Patent Application
20220184205
The present application includes a Sequence Listing which has been submitted electronically in an ASCII text format. This Sequence Listing is named 109007-23918US01_sequence listing.TXT was created on Nov. 8, 2021, is 7,251 bytes in size and is hereby incorporated by reference in its entirety.
The present disclosure relates to a system for and a method of incorporating SARS-CoV-2 genes and proteins into T4 phages. The present disclosure also relates to vaccine against SARS-CoV-2 containing recombinant T4 phages created using the method provided in the present disclosure.
Rapid discovery of safe and effective vaccines against emerging and pandemic pathogens such as the novel coronavirus SARS-CoV-2 requires a “universal” vaccine design platform that can be adapted to any infectious agent. It should be a multicomponent platform, allowing the incorporation of diverse targets, such as DNAs and proteins. Moreover, the platform would idealy also suitable for the development of multivalent vaccines, incorporating full-length proteins as well as peptides and domains in various combinations. Such a multiplex platform would not only compress the timeline for vaccine discovery but also offers critical choices for selecting the most effective vaccine candidate without going through iterative design cycles.
Though numerous vaccine platforms have been developed, most are limited to single vaccine target, require strong chemical adjuvants to boost immune responses, and lack sufficient engineering flexibility to generate multiplex vaccines. Here, a “universal” multiplex vaccine design is needed.
Tailed bacteriophages such as T4 are the most abundant and widely distributed organisms on Earth. T4 belongs to myoviridae family, infects Escherichia coli, and has served as an extraordinary model in molecular biology and biotechnology. As shown in
In addition to these essential components, the T4 capsid is coated with two nonessential proteins; Soc (small outer capsid protein) (118), which is a 9.1 kDa protein and Hoc (122), which is a highly antigenic outer capsid protein with the size of 40.4 kDa, In each capsid, there are 870 copies of Soc and 155 copies of Hoc. Soc is a trimer bound to quasi three-fold axes and acts as a “molecular clamp” by clasping adjacent capsomers. Hoc is a 170 Å-long fiber containing a string of four Ig-like domains with its N-terminal domain exposed at the tip of the fiber. Soc reinforces an already stable T4 capsid while Hoc helps phage to adhere to host surfaces. The structure of T4 is illustrated in
The above provides an ideal architecture to develop a universal vaccine design template. Therefore, exploration of CRISPR engineering of bacteriophage (phage) T4 into a potentially vaccine development platform that can be applied to any emerging pathogen is highly desirable.
According to a first broad aspect of the present disclosure, a universal vaccine design platform comprising: at least one bacterial phage; and at least one host cell comprising at least one CRISPR plasmid and at least one donor plasmid, wherein the bacterial phage can infect the host cell, wherein the CRISPR plasmid comprises a gene encoding at least one endonuclease that can be expressed within the host cell and create a cut in the genome of the bacterial phage, wherein the donor plasmid comprises at least one DNA segment that can be inserted into the genome of the bacterial phage at the cut created by the endonuclease encoded in the CRISPR plasmid, and wherein the genome of the bacterial phage comprising at least one inserted DNA segment from the donor plasmid can be packaged and released from the host cell is provided.
According to a second broad aspect of the present disclosure, a method of producing vaccine comprising: introducing at least one CRISPR plasmid and at least one donor plasmid into at least one host cell; infecting the host cell with at least one bacterial phage; and purifying the recombinant bacterial phage released from the host cell, wherein the CRISPR plasmid comprises a gene encoding at least one endonuclease that can be expressed within the host cell and create a cut in the genome of the bacterial phage, and wherein the donor plasmid comprises at least one DNA segment that can be inserted into the genome of the bacterial phage at the cut created by the endonuclease encoded in the CRISPR plasmid is provided.
According to a third broad aspect of the present disclosure, a vaccine produced using the method and platform above is provided.
According to a fourth aspect of the present disclosure, a vaccine comprising at least one recombinant bacterial phage, wherein the recombinant bacterial phage comprises at least one modification selected from the group consisting of: at least one gene encoding a component of SARS-CoV-2 inserted in the genome of T4 phage, at least one component of SARS-CoV-2 displayed on the surface of T4 phage, and at least one component of SARS-CoV-2 packaged in T4 phage but not inserted in the genome of T4 phage is provided.
Other aspects and features of the present disclosure will become apparent to those skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments of the invention, and, together with the general description given above and the detailed description given below, serve to explain the features of the invention.
Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. In the event that there is a plurality of definitions for terms herein, those in this section prevail. All patents, patent applications, publications and published nucleotide and amino acid sequences (e.g., sequences available in GenBank or other databases) referred to herein are incorporated by reference. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.
For purposes of the present disclosure, the term “comprising”, the term “having”, the term “including,” and variations of these words are intended to be open-ended and mean that there may be additional elements other than the listed elements.
For purposes of the present disclosure, directional terms such as “top,” “bottom,” “upper,” “lower,” “above,” “below,” “left,” “right,” “horizontal,” “vertical,” “up,” “down,” etc., are used merely for convenience in describing the various embodiments of the present disclosure. The embodiments of the present disclosure may be oriented in various ways. For example, the diagrams, apparatuses, etc., shown in the drawing figures may be flipped over, rotated by 90° in any direction, reversed, etc.
For purposes of the present disclosure, a value or property is “based” on a particular value, property, the satisfaction of a condition, or other factor, if that value is derived by performing a mathematical calculation or logical decision using that value, property or other factor.
For purposes of the present disclosure, it should be noted that to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about.” It is understood that whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations due to the experimental and/or measurement conditions for such given value.
For purposes of the present invention, the term “capsid” and the term “capsid shell” refers to the protein shell of a virus comprising several structural subunits of proteins. The capsid encloses the nucleic acid core of the virus.
For purposes of the present invention, the term “nucleic acid” refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art. The term should be understood to include, as equivalents, analogs of either DNA or RNA made from nucleotide analogs. The term should also be understood to include both linear and circular DNA. The term as used herein also encompasses cDNA, that is complementary, or copy, DNA produced from an RNA template, for example by the action of reverse transcriptase.
