Patent Grant
12054736
This application contains a Sequence Listing, which was submitted n ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on Nov. 11, 2020, is named SequenceListing.txt and is 91 KB in size.
Modified Vaccinia Ankara (MVA) is a genetically engineered, highly attenuated strain of vaccinia virus that does not propagate in most mammalian cells. This property minimally impacts viral or foreign gene expression because the ability of MVA to propagate in mammalian cells is blocked at late stage viral assembly. However, the DNA continues to replicate and therefore acts as an efficient template for RNA biosynthesis leading to high levels of protein synthesis. MVA also has a large foreign gene capacity and multiple integration sites, two features that make it a desirable vector for expressing vaccine antigens. MVA has a well-established safety record and versatility for the production of heterologous proteins. In fact, MVA-based vaccines for treatment of infectious disease and cancer have been developed and reached Phase I/II clinical trials.
MVA has an extensive history of successful delivery into rodents, Rhesus macaques, and other non-human primates, and more recently as a clinical vaccine in cancer patients. The original MVA virus was administered to 120,000 young and elderly in Europe in the 1970s. MVA is avirulent because of the loss of two important host-range genes among 25 mutations and deletions that occurred during its repeated serial passage in chicken cells.
MVA is appealing as a vaccine vector for CMV antigens in individuals who are both severely immunosuppressed and experiencing additional complications such as malignancy or organ failure, thereby requiring a transplant. CMV infection is an important complication of transplantation procedures and affects a wide variety of individuals including newborns and HIV patients with advanced disease. Human cytomegalovirus (HCMV) is a major risk factor for recipients of solid organ and hematopoietic stem cell transplants. Individuals who are previously CMV-infected or receiving a CMV-infected solid organ or stem cell allograft are candidates for a vaccine strategy that targets the cellular reservoir of the virus.
It has been reported that in vitro expression levels of foreign antigens by an rMVA vaccine are correlated with the rMVA vaccine's immunogenicity. However, after serial passage, the foreign antigen expression may be reduced, which can result in diminished immunogenicity. Thus, while MVA is an attractive viral vector for recombinant vaccine development, genetic instability and diminished immunogenicity are significant concerns after serial passage. The beneficial effect of high antigen expression levels and improved immunogenicity can be limited by the propensity of rMVA to delete genes unnecessary for its lifecycle.
A first generation “Triplex” vaccine was constructed to attenuate or suppress ongoing CMV viremia and its propagation. The first-generation Triplex includes three immunodominant proteins: pp65 (major tegument protein) and a fusion between immediate early proteins IE1 and IE2 (IEfusion). These antigens have previously been combined and expressed in a single MVA vector; however, the current assembly of these antigens within MVA is not optimal for mass production of a vaccine. Upon extended viral passage, a decrease in expression of IEfusion was observed. This vaccine was successfully evaluated in a Phase I safety and dose escalation trial in 24 healthy volunteers [31].
It will be advantageous to develop an rMVA vaccine with improved stable expression of foreign protein antigens and potent immunogenicity after extended serial passage, which will enable large scale manufacturing of MVA expressing certain HCMV antigens as a clinical vector for a broader portfolio of infectious pathogens and cancer antigens.
In one aspect, this disclosure is directed to an expression system for co-expressing two or more cytomegalovirus (CMV) antigens, e.g. human CMV antigens. The expression system includes a genetically recombinant modified Vaccinia Ankara (rMVA) vector inserted with two or more nucleic acid sequences encoding two or more CMV antigens or antigenic portions thereof. In some embodiments, the CMV antigens or antigenic fragments thereof include IE1 exon 4 (IE1/e4), IE2 exon 5 (IE2/e5), IEfusion (e.g. fusion of IE1/e4 and IE2/e5), and pp65. In various embodiments, pp65 can be co-expressed with IE1/e4, IE2/e5, or IEfusion. The expression system can co-express the CMV antigens simultaneously from a single vector. In some embodiments, the nucleic acid sequences encoding the two or more CMV antigens are inserted in one or more insertion sites including 044L/045L, IGR3, G1L/18R, and Del3. Additional insertion sites include those listed in Table 1.
