells. Virus titers were compared against the wildtype virus. The construct was sequenced to verify the stability of the mutations.
Vaccination and challenge of piglets: All procedures pertaining to animal experimentation were carried out with the approval and oversight of the Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC) regulations of N. Dakota (NDSU) and S. Dakota State Universities (SDSU). Twenty-seven, 3-4-week-old piglets which were serologically and PCR negative for PCV2 and other major swine pathogens such as PRRSV, SIV and Mycoplasma sp. were divided into 3 groups of 9 pigs each. Group I was administrated PBS, group II were administered a commercial, inactivated PCV2 vaccine as per label instructions (2 ml, intramuscular), and group III were inoculated with the rPCV2-Vac at 104 TCID50/ml, 2 ml intramuscular and 2 ml intranasally. Although the exact details regarding the antigen dose, formulation, and adjuvants present in the commercial vaccine are not publicly available, a commercial vaccine was selected as a control to represent current industry standards. On day 28 post vaccination (DPV) or day 0 post-challenge (DPC), all study animals were challenged with a heterologous PCV2d strain at 104TCID50, 2 ml intramuscular and 2 ml intranasally. Pigs were monitored daily for signs of porcine circovirus associated diseases (PCVAD) such as wasting, respiratory distress, jaundice, inappetence, or diarrhea. Body weights were assessed on DPC 0, 9, and 21. Serum samples were collected on day 0, and every 2 weeks thereafter to assess Ab responses. All animals were humanely euthanized on DPC 21 for evaluation of pathological lesions as described below.
Anti-PCV2 IgG responses: The measurement of binding IgG responses to PCV2 in vaccinated pigs was achieved with a commercial PCV2 ELISA kit (Ingezim Circovirus IgG kit, Ingenasa, Madrid, Spain), at the Iowa State University Veterinary Diagnostic Laboratory, following their standard operating procedures and the manufacturer's instructions. Signal to positive control (S/P) ratios produced as the assay output were used for further analysis of the data.
Virus neutralizing antibody responses: Functional antibody responses against the homologous PCV2b subtype and heterologous PCV2a and PCV2d subtypes were measured by a rapid fluorescence focus neutralization (FFN) assay, essentially as described before, except that the virus cultures were adjusted to 30-40 fluorescent focus units (FFU)/100 μl for consistent enumeration. Virus replication was assessed by an IFA, as described above. Four replicate values of the DPV 28 sera were obtained and used for analysis. The titers were expressed as the % reduction in viral replication compared to the virus only control, which was not treated with serum (
Antibody responses to the mutated epitopes: The abrogation of the immunodominant Ab response to the selected epitopes in vaccinated pigs was assessed by surface plasmon resonance on a Reichert SR7500DC instrument (Reichert Technologies, USA). Biotinylated peptides encoding the wildtype peptide sequences of epitopes A and B, as described above, were commercially synthesized (Biomatik, USA). Pooled sera collected at DPV 2S from the three treatment groups and from PCV2b infected pigs were used to purify IgG using a commercial kit (Melon gel IgG purification kit, Thermo Fisher, USA). The biotinylated peptides were immobilized on streptavidin coated carboxymethyl dextran sensor chips (Reichert Technologies, USA) by injecting 0.16 μg/μl peptide solution over the sensor chip at a flow rate of 25 μl/min. After an increase of about 300 μRU was observed, indicating immobilization of each peptide had occurred, the purified IgGs for the experimental groups were injected over the flow cells at a concentration of 20 μM in phosphate buffered saline with 0.005% Tween 20(PBST), at a flow rate of 25 μl/min for 240 secs. Binding of the IgGs to the peptides was assessed by the response in μ response units (μRU) (