For purposes of the present invention, the term “neck protein” and the term “tail protein” refers to proteins that are involved in the assembly of any part of the necks or tails of a virus particle, in particular bacteriophages. Tailed bacteriophages belong to the order Caudovirales and include three families: The Siphoviridae have long flexible tails and constitute the majority of the tailed viruses. Myoviridae have long rigid tails and are fully characterized by the tail sheath that contracts upon phage attachment to bacterial host. The smallest family of tailed viruses are podoviruses (phage with short, leg-like tails). For example, in T4 bacteriophage gp10 associates with gpl 1 to forms the tail pins of the baseplate. Tail-pin assembly is the first step of tail assembly. The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Once the heads are packaged with DNA, the proteins gp13, gp14 and gp15 assemble into a neck that seals of the packaged heads, with gp13 protein directly interacting with the portal protein gp20 following DNA packaging and gp14 and gp15 then assembling on the gp13 platform. Neck and tail proteins in T4 bacteriophage may include but are not limited to proteins gp6, gp25, gp53, gp8, gp10, gpl 1, gp7, gp29, gp27, gp5, gp28, gp12, gp9, gp48, gp54, gp3, gp18, gp19, gp13, gp14, gp15 and gp63.
For purposes of the present invention, the term “purified” refers to the component in a relatively pure state—e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.
For purposes of the present invention, the term “immune response” refers to a specific response of the immune system of a biological specimen to antigen or immunogen. Immune response may include the production of antibodies and cellular immunity.
For purposes of the present invention, the term “immunity” refers to a state of resistance of a biological specimen to an infecting organism or substance. It will be understood that an infecting organism or substance is defined broadly and includes parasites, toxic substances, cancer cells and other cells as well as bacteria and viruses.
For purposes of the present invention, the term “immunization conditions” refers to factors that affect an immune response including the amount and kind of immunogen or adjuvant delivered to a biological specimen, method of delivery, number of inoculations, interval of inoculations, the type of biological specimen and its condition. “Vaccine” refers to pharmaceutical formulations able to induce immunity.
For purposes of the present invention, the term “bind,” the term “binding” and the term “bound” refers to any type of chemical or physical binding, which includes but is not limited to covalent binding, hydrogen binding, electrostatic binding, biological tethers, transmembrane attachment, cell surface attachment and expression.
For purposes of the present invention, the term “vector”, “vehicle”, and “nanoparticle” are used interchangeably. These terms refer to a virus or a hybrid viral particle that can be used to deliver genes or proteins.
For purposes of the present invention, the terms “efficiency of plating” and “EOP” are used interchangeably. These terms refer to a relative number of plaques that a phage stock is capable of producing. in the present disclosure, EOP is determined by dividing the pfu produced from infection of E. coli containing a spacer by the input pfu, in which the input pfu is the count of phages initially infect E. coli.
For purposes of the present invention, the term “reactogenic” refers to the property of a vaccine of being able to produce common, “expected” adverse reactions, especially excessive immunological responses and associated signs and symptoms, including fever and sore arm at the injection site. Other manifestations of reactogenicity typically identified in such trials include bruising, redness, induration, and swelling. The reactogenic effects of vaccine are often caused by adjuvant.
For purposes of the present invention, the term “codon optimization” refers to a process used to improve gene expression and increase the translational efficiency of a gene of interest by accommodating codon bias of the host organism.
For purposes of the present invention, the term “plaque” refers to clear zones formed in a lawn of cells due to lysis by phage. At a low multiplicity of infection (MOI) a cell is infected with a single phage and lysed, releasing progeny phage which can diffuse to neighboring cells and infect them, lysing these cells then infecting the neighboring cells and lysing them, etc, ultimately resulting in a circular area of cell lysis in a turbid lawn of cells.
For purposes of the present invention, the term “recombination frequency” refers to a measure of genetic linkage. Recombination frequency is the frequency, with which a single chromosomal crossover will take place between two genes. In the present disclosure, recombination frequency is determined by dividing the pfu when both plasmids are presented in the host cells by the pfu when only donor plasmid is presented in the host cells.
For purposes of the present invention, the terms “plaque-forming unit” and “pfu” are used interchangeably. These terms refer to a measure used in virology to describe the number of virus particles capable of forming plaques per unit volume. It is a proxy measurement rather than a measurement of the absolute quantity of particles.
For purposes of the present invention, the term “protective effecacy” refers to measured in a controlled test and is based on how many individuals who got vaccinated developed the ‘outcome of interest’ (usually disease) compared with how many individuals who got the placebo (dummy vaccine) developed the same outcome. In the present disclosure, the ‘outcome of interest’ include but not limited to weight loss of subject, such as an amimal, and death caused by a disease.
Genetic, biochemical, and structural studies on phage T4 including the recently developed CRISPR phage engineering constitute an extraordinary resource for creating universal vaccine development platform using T4. The atomic structures of all the capsid proteins including Soc, Hoc, as well as the entire capsid have been determined. It has also been demonstrated that Soc and Hoc can be used as efficient adapters to tether foreign proteins to T4 capsid. Both Soc and Hoc have nanomolar affinity and exquisite specificity, allowing up to 1,025 molecules of full-length proteins, domains, and peptides to be arrayed on capsid. T4 capsids so decorated with pathogen epitopes mimic PAMPs (pathogen-associated molecular patterns) of natural viruses and stimulate strong innate and as well as adaptive immune responses.
The large amount of nonessential genetic space available in T4 genome is usefule in developing a universal vaccine design template. SARS-CoV-2 (110) comprises nucleocapsid protein (NP) molecules (102), genome RNA (106), spike trimer (108) and envelope (E) epitopes (104), as shown in
In one embodiment, A number of SARS-CoV-2 components were incorporated into T4 phage by CRISPR engineering. The design was such that each of these components occupied an appropriate compartment in the phage nanoparticle as shown in
In one embodiment, when tested in mouse and New Zealand White rabbit models, T4 phage decorated with S-trimers elicited robust ACE-2 receptor blocking and virus neutralization established a new bacteriophage nanovaccine framework for rapid and multiplex design of effective vaccine candidates potentially against any emerging pathogen in the future.
Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering
In one embodiment, a series of CRISPR-E. coli strains were constructed to insert SARS-CoV-2 gene segments into T4 phage genome.