In some embodiments, two or more nucleic acid sequences are operably linked to and under the control of a single promoter, such as the mH5 promoter. In other embodiments, each nucleic acid sequence is operably linked to and under the control of a separate mH5 promoter. Additionally, other poxvirus promoters can be used and the use of an mH5 promoter is not required. In some embodiments, one or more nucleic acid sequences are codon optimized to remove consecutive cytosines or guanines while expressing without alteration of the same amino acids. In some embodiments, the amino acid sequences of the CMV antigens comprise one or more mutations to improve the genetic stability of the rMVA upon viral passaging. In some embodiments, IE1 and IE2 or antigenic fragments thereof are expressed as an IE fusion protein such as a fusion of IE1/exon 4 and IE2/exon 5. In some embodiments, the MVA expressing the CMV antigens is genetically stable for at least 10 passages.
Another aspect of this disclosure is directed to a vaccine comprising an immunologically effective amount of the recombinant modified vaccinia Ankara (rMVA) disclosed herein which is genetically stable after at least 10 passages.
Another aspect of this disclosure is directed to a method of eliciting or modifying an immune response and clinical protection against viremia and diseases caused by uncontrolled propagation of CMV in a subject by administering a vaccine composition as described above to the subject. In some embodiments, the subject is a mammal, such as a human.
Yet another aspect of this disclosure is directed to a method of improving the stability upon passage of an rMVA expressing two or more CMV antigens or antigenic fragments thereof by incorporating one or more of the following modifications: (1) inserting one or more nucleic acid sequences encoding the CMV antigens or antigenic fragments thereof into one or more insertion sites including 044L/045L, IGR3, G1L/18R, and Del3, as well as additional insertion sites listed in Table 1, not including Del2; (2) codon optimizing the nucleic acid sequences encoding the CMV antigens by removing consecutive cytosines or guanines; and (3) introducing one or more mutations in the amino acid sequences of the CMV antigens. In some embodiments, the CMV antigens or antigenic fragments thereof include IE1 exon 4 (IE1/e4), IE2 exon 5 (IE2/e5), IEfusion (e.g. fusion of IE1 and IE2 or IE1/e4 and IE2/e5), and pp65.
The current Triplex vaccine formulation includes three immunodominant proteins: pp65 and a fusion of immediate early proteins IE1 and IE2, but has restrictive manufacturing properties: 1) it must undergo limited passaging to maintain the stability of the IEfusion insertion; 2) restricted growth conditions to allow virus propagation without IEfusion instability; and 3) for mass production of large scale clinical lots, the current Triplex formulation is not the most efficient, long-term production strategy.
Utilizing the modified vaccinia Ankara (MVA) vaccine platform in combination with the bacterial artificial chromosome (BAC) technology, a new form of Triplex that stably expresses both IE1 and IE2 proteins in separate insertion sites over ten passages is generated. MVA is a well-characterized and clinically well-tolerated vaccine vector that is widely used for developing therapeutic vaccine strategies to treat or prevent infectious diseases or cancer. Induction of cellular immune responses by HCMV antigens IE1, IE2, and pp65 is thought to be imperative for the construction of a vaccine candidate to prevent infection or re-infection of individuals that have or will undergo hematopoietic stem cell or solid organ transplants. Disclosed herein are the construction of MVA vectors simultaneously expressing multiple HCMV antigens with insertion sites within MVA, modifications to the IE1 and IE2 components of IEfusion, and splitting IEfusion into its individual components of IE1 (exon 4) and IE2 (exon 5). The inserted HCMV antigen sequences are based on their natural HCMV DNA sequences or have been codon-optimized for efficient vaccinia virus expression. The individual HCMV antigens are separately inserted into three unique MVA insertion sites. There are four candidate insertion sites that include MVA deletion site III (Del3), a site between MVA essential genes 18R and G1L, intergenic region IGR3, and MVA 044/045L site. An ectopically inserted modified promoter H5 induces expression of the HCMV antigens from the MVA vector. Furthermore, a His to Ala amino acid substitution on the C-terminal DNA-binding domain of IE2 has aided in the stable expression of IE2 over a minimum of ten passages. Therefore, His to Ala substitutions were inserted via site-directed mutagenesis to further stabilize IE2. These mutations have helped stabilize expression of IE2 through ten passages.