Antibody responses to the DIVA marker: The selected peptide from the N. caninum SRS2 protein was cloned into a bacterial expression vector (pETSumo Thermo Fisher Scientific, USA) using the Q5 site directed mutagenesis kit (New England Biologicals, USA). The protein was expressed with a HIS tag and purified by nickel affinity chromatography (His-spin protein miniprep, Zymo research, USA), following the manufacturer's instructions. The identity of the purified protein was verified by Western blotting with an anti-HIS tag specific monoclonal Ab (
Measurement of vaccine viral replication by qPCR: Replication of the rPCV2-Vac virus following immunization was quantified by a TaqMan quantitative PCR (qPCR), using a SRS2 marker specific primer and probe combination, and serum collected on DPV 0, 14, and 28. Samples were assessed in duplicate. Viral DNA was extracted using the QiaAmp DNA mini Kit (Qiagen, USA) according to manufacturer's instruction. Primer pairs with sequences of 5′-AAGTGGGAGGTTTGCCTTTGT-3′ (SEQ ID NO: 28) and 5′-ATGGCCCAATCCTCGGAGAA-3′ (SEQ ID NO: 29) and a probe with a sequence of 5′-TACCTGTTCCCCGTCGCGT-3′ (SEQ ID NO: 30) were used. Briefly, 2.0 μl of extracted DNA, 0.4 μM of primers, 0.1 μM probe, and a Tm of 67° C. were used in combination with the QuantiFast Probe PCR Kit (Qiagen, USA) and cycled in a qPCR thermocycler (CFX96 Touch, Bio-Rad, USA). The obtained Ct values were converted to log copy numbers using a standard curve generated with plasmid DNA encoding the SRS2 DIVA marker. The specificity of the assay was evaluated using the infectious clones for the wildtype PCV2b and heterologous PCV2a and PCV2d. The lowest limit of detection of the assay was 2000 genomic copies per ml of serum.
Detection of challenge viral replication: A qPCR assay which is specific to the PCV2d subtype was designed after analysis of PCV2a, PCV2b and PCV2d sequences to identify regions unique to PCV2d (
Assessment of pathological lesions: Evaluation of tissue pathology was carried out as described previously. Macroscopic evaluation of the major organs for gross lesions in the major organs was conducted by assessing lungs for the presence of lesions scored as the percentage of lung parenchyma affected from 1-100%. Inguinal lymph node enlargement was scored from 0-3, where 0 was no enlargement, 1, 2 and 3 were two, three or four times the normal size. Sections of the major organs including the lung, liver, kidney, spleen ileum, tonsils, tracheobronchial and mesenteric lymph nodes were fixed in 10% buffered formalin for 48 h and then transferred to 70% ethanol for sectioning. Slides were examined by hematoxylin and eosin (H&E) staining for microscopic lesions and immunohistochemistry (IHC) to detect viral antigen, following the standard operating procedures of the Iowa State University Veterinary Diagnostic Laboratory. The slides were assigned scores ranging from 1-4 in a blinded fashion by a board-certified veterinary pathologist as follows; 1=single follicle or focus staining, 2=rare to scattered staining, 3=moderate staining, 4=strong widespread staining (
Statistical analysis: A significance level of p<0.05 was used for all statistical analysis. Analysis was conducted using the Minitab 19 software (Minitab, State College USA) or Microsoft excel. Where data was not normally distributed, non-parametric analysis was used. Serological and qPCR data were analyzed by a Student's T test. The lesion scores and body weight data were analyzed by the Mann Whitney U test. The consolidated values, statistical significance and standard deviation are represented in the figures.