In one embodiment, four nonessential regions of the T4 genome were chosen for insertion of various SARS- CoV-2 genes, as illustrated in
In one embodiment, the recombinant phages were constructed by deleting certain known “nonessential” segments of the phage genome. The “nonessential” segments deleted included about 18 kb FarP, about 11-kb 39-56, or both (about 29 kb), that created space for CoV-2 insertions. The locations of segment FarP and segment 39-56 on T4 genome are illustrated in
In another embodiment, the recombinant phages were constructed by deleting shorter segments, since yield is critical for vaccine manufacture. The shorter segments deleted included about 675 bp SegF within 39-56 and about 7 kb segment within FarP. The structure of T4 genome is illustrated in
In one embodiment, three SARS-CoV-2 spike gene variants corresponding to i) 1273 aa WT full-length (S-fl), ii) 1208 aa ectodomain (S-ecto, aa 1-1208), and iii) 227 aa receptor binding domain (RBD, aa 319-545) were engineered as expressible cassettes and inserted into 7del.SegFdel. T4. The three spike gene variants and their respective location of insertion are illustrated in
In one embodiment, the spike gene variants were codon-optimized and kept under the control of a strong mammalian expression promoter, either CMV or CAG, and a human CD5 signal peptide fused to the N-terminus for efficient secretion. The S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome are illustrated in
In one embodiment, CRISPR E. coli cells containing the Cas9/Cpf1-spacer plasmid but lacking the spike gene donor plasmids was created as a control. The control CRISPR E. coli cells gave very few or no plaques when infected with 7del.SegFdel phage.
In one embodiment, the phage plaques formed by phages obtained from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer combined with S-ecto donor were compared. As shown in
In one embodiment, the EOP of different Cpf1-FarP7K and Cpf1-SegF spacers were different. As shown in
In one embodiment, the recombination frequency of using the CRISPR E. coli cells containing both the Cas9/Cpf1-spacer plasmid and donor plasmid is upto about 4.5%. The recombination frequency is the pfu when both plasmids are presented in the CRISPR E. coli cells divided by the pfu when only donor plasmid is presented in the CRISPR E. coli cells.
In one embodiment, at least 95% of recombinant T4 phages contained the correct insertion of spike genes.
In one embodiment, the phages with an insertion of spike genes have similar plaque forming ability as the WT phage.
In one embodiment, a similar CRISPR strategy as described above was used for creating deletions and/or insertions at the other sites, indlucing IPIII, IPII, Hoc, and Soc. The location and structure of IPIII, IPII, Hoc, and Soc are illustrated in segments III and IV of
SARS-CoV-2 infected patients have been reported to generate robust NP-specific immune responses including cytotoxic T cells that might be important for protection and virus clearance2.
In one embodiment, NP was incorporated into the T4 nanoparticle by designing a CRISPR strategy that packaged NP protein molecules inside the phage capsid along with the genome. As NP is a nucleic acid binding protein, the packaged phage genome might provide an appropriate environment to localize this protein.
During T4 phage morphogenesis, the major capsid protein gp23 assembles around a scaffolding core formed by a cluster of proteins including three nonessential, highly basic, “internal proteins”; IPI, IPII, and IPIII. Following assembly, most of these scaffold proteins are degraded to small peptides and expelled from the capsid creating space for genome packaging. This assembly process is illustrated in
Previous studies showed that when the IPs are replaced with foreign proteins fused to N-terminal CTS, the foreign proteins are targeted to the core and remain in the capsid interior following CTS removal4.
In one embodiment, IPs were replaced with CoV-2 NP.
In one embodiment, an amber mutation was introduced into the CTS sequence, changing the TTT corresponding to Phe at site aa 7 to TAG corresponding to amber, because, for unknown reasons, the donor plasmid containing the WT CTS sequence was found to be toxic to E. coli. This CTSa-NP phage when grown on amber suppressor E. coli B40 (Sup1) or BL21-RIPL (Sup1) expressed the nucleocapsid protein and encapsidated it as demonstrated by SDS-PAGE shown in
In one embodiment, the copy number of NP is about 70 NP molecules per phage capsid.
In one embodiment, SARS-CoV-2 antigens were incorporated onto the nanoparticle surface. In order to incorporate SARS-CoV-2 antigens on the surface of T4, Hoc and Soc genes were deleted from each of the above spike & NP phage genomes to create T4-SocΔ-HocA phages and then Hoc- and Soc-fused CoV-2 genes under the control of their respective native promoters were inserted. In one preferred embodiment, the inserted Hoc-fused CoV-2 gene encodes E epitope and the resulting recombinant T4 phages are T4-SocΔ-(E epitope-Hoc) phages. The inserted Hoc- and Soc-fused CoV-2 genes, upon phage infection, would express and assemble the epitopes encoded by the inserted CoV-2 genes on T4 capsid surface. The steps of constructing T4-SocΔ-HocA and T4-SocΔ-(E epitope-Hoc) phages are illustrated in
In one embodiment, the inserted E epitope was constructed by fusing the gene segments corresponding to the N-terminal 12-aa ectodomain peptide (Ee) or the 18-aa peptide from the C-terminal region (Ec) of CoV-2 envelope (E) protein fused to the N-terminus of Hoc. The pentamieric structure of CoV-2 envelope (E) protein and the Ee and Ec domains are illustrated in
These peptides are predicted to be exposed on the SARS-CoV-2 virion and shown to elicit T cell immune responses in humans5. In one embodiment, by virtue of fusion to the N-terminus of Hoc, these epitopes would be exposed at the tip of the ˜170 Å-long Hoc fiber. The Ee and Ec recombinant phages indeed showed an upward shift of the Hoc band upon SDS-PAGE, consistent with the increase of mass of the fused peptides. The SDS-PAGE is shown in
In one embodiment, the 12-aa Ee peptide was displayed at the maximum copy number, up to about 155 copies per capsid, without significantly affecting phage yield.
In one embodiment, the 18-aa Ec peptide showed lower epitope copies, as indicated by the fade Hoc band in the T4-S-NP-SocΔ-(Ec-Hoc) column of
In one embodiment, CoV-2 receptor binding domain (RBD) was displayed on the capsid surface as a Soc-fusion, using a similar strategy as described above.
In one embodiment, the copy number of the displayed RBD was very low, due to inefficient in vivo display of E. coli-expressed Soc-RBD on T4 phage.
RBD contains ˜82.5% non-hydrophilic residues. In one embodiment, RBD formed insoluble inclusion bodies when expressed from a strong promoter. The insolubility of RBD was confirmed in the solubility analysis shown in
In one embodiment, numerous N- and C-terminal truncations of RBD, one of which is the shortest receptor binding motif of 67 aa, were constructed, however none showed a significant improvement in solubility and copy number (
In one embodiment, E. coli expressing Soc-RBD from a plasmid under the control of the phage T7 promoter constructed. Under the control of the phage T7 promoter, the pre- expression of Soc-RBD can be kept at low level.