In one aspect, this disclosure relates to improving the stability upon extended passage of Triplex and to retaining immunogenicity while maintaining all three antigens needed for an effective vaccine formulation. For example, one or more modifications can be made to yield an MVA that stably expresses IE1, IE2, and pp65 for efficient viral vaccine production, including but not limited to: 1) use of multiple, unique gene insertion sites in MVA that could be the preferred environment for gene stability; 2) removal of DNA mutation “hot spots” within the gene sequence that have been previously identified to include mutations at the codon “wobble” position thereby disrupting consecutive C or G nucleotides; and 3) pox virus codon-optimization for increased protein expression. In some embodiments, IEfusion is inserted into other sites within MVA. Candidate sites include Del3 [5, 6], G1L/18R [7, 8], IGR3 [9], and 044L/045L [10]. Additional insertion sites are listed in Table 1. In some embodiments, the insertion sites do not include Del2. In some embodiments, 3 or more, 4 or more, 5 or more, 6 or more consecutive C or G nucleotides in the gene sequence are disrupted by wobble base substitution that maintain identical amino acid identity.
Disclosed herein are the most stable combinations of insertion sites and gene modifications to generate an MVA that stably expresses all three CMV antigens at a minimum of 10 passages for large-scale propagation of the vaccine. Various combinations are contemplated to find the most stable combination of insertion sites that allows stable expression of IE1, IE2, and pp65: 1) splitting IEfusion into its IE1 and IE2 gene components; 2) inserting all three genes into separate insertion sites in MVA and using variant gene sequences of the inserts; and 3) explore new insertion sites in MVA. Some examples of the insertion sites are provided in Table 1:
Various modifications and/or insertion sites selection are made with the purpose of increasing the stability of Triplex simultaneously expressing IE1, IE2 and pp65 in a single MVA vector, as illustrated in
An “immunologically effective amount” as used herein means an amount that is both safe to a subject (animal or human) to be immunized and sufficient to improve the immunity of the subject. The immunologically effective amount can vary and can be determined by means of known art through routine trials.
In another embodiment, a CMV vaccine containing an immunologically effective amount of rMVA virus, which is genetically stable after serial passage can be produced by the methods disclosed herein, incorporating one or more modifications described above.
A CMV antigen can be a CMV protein antigen, a fragment of a CMV protein antigen, a modified CMV protein antigen, a fragment of a modified CMV protein antigen, a mutated CMV protein antigen or a fusion CMV protein antigen. Examples of CMV protein antigens and CMV fragments may include pp65, IE1 exon 4 (IE1/e4), IE2 exon 5 (IE2/e5), and antigenic fragments thereof. Examples of modified CMV protein antigens and fragments thereof may be found in U.S. Pat. No. 7,163,685 to Diamond et al. and is incorporated herein by reference in its entirety. Examples of mutated CMV protein antigens may be found in U.S. Pat. No. 6,835,383 to Zaia et al. and is incorporated herein by reference in its entirety. Moreover, all ranked antigens established by assessing immune response in healthy adults can be added up until reaching the maximal capacity of the MVA vector for gene insertions (see
Fusion CMV protein antigens may comprise two or more CMV proteins, modified CMV proteins, mutated CMV proteins or any antigenic fragments thereof. In one aspect, an exemplar fusion protein is a fusion of IE1 exon 4 (IE1/e4) and IE2 exon 5 (IE2/e5), IE1/e4-IE2/e5 (“IEfusion”). In one embodiment, the use of fusion proteins involves creating an IEfusion protein that comprises exon4 from IE1 and exon5 from the IE2 gene into a single gene without additional genetic material. The IEfusion protein comprises a unique representation of the immediate-early antigens than either protein alone. In another embodiment, the nucleic acid sequence encoding the IEfusion is codon optimized. In yet another embodiment, the amino acid sequence of the IEfusion protein comprises one or more His to Ala mutations in the C-terminus of IE2.
The term “genetic stability” as used herein refers to a measure of the resistance to change, with serial passage of virus, of the DNA sequence of a gene, the expression level of the gene, or both. The genetic stability of the target gene in an rMVA vector is a concern in the development of a vaccine. A reduction of the genetic stability of the target gene may have the effect of reducing the immunogenicity of the rMVA vector due to changes in gene sequence or expression level. Genetic instability of the insert gene sequence can lead to alterations of the sequence flanking the gene insertion. Suppressing the instability of the insert gene seems to curtail instability of the flanking virus DNA sequence.