Results:
The rPCV2-Vac was successfully rescued and expressed the DIVA peptide: The reverse genetics approaches were used to mutate the selected immunodominant linear B cell epitopes in the PCV2 capsid protein enable the successful rescue of the recombinant rPCV2 Vac virus. There were no significant differences between the titers of the wildtype PCV2b 41513 and the rPCV2 Vac virus cultures generated by transfection with the respective infectious clones (
The rPCV2-Vac induces binding antibody responses in vaccinated pigs: Measurement of anti-PCV2 IgG responses in the study animals using a commercial PCV2 ELISA kit showed an increase in titers after 14 DPV in both the vaccine groups, with the differences between rPCV2-Vac and unvaccinated control group being significantly different at DPV 28 and DPC 09. Although a direct comparison between rPCV2-Vac and the commercial control cannot be drawn due to differences in vaccine formulation, the magnitude of the IgG response to the commercial vaccine remained consistently higher than that of the rPCV2-Vac. As expected, antibody responses in the unvaccinated controls remained low until DPC 9, after which significant differences were not noted between the groups at DPC 21 (
The rPCV2-Vac elicits broad virus neutralization responses: To determine if the mutation of immunodominant, non-protective epitopes would improve the cross-neutralization response to heterosubtypic strains, virus neutralizing responses were measured against the homologous PCV2b subtype as well as heterologous PCV2a and PCV2d subtypes using a rapid fluorescence focus reduction assay. Both MLV-I and MLV-II were highly effective in neutralizing all three PCV2 subtypes tested. Despite the fact that the commercial vaccine has an adjuvant and has undergone extensive dose optimization, neutralization responses elicited by the rPCV2-Vac against the PCV2a subtype was comparable in kinetics and magnitude to that of the commercial vaccine, which contains the PCV2a capsid antigen. Similarly, neutralizing responses against the currently predominant PCV2d subtype in the rPCV2-Vac group were higher than that of commercial vaccine by DPV14, with the difference becoming statistically significant at DPV28. As expected, neutralizing responses elicited by the rPCV2-Vac against its homologous PCV2b strain were robust. However, the commercial vaccine was significantly less effective than rPCV2-Vac in neutralizing PCV2b. Overall, the data supports the conclusion that rPCV2-Vac was more effective in neutralizing heterologous subtypes than the PCV2a based commercial vaccine (
Mutation abrogates antibody responses to the selected epitopes. As expected, antibody responses to epitope A and B were not detected in the serum of rPCV2-Vac immunized pigs by a qualitative SPR analysis, while the responses in pigs infected with the wildtype virus were strong. For epitope 1A, the response in pigs administered the rPCV2-Vac was similar to that of the unvaccinated pigs. The response in the pigs administered the commercial vaccine was of a lesser magnitude than that of the pigs infected with the wildtype virus. In the case of epitope B, strong responses were noted pigs infected with the wildtype virus as expected, but the differences between the other three groups were not significant (
Vaccinated pigs mount DIVA tag specific Ab responses: Assessment of the antibody responses to the DIVA marker by an ELISA specific to the peptide selected from the N. caninum SRS2 protein showed that pigs in the vaccinated groups mounted detectable Abs responses to the DIVA marker by DPV14, with the magnitude of the responses increasing until DPV 28. As expected, the unvaccinated pigs and pigs administered the commercial vaccine did not mount significant antibody responses to the DIVA marker (
Vaccination protects against challenge viral replication: Replication of the heterologous PCV2d challenge virus was not detected in either of the vaccine groups at DPC 9 or DPC 21. As expected, robust challenge viral replication was detected in the unvaccinated pigs, with the titers increasing by about 1 log between day 9 and day 21 post-challenge. In contrast, challenge viral replication was not detected in any of the vaccinated pigs, including those administered the commercial vaccine, indicating that the experimental vaccine induced sterilizing immunity. The values for both vaccine groups were significantly different from the unvaccinated control group at both the time points tested (
Protection against gross and histological lesions: Except for the lungs, gross lesions were not observed in any of the other major organs for all experimentally challenged pigs (
Vaccination protects against weight loss due to challenge: As is commonly encountered in experimental models, severe clinical signs of PCVAD were not observed in any of the experimental groups during the 21 days post-challenge observation period. However, the post-challenge weight gain in both vaccination groups were significantly higher than the unvaccinated control group at DPC 21, but not DPC 14. There were no significant differences between the two vaccine groups during the post-challenge observation period.