In one embodiment, a low level pre-expression of Soc-RBD would keep it in soluble form that could then assemble on capsids produced during phage infection.
In one embodiment, the well-established spytag-spycatcher technology was deployed to display RBD on T4 phage. The optimized spycatcher and spytag from Streptococcus pyogenes, interact with eath other at least picomole affinity, indicating approaching “infinite” affinity with second-order rate constant: 5.5×105M−1 s−1, and exquisite specificity that then leads to a covalent bond formation7. In one embodiment, to display RBD, phage decorated with the ˜12.6 kDa soluble spycatcher was produced by growing the T4-Spike-Ee-NP-SocΔ phage on E. coli expressing Soc-spycatcher fusion protein from the T7 expression plasmid. The expression of the Soc-spycatcher fusion protein is illustrated in
In another embodiment, RBD was expressed as Sumo-RBD-Spytag fusion protein in E. coli. The Sumo domain was supposed to enhance the expression and solubility of RBD but it resulted in only a small improvement, as shown in
In one embodiment, the sRBD and rRBD phages produced as above, such as T4-Spike-Ee-CTSam-NP-sRBD and T4-Spike-Ee-CTSam-NP-rRBD, bound to human ACE2 receptor protein.
In one embodiment, rRBD phages also bind to some of the RBD-specific monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) but not to all, as shown in
Decoration of Phage T4 Nanoparticles with Spike Ectodomain Trimers
In one embodiment, spike ectodomain (S-ecto) trimers (433.5 kDa) were displayed on T4-Spike-Ee-NP-SocΔ phage. The pre-fusion stabilized hexa-Pro S-ectodomain construct described as above was fused to a 16-aa spytag at the C-terminus and expressed in ExpiCHO cells. The ectodomain trimers secreted into the culture medium were purified by HisTrap affinity chromatography and size-exclusion chromatography. The successful construction of the trimers were confirmed by Size-exclusion chromatography (SEC) as shown in
In one embodiment, the trimers-decorated T4 phage efficiently bound to human ACE2 receptor.
In one embodiment, the T4-CoV-2 vaccine candidates generated as above by sequential engineering.
1. T4-Wild type
In one embodiment, the vaccine candidates obtained using the sequential engineering method above can be screened for their immunogenicity and protective efficacy in a mouse model. Schematic diagram in
In one embodiment, the vaccine candidates produced using the above method can induce immune responses, as evidenced by the SARS-CoV-2-specific antibody titers. The SARS-CoV-2-specific antibody titers were determined by ELISA using purified proteins, including S-ecto, RBD, NP, or E as coating antigens.
In one embodiment, recombinant phages that delivered CoV-2 DNA alone did not induce significant titers of spike-specific antibodies.
In one embodiment, recombinant phages that delivered CoV-2 DNA did not elicit anti-RBD antibody titers after boost-1 with same particles (CoV-2 DNA). But they were able to elicit significant antibody titers if boost 2 phage nanoparticles were displayed with ectodomain trimers.
In one embodiment, strong antibody titers were elicited against T4 nanoparticle-delivered protein or peptide epitopes, either displayed on surface or packaged inside. These include E-specific antibodies, NP-specific antibodies, RBD-specific antibodies, and spike-specific antibodies, as shown in
In one embodiment, antibodies elicited were specific to the conformation of the displayed protein. For instance, the antibodies elicited against sRBD or rRBD displayed on phage reacted poorly with the mammalian expressed S-ecto trimer or RBD, as evidenced by the lower antibody titers of groups 6 and 7 (G6 and G7) in
In one embodiment, the endpoint titers elicited by phage-delivered trimers without any adjuvant were as high as those generated using Alhydrogel as an adjuvant.
In one embodiment, IgG subclass specificity data indicated that phage nanoparticles stimulated both humoral (TH2) and cellular (TH1) arms of the immune system. In mice, IgG2a subclass represents TH1 response whereas IgG1 class reflects TH2 response. The adjuvant-free T4 nanoparticles (G8 and G9) elicited high levels of both IgG1 and IgG2a classes against all three SARS-CoV-2 antigens, including spike/RBD, E, and NP, as shown in
In one embodiment, phage nanoparticles gave slightly higher TH1-derived IgG2a antibodies than the TH2 derived IgG1 class antibodies, while the alum-adjuvanted mice elicited two orders of magnitude lower IgG2a antibodies, as showin in
In one embodiment, the T4-stimulated spike-specific antibodies blocked binding of RBD to human ACE-2 expressed on HEK293 cells in a dose-dependent manner.
In one embodiment, these antibodies elicited by the engineered phage vaccine candidates described above also exhibited strong virus neutralizing activity as determined by Vero E6 cell cytopathic assay using the live SARS-CoV-2 US-WA-1/2020 strain (BSL-3).
In one embodiment, the neutralization titers correlated with protective efficacy when mice were challenged with mouse-adapted SARS-CoV- 2 MA10 virus12. The protective efficacy can be reflected by the body weight changes of immunized animal and the rate of survival.
In one embodiment, the most effective vaccine candidate down-selected from the mouse study, the T4 phage-decorated trimers, was evaluated for its ability to induce virus neutralizing titers in a different animal model, including the New Zealand White rabbit. The immunization of rabbits followed the formulations, groups, and prime-boost immunization scheme shown in
In one embodiment, the phage vaccine candidate selected using the presently disclosed method was highly effective, which generated robust virus neutralization titers in two different animal models, mouse and rabbit, which also resulted in complete protection against acute viral infection in mice.
In one embodiment, a “universal” vaccine platform was developed centered around the bacteriophage T4 nanoparticle. As demonstrated in the present disclosure, a number of special features would make this a powerful platform to rapidly generate vaccine candidates against any emerging and pandemic pathogen in future.
In one embodiment, a series of recombinant phages containing SARS-CoV-2 gene insertions were generated in mere days by CRISPR genome engineering using a combination of type II Cas9 and type V Cpf1 nucleases. This combination provides built-in choices for spacers as well as for efficient cleavage of T4 genome that is extensively modified by cytosine hydroxymethylation and glycosylation, for attaining near 100% success13, 14.