Genetic stability of recombinant virus can be measured or assessed by numerous methods known in the art, e.g., testing foreign protein expression levels at each passage by Western blot (WB) or immunostaining virus plaques and calculating the percentage of foreign protein producing foci before and after serial passage. An alternative means to assess genetic stability is by real-time quantitative PCR (RT-qPCR) method, which amplifies isolated MVA genomic DNA and calculates the copy numbers of the inserted gene of interest and MVA vector after each passage. The ratio of the gene of interest copy number versus the MVA backbone vector copy number is used to determine the genetic stability of the gene or the MVA vaccine carrying the gene. A higher ratio of the gene of interest copy number to the MVA backbone vector copy number reflects a higher genetic stability, with the highest ratio=1 means approximately 100% gene expression remains after serial passage. RT-qPCR is more sensitive, high-throughput and provides highly reproducible results relative to other methods, such as Western blot or immunostaining. The method of RT-qPCR can be performed following well-known procedures in the art or the manuals of commercially available RT-qPCR kit. However, this method may not detect single nucleotide changes without accompanying sequence information. Disruptions of the coding sequence of the IE1 or IE2 inserts can prevent recognition by monoclonal antibodies that recognize intact forms.
An rMVA vaccine carrying a gene of interest is genetically stable when the DNA sequence of the gene and the expression of the gene is substantially unchanged during serial passage of the vaccine, particularly, after 10 or more passages.
Another aspect is a method for the prevention or treatment of infections or cancer in a mammal subject by administering to the subject a genetically stable rMVA vaccine disclosed herein, wherein the rMVA vaccine contains two or more CMV antigens under control of a mH5 or other poxvirus promoters, including IE1, IE2, and pp65 or antigenic fragments thereof. In some embodiments, the mammal subject is a human subject.
The nucleic acid sequences and amino acid sequences of certain IEfusions, IE proteins, and variants thereof are disclosed below.
Having described the invention with reference to the embodiments and illustrative examples, those in the art may appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in numerous publications. All references mentioned herein are incorporated in their entirety.
Materials and Methods
DATABASE SEARCHING: Tandem mass spectra (MS/MS) were extracted from a gradient 4-20% SDS-PAGE gel (Bio-Rad, USA) via in-gel trypsin digestion and subsequent peptide extraction. Charge state de-convolution and de-isotoping were not performed. All MS/MS samples were analyzed using Sequest (XCorr Only) (Thermo Fisher Scientific, San Jose, CA, USA; version IseNode in Proteome Discoverer 2.1.0.81). Sequest (XCorr Only) was set up to search crap_ncbi.fasta; Heidi_20170828.fasta; human_refseq.fasta (unknown version, 73204 entries) assuming the digestion enzyme non-specific. Sequest (XCorr Only) was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Sequest (XCorr Only) as a fixed modification. De-amidation of asparagine, oxidation of methionine and acetyl of the N-terminus were specified in Sequest (XCorr Only) as variable modifications.
CRITERIA FOR PROTEIN IDENTIFICATION: Scaffold (version Scaffold_4.8.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 36.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 98.0% probability to achieve an FDR less than 1.0% and contained at least 5 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [33]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
In this study, “stability” was assessed by the integrity of the gene of interest within MVA being monitored via polymerase chain reaction (PCR), DNA sequencing and western blot to ensure the full-length gene is present and full-length protein is present. The original design of Triplex contained a pSyn promoter that, upon serial passaging, caused instability resulting in greatly reduced protein expression. The pSyn promoter was previously replaced with a modified vaccinia virus H5 (mH5) promoter (
Therefore, the first-generation Triplex might not be acceptable to the Food and Drug Administration (FDA) as a Phase 3 manufacturing solution without rigorous validation.
Although the genomic architecture of the first-generation Triplex and the new Triplex constructs disclosed herein are similar, IEfusion inserted at other sites in MVA (Scheme I shown in
As shown in Table 2, some versions of IEfusion (IEfus) were inserted into the following sites in MVA: Del2, G1L, IGR3, or 044L/045L. After inserting IEfusion into Del3, G1L/18R, IGR3, and 044L/045L, some sites did aid in stabilizing gene expression to some extent (data not shown), but likely insufficient to meet FDA standards for late stage clinical evaluation.
Mutation hot spots were removed by disrupting the runs of consecutive C or G nucleotide bases [15], followed by vaccinia virus codon (designated as VacO) optimizing the DNA sequence of IEfusion. Constructs shown in Table 2 marked with an “X” were analyzed for stability via PCR and by western blot to monitor the integrity of the gene within its insertion site and expression after passage. This type of analysis provides insight regarding instability at either the DNA or protein levels.
Based on the knowledge that removal of spontaneous mutation hot spots minimizes instability, all IEfusion constructs shown in Table 2 were evaluated.