The rPCV2-Vac is safe and stable: In contrast to wildtype PCV2 viruses, which can be easily detected by qPCR by DPC 9 (
Discussion
The phenomenon of “original antigenic sin” or ability to elicit memory responses to antigens and specific epitopes is critical to the success of vaccination. On the other hand, the preferential clonal expansion to immuno-dominant but non-protective epitopes encountered by the host on challenge, coupled with minor sequence variation leading to escape variants, is an elegant immuno-subversion strategy the present inventors termed “deceptive imprinting.” Strategies to counter deceptive imprinting in vaccine design include “dampening” the response to the immuno-dominant, non-protective epitopes. The immune refocusing strategy has been successfully applied to several viruses such as human immunodeficiency virus (HIV), influenza, and dengue virus, among others. Unlike structurally complex pathogens, where protection is mediated by multiple antigens, the requirement for a single protective antigen makes PCV2 both a simple and elegant model for studying the effects of immunodominance on vaccine design. This Example explored the hypothesis that alteration of the immunodominance properties of the PCV2 capsid protein will result in the improvement of vaccine efficacy.
The PCV2 capsid protein contains four major immunodominant regions. Within these regions, 4 putative immunodominant, non-protective linear B cell epitopes have been identified. As the PCV2 capsid protein is relatively small (233 amino acids), and incapable of tolerating large sequence changes, only two of the identified decoy epitopes were selected for mutation in this Example. It was previously demonstrated that mutation of an immunodominant HIV-1 epitope located in proximity to a neutralizing epitope can direct the response towards the neutralizing epitopes, possibly due to alteration of steric constraints. As both epitope A and B were flanked by putative neutralizing epitopes they were selected for analysis. To minimize the risk of introducing lethal mutations, the present inventors elected not to delete residues but rather replace them with other residues with a low penalty score on a point accepted mutation (PAM) matrix and were able to successfully rescue the recombinant virus harboring mutations in the selected epitopes (
As anticipated, the introduced changes to the amino acid sequences of the PCV2 capsid protein resulted in the loss of immunodominance of epitope A and B as assessed by SPR (
Several other factors such as glycosylation, hypervariability, and proximity to MHC-II epitopes or other neutralizing epitopes could also potentially influence the outcomes of this Example. While a detailed experimental characterization of the above listed parameters is not within the scope of the Example, they are discussed below. Hyper-glycosylation is a strategy which has been previously used to dampen the Ab response to immunodominant epitopes. While not the primary strategy targeted in this Example, the replacement of a threonine (T) with an asparagine (N) residue in epitope A resulted in the introduction of a putative N-linked glycosylation sequon (N×S) (Table 3). Epitope B naturally contained a predicted N-linked glycosylation site (Table 3), and was not altered for glycosylation properties. As immunodominance is influenced by the successful competition for the recruitment of antigen specific T cells in early infection, the presence of a helper T cell epitopes overlapping or adjacent to a B cell epitope can influence the strength of the Ab response elicited. Epitope A contained a predicted (Propred MHC-II server), but non-conserved, MHC-II epitope 124 ILDDNFVT 131 (SEQ ID NO: 34) in the rPCV2-Vac backbone, which was altered by the mutation of the residue T to an N. Two conserved, predicted MHC-II epitopes, 161 FTPKPVL 167 (SEQ ID NO: 35) and 174 FQPNNKRNQL 184 (SEQ ID NO: 36) overlapped with epitope B. The second predicted MHC-II epitope within epitope B was also altered by the mutations introduced. It is possible that mutation of these T helper epitopes could have enhanced the loss of immunodominance of Epitopes A and B.
Hypervariability is a common property of decoy epitopes, and is an effective immuno-subversion mechanism. However, epitopes A and B were conserved between the first discovered PCV2a and PCV2b subtypes (Table 3,
The lack of challenge viral replication resulting in sterilizating immunity (
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:
This application claims the benefit of U.S. Provisional Application Ser. No. 62/879,016, filed Jul. 26, 2019, the entire disclosure of which is incorporated herein by this reference.
This invention was made with government support under grant nos. 2014-31100-06038, 2015-67016-23318, and NI18HMFPXXXXG008 awarded by the United States Department of Agriculture/National Institute of Food and Agriculture (USDA/NIFA). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/043770 | 7/27/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62879016 | Jul 2019 | US |