In another embodiment, a large amount of genetic and structural space available in phage T4 was exploited to incorporate CoV-2 DNAs, peptides, proteins, and complexes into the same nanoparticle. In a preferred embodiment, at least 6 kb full-length spike gene expression cassette, 2.7 kb RBD gene expression cassette, and about 1.3 kb nucleocapsid gene were inserted into the same genome by replacing certain nonessential genetic material. In one embodiment, the genetic space was further expanded by inserting Hoc and Soc fusions, and/or replacing additional nonessential segments that span across the genome. In another embodiment, up to 155 copies of a 12-aa Ee peptide and ˜100 copies of 433.5 kDa S-trimers were displayed on the same nanoparticle while ˜70 molecules of 50-kDa NP were packaged inside the structure. These represent an extremely large payload carried by any vaccine delivery vehicle reported thus far.
In one embodiment, different areas of phage nanostructure were utilized for placing different vaccine cargos. In a preferred embodiment, the tips of Hoc fibers with ˜170 Å reach were used to display short 12-aa Ee peptide epitopes as this would allow efficient capture by antigen presenting cells, B cells, and T cells15. In one embodiment, these vaccine candidates resulted in strong antibody titers. At the same time, the nucleocapsid protein was co-packaged with phage genome as this mimics NP's natural environment as an RNA binding protein.
In one embodiment, the T4 platform could be readily adapted to mammalian-expressed proteins, which might be essential for proper folding and glycosylation, as in the case of spike trimers, through formation of highly efficient spycatcher-spytag bridges. As the Cryo-EM structure showed, such spikes anchored to capsid with their RBDs well-exposed in some respects mimic the spikes present on SARS CoV-2 virion and might provide a more native context for stimulating effective immune responses.
In one embodiment, alternative strategies such as pre-expression from a plasmid and display through subsequent phage infection provided additional advantages such as enhancing copy number and better control over folding. As demonstrated, this approach resulted in 3-6 fold higher copy number of Soc-spycatcher on phage capsid. However, RBD still remained poorly folded.
In one embodiment, sequential engineering generated a pipeline of SARS-CoV-2 vaccine candidates in mere weeks, and allowed down-selection of the best vaccine candidate, phage decorated with trimers, in a single animal experiment. However, the DNA-alone vaccines failed to elicit immune responses.
In one embodiment, unlike the DNAs, the peptide/protein epitopes, either displayed on surface or packaged inside, generated robust immune responses. The strong virus neutralization titers and ACE-2 blocking titers elicited by T4-delivered trimers in two different animal models, mouse and rabbit, are particularly noteworthy. These responses correlated with protective efficacy where all the vaccinated mice were protected from acute viral infections.
In one embodiment, the T4 phage vaccines generated balanced TH1 and TH2 derived antibody responses against all three CoV-2 antigens tested. In fact, T4 seems to have a slight TH1-bias, a desirable property that distinguishes this platform from other subunit vaccine platforms and adjuvant systems. The TH1-bias might be because the phage-decorated antigens are recognized as PAMPs present on natural viruses thereby triggering host responses through Toll-like receptors (TLR) 2 mediated innate immunity pathways. In one embodiment, TLR2 and TLR4 pathways are stimulated by the engineered phage vaccines in in vitro cultured cells (data not shown).
In one embodiment, the T4 nanoparticle vaccine does not require an adjuvant to stimulated robust anti-CoV-2 immune responses, as demonstrated in two different animal models. Therefore, in addition to reducing cost and manufacturing complexity, the adjuvant-less T4 vaccine formulations are less reactogenic that is often associated with the chemical adjuvants used in traditional vaccine formulations. In numerous immunizations with T4 phage performed in a variety of animal models including mouse, rat, rabbit, and macaque, no significant reactions were noted.
In one embodiment, one of the best T4 vaccine candidates elicits, in addition to spike-specific antibodies, broad antibody responses against two additional virion components, E that is exposed on the surface and NP that is abundantly present on CoV-2 infected host cells. In one embodiment, the spycatcher phage could serve as a backbone to capture ectodomain trimers from other coronaviruses, or any spytagged antigen(s) from other infectious agents. Thus, it is conceivable that different vaccine formulations can be “created” at the site of administration by mixing the spycatcher T4 phage with the desired antigen(s) combinations. Co-carrying multiple and distinct antigens on the same nanoparticle can increase the breadth of the elicited immune responses16,17. Since the S-ecto, Ee and NP are conserved in other coronaviruses such as SARS-CoV-1, the T4 vaccine platform might be considered for expanded protection.
In one embodiment, a versatile “universal” vaccine design template by which vaccine candidates for any emerging or pandemic infectious agent can be rapidly generated, was developed and evaluated in animal models. As T4 is a highly stable nanoparticle, has good safety profile and no pre-existing antibodies in humans, manufactured for mass production at a relatively low cost, it provides a robust platform to rapidly generate effective vaccines against epidemic- and pandemic-causing pathogens in the future, particularly when multivalent vaccine candidates are essential to protecting global communities.
Having described the many embodiments of the present disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure, while illustrating many embodiments of the invention, are provided as non-limiting examples and are, therefore, not to be taken as limiting the various aspects so illustrated.
The expression vector pET28b (Novagen®, MA) was used for donor plasmid construction and protein expression plasmid construction. LbCpf1 and SpCas9 plasmids were constructed for spacer cloning13, 14. Briefly, SpCas9 plasmids were constructed by cloning spacer sequences into the streptomycin-resistant plasmid DS-SPCas (Addgene® no. 48645). Sequences of the spacers are shown in table below. LbCpf1 plasmid was constructed by replacing Cas9 and its spacer cassette in SpCas9 plasmid with Cpf1 and spacer cassete.
The DNA fragment containing NP and RBD were codon-optimized for E.coli expression and synthesized by GeneArt (Thermo Fisher®). The plasmids containing wild-type SARS-CoV-2 Spike (S) gene and S-ecto-6P gene were provided by Dr. Kizzmekia S. Corbett (National Institutes of Health) and Dr. Jason S. McLellan (University of Texas, Austin). The RBD gene for mammalian expression was amplified from the wild-type Spike (S) gene. SpyCatcher/Spy-tag and SUMO containing plasmid were purchased from Addgene® (#133449 and #111560).
E. coli strain DH5a cell (hsdR17 (rK−mK+) sup2) (NEB®) was used for all the clone construction. The E. coli BL21-CodonPlus (DE3)-RIPL (Novagen®, MA) was used for the expression of recombinant proteins. P301 (sup0) and B834 (hsdRB hsdMB met thi sup0were used for the propagation and recombination of phages without amber mutations. The BL21 (DE3) RIPL transformed with amber-suppressor plasmid and E. coli B40 (sup1) were used for the propagation and recombination of phages with amber mutations (CTS-amber-NP). Wild-type T4 phage was used as a starting phage of CRISPR engineering and propagated on E. coli P301 or B834.