IEfusion 4nt inserted in IGR3 (
When the integrity of the full-length gene was compromised during passaging, non-specific PCR products would emerge and aberrant DNA sequencing results were observed around passage 3 (P3) or products similar to the size of the negative control (
The goal was to find the most stable combination of insertion sites and gene modifications to generate an MVA that stably expresses all three antigens at a minimum of 10 passages for large-scale propagation of the vaccine virus. A new vaccine construction strategy was initiated considering three main points to find the most stable combination of insertion sites that allows stable expression of IE1, IE2, and pp65: 1) splitting IEfusion into its IE1/IE2 components; 2) inserting variant gene sequences of all three genes into separate insertion sites in MVA; and 3) explore new insertion sites in MVA. Since there was limited success in enhancing stability with IE1 and IE2 as a fusion protein post nucleotide modifications, IE1 and IE2 were separated and each gene was inserted in separate insertion sites, and then stability of each gene in each of the new sites was analyzed. The original IEfusion construct was used as a template to split IE1 and IE2, with each component under the control of separate mH5 promoters [1]. Table 2 shows all the constructs generated in an attempt to give rise to an MVA that stably expresses IE1 and IE2 over ≥10 passages. Furthermore, other modifications, such as removal of consecutive C and G nucleotides as well as codon-optimization of the genes as was done for IEfusion, were incorporated. Different sequence modifications of IE1 and IE2 genes were analyzed for stability in CEFs, also using BAC technology to generate the various MVA constructs. Because instability in Del2 has been observed [15], Del2 site was not further pursued as a candidate site.
Based on the experimental setup to evaluate stability of the genes in CEFs, five potential candidates for the IE1 gene to be expressed in either G1L or IGR3 were obtained (Table 2). Only IE1 (non-codon optimized (NCO), four nucleotide optimization (4nt), and vaccinia optimized (VacO)) showed stability in G1L (
Although none of the IE2-expressing MVA constructs produced full-length IE2 protein, one construct, MVA:IE2 (044L/045L) (Table 2), expressed IE2-related products throughout all ten virus passages (
As observed in
IE1 has properties at the nucleotide level that render it unstable in some locations; inserting IE1 into a different site in MVA mitigated instability. IE2 instability could not be resolved solely by this method. Putative IE2 functional domains have been reported [20]. The C-terminus of IE2 has been described as part of a “core” domain, important for DNA binding, transactivation, and autorepression (
There were challenges to find a location and identify a sequence for IE2 that would render it “stable” for expression in MVA. Similar to the original construction of Triplex containing IEfusion, N-terminal signal peptide, nuclear localization sequences, as well as activation domains (exons 1, 2, and 3; amino acids 1-85) were omitted when generating MVA expressing IE2 [18, 26]; hence, these deletions changed the amino acid residue numbers from His446 and His452 to His363 to His369, respectively. The following single and double mutants of IE2 were generated for insertion into the 044/045L site on MVA: H363A, H369A, and H363A/H369A (Table 5). In Table 5 below, X □=unstable; ✓□=stable. Roman numeral identifies the iteration and mutation of IE2.
(i)
✓(ii)
✓(iii)
✓(iv)
✓(v)
✓(vi)
✓(vii)
✓(viii)
After transfection and viral reconstitution, all constructs were passaged in CEFs. It was observed that upon serial passaging, IE2 expression was stable based on Western blot analysis (
Identifying IE2 as a major contributor of instability, IEfusion was reassessed. The corresponding His was mutated to Ala residues on the C-terminus of IEfusion. Based on previous data (
Two constructs were identified to stably express both IE1 and pp65: (A) MVA BAC:IE1 4nt (IGR3):: pp65 (Del3) and (B) MVA BAC:: IE1 VacO (IGR3):: pp65 (Del3). Once the effect of mutating H363A and/or H369A on IE2 stability was evaluated, various mutant IE2 versions were inserted into MVA site 044L/045L of either of the aforementioned constructs. Based on the previous studies evaluating the stability of IE2, it became apparent that, although IE1 has properties at the nucleotide level that render it unstable in some locations, the instability was mitigated by inserting IE1 into a different site in MVA. In contrast, IE2 was difficult to find a location and sequence that would render it “stable” for expression in MVA. Upon mutation of C-terminal His, the gene and protein stability within the 044L/045L site was improved. Identifying IE2 as a major contributor of instability, IEfusion was reassessed. Mutants of IEfusion were generated, including mutagenizing the His residues that lie on the C-terminus of the IE2 portion. However, due to the instability of genes inserted in Del2, inserting genes within that site was not pursued. Either H363A and/or H369A mutants of IEfusion, IEfusion 4nt, and IEfusion VacO were generated. These variants of IEfusion were either inserted in IGR3 or 044L/045L, while also containing pp65 in Del3. Upon completion of the Triplex variants, vaccination of transgenic HLA-expressing mice can be used to compare immunogenicity generated by IEfusion mutants versus re-derived Triplex with separated IE1 and IE2 genes, all with His mutations as described in
Furthermore, mutation of C-terminal His prolonged gene and protein stability within the 044L/045L site for IE2 and IGR3 site for IEfusion. Three constructs (IE2 NCO H363A (i); IE2 4nt H369A (v); IE2 4nt H363A/H369A (vi)) in the context of (A) appeared stable up to P10 with all three antigens (IE1, IE2, pp65) being expressed from a single MVA (
Mutants of IEfusion were also characterized for stable expression over ten serial virus passages (
Upon complete construction of the new Triplex variants, immunogenicity studies took place to compare immunogenicity generated by IEfusion variant mutants and re-derived, second-generation Triplex with separated IE1 and IE2 variants, compared to first-generation Triplex. Transgenic C56BL/6 mice expressing HLA-B HLA-B*0702 (B7) or HLA-A*0201 (HHD-II) class I molecules were immunized with six second-generation Triplex constructs (A(i), A(v), B(i), B(iii), B(vii), IEfusion 4nt H363A (IGR3):: pp65 (Del3)) in addition to Triplex. Mice were vaccinated two times in 3-week intervals with the various constructs by the intraperitoneal (i.p.) route with either 2.5×107 PFU (for B7 mice) or 5×107 PFU (for HHD-II), followed by splenocyte isolation. Human MHC-restricted T-cell responses elicited by second-generation Triplex were compared to original Triplex and an unvaccinated, naïve group as assessed by ELISpot (Table 6). For Table 6, transgenic C57BL/6 mice expressing HLA-B*0702 (B7, top) or HLA-A*0201 (HHD-II, bottom) class I molecules were immunized with various constructs expressing either IEfusion/pp65 (IEFus) or IE1/IE2/pp65. Antigen-specific T-cell responses were determined by IFN-γ Enzyme-linked immune absorbent spot (ELISpot) assay using pp65-, IE1-, and IE2-specific libraries, HLA-B*0702- or HLA-A*0201-restricted immunodominant epitopes of pp65 and IE1. DMSO was used as a negative control. Mean and standard error of the mean (SEM) values were calculated from (N) number of either HLA-B7 (top) or HHD-II (bottom) mice. SFC: cytokine-specific spot-forming cells.
T-cell stimulation from splenocytes isolated from immunized mice was also performed to evaluate antigen-specific T-cell responses, as analyzed by FACS analysis (Table 7). For Table 7, transgenic C57BL/6 mice expressing HLA-B*0702 (B7, top) or HLA-A*0201 (HHD-II, bottom) class I molecules were immunized with various constructs expressing either IEfusion/pp65 (IEFus) or IE1/IE2/pp65. Antigen-specific T-cell responses were evaluated by intracellular cytokine staining (ICS) following stimulation with pp65-, IE1-, and IE2-specific libraries or HLA-B*0702- or HLA-A*0201-restricted immunodominant epitopes of pp65 and IE1. DMSO was used as a negative control. Percentages of IFN-γ-secreting CD8+-T cells following stimulation of splenocytes from B7 or HHD-II-immunized mice with different stimuli are shown. Mean and standard error of the mean (SEM) values were calculated from (N) number of either HLA-B7 (top) or HHD-II (bottom) mice.
Increased stability of IE2 expressed in MVA has been observed upon mutation of one or two His residues that reside within the C-terminus of IE2 protein. To examine the effect of IE2 mutants on overall IE2 stability, an MVA was constructed to harbor two copies of IE2: IE2 NCO (wild-type) in G1L and the other in the 044/045L site harboring an IE2 mutant. MVA constructs harboring two copies of IE2 were passaged to P5 in baby hamster kidney (BHK) cells (
The references listed below, and all references cited in the specification are hereby incorporated by reference in their entirety.
This application is a continuation of International Application No. PCT/US2019/031866, filed May 10, 2019, which claims priority to U.S. Provisional Application No. 62/670,656, filed on May 11, 2018, both of which are incorporated by reference herein in their entirety, including drawings.
This invention was made with government support under Grant Number CA077544, awarded by the National Institutes of Health. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20210062221 A1 | Mar 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62670656 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/031866 | May 2019 | WO |
| Child | 17095300 | US |