CRISPR-LbCpfl/SpCas9 plasmid was constructed based on the streptomycin-resistant plasmid DS-SPCas (Addgene® no. 48645)13, 14. The spacer containing fragments were prepared by annealing and extension of two amplified DNA fragments containing 26 bp complementary nucleotides (overlap extension PCR). The spacer fragment digested by restriction enzymes XhoI&EagI was cloned into linearized LbCpf1/SpCas9 plasmid. Sequences of the spacers are shown in the table above in Example 1.
The donor plasmids for deletion/insertion, including Hoc-del, Soc-del, FarP7K-del, FarP18K- del, 39-56 11K-del, SegF-del, IPIII-del, IPII-del, E insertion (Hoc site), Soc-SpyCatcher/RBD insertion (Soc site), CTS-amber-NP insertion (IPIII site), CAG-S-fl/S-ecto insertion (FarP7K site), and CMV-RBD insertion (SegF site), were constructed using overlap extension PCR. Briefly, the corresponding ˜500 bp homology arms (left and right) were amplified from T4 genome DNA, stitched, and cloned into pET28b linearized with BglII and XhoI to generate the deletion donor plasmid. For the insertion donor plasmid, the insertion fragment, left homology arm, and right homology arm, which contains 25 bp complementary nucleotides each other, were stitched by annealing and extension. The BglII&XhoI digested donor fragment was cloned into pET28b.
The Soc-fused expression plasmids, including Soc-SpyCatcher, Soc-RBD, RBD-Soc, SUMO- Soc-Spy, and Soc-truncated RBD (RBD67, RBD106, RBD135, RBD162, RBD181, and RBD197), were constructed by two rounds of cloning. First, the MCS (NcoI, NdeI, NheI, and BmtI)-linker (4GGS)-Soc-linker (2GGGGS)-MCS (HindIII, EagI, NotI, and (hop was amplified using Soc- plasmid template, and cloned into the pET28b DNA linearized with Ncol and XhoI restriction enzymes to generate pET28b-MCS-L-Soc-L-MCS. Second, SpyCatcher, SUMO, Spy, and/or various RBDs were amplified and inserted to 3′- or 5′- MCS of pET28b-MCS-L-Soc-L-MCS as needed. CTS-NP and Ee-Hoc expression fragments were amplified using synthesized NP and T4 genomic DNA respectively, and cloned into Ncol&XhoI-linearized pET28b.
The plasmids for expression in mammalian cell, including pCMV-CDS-RBD, pCAG-CDS-S-fl, pCAG-CDS-S-ecto-6P, and pCAG-CDS-S-ecto-6P-Spytag, were constructed. The RBD fragment was amplified using the wild-type spike gene and CD5 secretion leading peptide (MPMGSLQPLATLYLLGMLVASVLA) was added to the N terminus of RBD by PCR. The CD5-RBD was cloned into pAAV vector (Cell Biolabs) using HindIII and XhoI to construct pCMV-CD5-RBD. For the construction of pCAG-CD5-S-fl, pCAG-CD5-S-ecto-6P, and pCAG-CD5-S-ecto-6P-Spytag, plasmids pCAG-S-fl and pCAG-S-ecto-6P were used as template and backbone. The CD5 fragment was cloned into N terminus of S-fl or S-ecto-6P using KpnI and EcoRI. Similarly, Spytag (RGVPHIVMVDAYKRYK) was cloned into the C terminus of S-ecto using BamHI and XhoI. All the constructed plasmids were sequenced to confirm correct fragment insertion (Retrogen®, CA).
Plaque assay was applied to determine the efficiency of the individual spacers to restrict T4 phage infection. The CRISPR-Cpfl/Cas9 spacer plasmid was transformed into E. coli strains B834 or B40. The serial diluted T4 phages, which was in the range of 101 to 107 in 100 μl PI-Mg buffer (26 mM Na2HPO4, 68 mM NaCl, 22 mM KH2PO4, 1 mM MgSO4, pH 7.5), was mixed with 350 μl of spacer-containing E. coli (108 cells/ml). E. coli cells without spacer were used as control. After incubation at 37° C. for 7 min, 3.5 ml of 0.75% top agar with spectinomycin (50 μ/mL) was added into each tube, mixed, and poured onto LB plate. The plates were incubated at 37° C. overnight to produce plaques. The plaque-forming units (pfu) were counted on each plate and the efficiency of plating (EOP) was determined by dividing the pfu produced from infection of E. coli containing a spacer by the input pfu.
The CRISPR-Cpfl/Cas9 spacer plasmid and the corresponding donor plasmid were co-transformed into E. coli strains, either B834/P301 without amber suppressor or B40/RIPL with amber suppressor as needed. Single-plasmid transformed E. coli cells, either with the donor plasmid or with the CRISPR spacer plasmid, were used as controls. An appropriate amount of T4 phages, which were determined by the EOP as described above, were added to E. coli cells containing spacer&donor and incubated for 7 min at 37° C. After adding 3.5 ml 0.75% top agar with 50 μg/ml spectinomycin and 50 μg/ml kanamycin, the infection mixture was poured onto LB plate and incubated overnight. Single plaque, which was named G1 (Generation 1) was picked using a sterile Pasteur glass pipet and transferred into a 1.5 ml Eppendorf tube containing 200 μl of PI-Mg buffer. After 20 min incubation at room temperature with gently vortexing every 5 mins, serial diluted G1 phages were used to infect spacer-containing E. coli cells (50 μg/ml spectinomycin). The resultant single G2 plaque was picked and used to infect E. coli cells (without spacer or donor) to produce G3 phages. Single G3 plaque was picked into 200 μl of PI-Mg buffer. PCR analysis was applied to check T4 DNA deletion or foreign gene insertion. One microliter G3 phages were denatured at 94° C. for 8 min and used as a template for PCR using Phusion High-Fidelity PCR Master Mix (Thermo Fisher®). The amplified DNA fragment was purified using QlAquick® Gel Extraction Kit (Qiagen®) and then was sequenced (Retrogene®). The sequencing confirmed G3 phages were added a few drops of chloroform, and stored at 4° C. as zero stocks for the next study. More rounds of CRISPR gene editing can be similarly introduced into the same phage as described above.
E. coli strains B40 or B834 were used for the production of amber-phage or non-amber-phage, respectively. Fresh overnight-cultured E.coli cells were inoculated in 1 L of Moore's media (20 g tryptone, 15 g yeast extract powder, 2 g dextrose, 8 g NaCl, 2 g Na2HPO4, 1 g KH2PO4, dissolved in 1 L MQ water) at 1/50 dilution, and then cultured at 37 C for 2-2.5 hrs in a shaker incubator at 200 RPM. When the cells reached the density of ˜4×108/ml, phages were added at a multiplicity of infection (MOI) 0.5. The infection mixture was cultured at 37 C, 200RPM, for another 2.5 -3 hours and periodically checked under the microscope. The shape of phage-infected cells usually is changed from long bacilli to short dumbbell shape.
When the cell number started dropping, the culture was transferred to Sorvall GSA bottle and centrifuged at 27,504 g for 1 hr at 4 C. The supernatant was discarded and the pellet was resuspended in 50 ml PI-Mg buffer containing 10 μg/ml DNase I and one tablet of protease inhibitor cocktail. The resuspended pellet was added with 5 ml chloroform and incubated at 37 C for 1 hr to lyse the bacteria and release the phage. Then the debris was removed by low-speed centrifugation at 4,302 g for 10 min. The phage-containing supernatant was transferred to a sterilized falcon tube and the pellet was discarded. The titer of this phage stock was determined using B40 or B834. The working stock of produced phages can be stored at 4° C. for long periods of time (a few drops of chloroform added), or used as seed phage to make Spy-Catcher or Soc-RBD induction-displayed phage, or purified as vaccine candidate for animal study as described below.
The produced ˜50 ml phage stock was distributed into 5 Corex glass tubes (10 ml each) and seal with parafilm. The phage stock was centrifuged at 34,540 g for 1 hr using a Sorvall® SS34 rotor. The supernatant was discarded and the phage-containing pellet was resuspended with 1-3 ml PI-Mg buffer plus 5 ul Benzonase overnight at 4 C. Next, the resuspended phages were loaded on CsC1 gradient solution and centrifuged at 152,000 g for 1 hr in a Beckman® ultracentrifuge using a SW 55 Ti swinging bucket rotor. The purified phage band localizes between CsC1 densities 1.46 and 1.55, and was collected by inserting a syringe right below the phage band and aspirating the band. The phages were dialyzed in high-salt Tris-Mg buffer (10 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM MgCl2) for 4 hrs followed by low-salt Tris-Mg buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgC12) overnight. The second-round CsC1 centrifugation and dialysis of purified phages were applied to obtain purer phages. Two-round- CsCl-purified phages were further purified by passing through a 0.22 um filter unit to remove any minor contaminants. The phage concentration and copy numbers of displayed antigens were examined by 4-20% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The phage genomic DNA was released and digested by treatment with frozen-thaw and Benzonase before SDS-PAGE.
E. coli BL21 (DE3) RIPL transformed with T7-Soc-Spycatcher (or Soc-RBD) plasmid was used for Soc-Spycatcher (or Soc-RBD) displayed phage production. E. coli BL21 (DE3) RIPL co-transformed with T7-Soc-Spycatcher (or Soc-RBD) plasmid and amber suppressor plasmid was used for the production of Soc-Spycatcher (or Soc-RBD) displayed and NP protein packaged phage. Briefly, the RIPL cells were inoculated in 1 L of Moore's Media at 1/50 dilution with appropriate antibiotics as needed (RIPL-Soc-Spycatcher: 50 μg/ml Kanamycin+37 μg/ml Chloramphenicol; RIPL-Soc-Spycatcher-Amber Suppressor: 50 μg/ml Kanamycin +37 μg/ml Chloramphenicol +100 μg/ml Ampicillin). The culture was incubated at 37° C., 200 RPM, for 2.5-3 hrs. When the cells reached the density of ˜4×108/ml, 0.5 mM IPTG was added for induction. At 10 min post IPTG addition, the corresponding phages (Hoc-del/Soc-del/IPII-del/IPIII-del) were added to infect cells at MOI 0.5. The culture was further incubated at 37 C, 200 RPM, for 3 hours. The following production and purification procedures are the same as described above.
The LAL chromogenic endotoxin quantitation kit (Thermo Fisher®) was used to measure the amount of endotoxin in phage sample using the Limulus Amebocyte Lysate (LAL) assay according to the manufacturer's instructions. Phage samples were diluted with a 2-fold dilution series beginning with an initial 1010 particles/50 μl in endotoxin-free water. LAL reagent was added and incubated at 37 C, followed by the addition of chromogenic substrate solution. After 6 mins incubation, stop reagent was added and the absorbance was measured at 405 nm. The endotoxin concentration of phage sample was determined using the formulated standard curve. The endotoxin threshold for phage immunization was <0.5 EU/1010 particles.
Plasmid pCAG-CDS-S-ecto-6P-Spytag was transiently transfected into ExpiCHO cells using ExpiFectamine CHO transfection kit (Thermo Fisher®). After 18-22 hours of transfection cells were supplemented with ExpiCHO Feed and Enhancer and grown at 32° C. according to the manufacturer's High Titer protocol. Cultures were harvested 8-10 days after transfection by centrifuging the cells at 3000 g for 20 minutes at 4 C. The supernatant was clarified through a 0.22 μm filter and then loaded on a HisTrap HP column (Cytiva®) previously equilibrated with wash buffer (50 mM Tris-HC1, pH 8.0, containing 300 mM NaCl and 20 mM imidazole), at a flow rate of 1 ml/minute, using AKTA® Prime-Plus liquid chromatography system (GE® Healthcare). Protein-bound column was washed with wash buffer until the UV absorbance reached the baseline to remove non-specifically bound proteins. The trimers were eluted using a 20 mM-300 mM linear gradient of imidazole. HisTRAP eluted peak fractions were pooled and applied to a Hi-Load 16/600 Superdex-200 (preparation grade) size exclusion column (GE® Healthcare) equilibrated with the gel filtration buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl) to obtain further purified trimers, using the AKTA® FPLC system (GE® Healthcare). Eluted fractions were collected, filtrated by 0.22 um filter unit, flash-frozen, and stored at ˜80° C. until use for the Soc-spycatcher mediated display on various phage vaccine candidates.
S Trimer or rRBD Display on T4-SpyCatcher Phage
In vitro display of S-trimer/rRBD on the T4-SpyCatcher phage was assessed by the co-sedimentation18, 19. Briefly, two-round CsC1 purified and 0.22 um filtered phage particles were sedimented for 45 min at 34,000 g in Protein-LoBind Eppendorf tubes, washed twice with sterilized phosphate-buffered saline (PBS) buffer (pH 7.4), and resuspended in PBS buffer (pH 7.4). S-trimer/rRBD was sedimented for 25 min at 34,000 g to remove possible aggregates. T4-SpyCatcher phages were incubated with S-trimer/rRBD proteins at 4° C. for 1 hr. The mixtures were sedimented by centrifugation at 34,000 g for 45 min, and unbound proteins in the supernatants were removed. After washing twice with excess PBS to further remove the unbound protein and any other minor contaminants, the phage pellets containing the displayed proteins were incubated at 4° C. overnight and then resuspended in PBS. For rabbit animal studies, fifty microliters of phage-trimer particles were added to blood agar (TSA with sheep blood) to examine any contamination of a wide variety of fastidious microorganisms. LPS level was also examined using LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher®). The resuspended pellets were analyzed using Novex 4-20% SDS-SDS-PAGE mini gel (Thermo Fisher Scientific®, Waltham, Mass.) to quantify the S trimer/rRBD copies. After Coomassie Blue R-250 (Bio-Rad®, CA) staining and destaining, the protein bands on SDS-PAGE gels were scanned and quantified by ChemiDoc™ MP imaging system (BioRad®) and image J. The copy numbers of SpyCatcher and displayed S-trimer/rRBD molecules per capsid were calculated using gp23 or gp18 as the internal control (930 copies of gp23 and 138 copies of gp18 per capsid) and S-trimer protein standard.
The E. coli expression, denaturing, refolding, and purification of SUMO-RBD-Spy (rRBD) were performed20. Briefly, the BL21-CodonPlus (DE3)-RIPL cells containing PET28b-SUMO-RBD-Spy were induced with 0.5 mM isopropyl--D-1-thiogalactopyranoside (IPTG) for 3 h at 28° C. Cells were harvested and resuspended in buffer A (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 5 mM β-mercaptoethanol, pH 7.9) containing protease inhibitor cocktail (Roche®, USA, Indianapolis, Ind.) and Benzonase. After the cells were lysed using a French press (Aminco®, Urbana, Ill.) and centrifuged, the pellet containing the inclusion body proteins was resuspended and washed with buffer B (buffer A+0.5% Triton X-100). Then, the inclusion bodies were solubilized in buffer C (Buffer A+8 M urea) by incubating/stirring at 4° C. overnight, followed by centrifugation and clarification. The supernatant containing denaturing protein was loaded on HisTrap column (AKTA®-prime; GE® Healthcare) followed by buffer C washing. The rRBD was refolded on HisTrap column using a linear gradient urea buffer D (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 1 mM GSH, 0.1 mM GSSG, 20% glycerol, pH 7.9) between 8 and 0 M. Finally, the column was washed with buffer E (20 mM Tris-HCl, 500 mM NaCl, 100 mM imidazole, 20% glycerol, 5% glucose, pH 7.9), and the refolding rRBD was eluted with buffer F (20 mM Tris-HCl, 500 mM NaCl, 800 mM imidazole, 20% glycerol, 5% glucose, pH 7.9) and dialyzed to remove imidazole. The proteins were then quantified, aliquoted, and stored at −80° C. until use.
After treatment with multiple freeze-thaw cycles and Benzonase, phage particles were boiled in SDS loading buffer for 10 min, separated by 4-20% SDS-PAGE, and then transferred to nitrocellulose membrane PVDF (Bio-Rad®). The PVDF was then blocked with 5% bovine serum albumin (BSA)-PBS (pH 7.4) buffer at RT for 1 hr with gentle shaking. Anti-NP or anti-RBD primary antibodies were added to the blots and incubated overnight at 4° C. in PBS-5% BSA, followed by five times rinsing in PBST buffer [1 xPBS (pH 7.4) and 0.05% Tween 20]. Goat-anti-mouse or goat-anti-rabbit HRP-conjugated antibody (Thermo Fisher®) was applied at a 1:5000 dilution in 5% BSA-PBST for 1 hour at RT with gentle shaking. After rinsing five times in PBST, signals were visualized with an enhanced chemiluminescence substrate (Bio-Rad®) using the Bio-Rad® Gel Doc XR+ System and Image Lab software according to the manufacturer's instructions (Bio-Rad®).
The T4-SocA, T4-SpyCatcher, and T4-S trimer phages were applied to the carbon grid for 5 min at RT. The phage-loaded grid was frozen in liquid nitrogen using Gatan CP3 cryo-plunger. The cryo-electron microscopy images were collected and reconstructed by Zhiqing Wang at Purdue University using a Titan Krios microscope equipped with a charge-coupled device camera.
HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco®) supplemented with 1% antibiotics (Thermo Fisher®), 1×HEPES (Thermo Fisher®), and 10% fetal bovine serum (Thermo Fisher®). Cells were passaged with 0.25% (w/v) trypsin/0.53 mM EDTA at a subcultivation ratio of 1:5 at 80 to 90% confluence. Cultures were incubated in a humidified atmosphere at 37° C. and 5% CO2. The plasmid containing human ACE2 (Addgene® #1786) was transfected into HEK293T cells using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher®) according to the manufacturer's instructions. Two days after ACE2 plasmid transfection, the cells were used for RBD or phage binding assay.
Human ACE2 transfected HEK293T cells were washed with PBS twice and then fixed with 4% formaldehyde for 15 mins at RT. After rinsing twice in PBS for 5 mins each, cells were blocked in blocking buffer (5% BSA-PBS) for 1 hr at RT. Recombinant SARS-CoV-2 RBD protein (Sino Biological®) was added to the cells to a final concentration of 0.2 μg/ml in the presence or absence of the sera with a series of dilutions. The unbound RBD was removed by washing the cells five times in PBST (PBS+0.1% Tween 20) for 5 mins each. The 1/1000 diluted human anti-RBD monoclonal antibody (Thermo Fisher®) was added to cells and incubated in a humidified chamber for 1 h at RT or overnight at 4° C. After rinsing five times in PBST for 5 mins each, Alexa® 488- or Rhodamine-conjugated goat anti-human secondary antibody was added (1/500 dilution) (Thermo Fisher®), and incubated for 2-3 hrs at RT in the dark. The cells were then rinsed five times in PBST for 5 mins each and counter-stained on 1 μg/ml Hoechst 33342 (Thermo Fisher®) for 5 mins. The fluorescent signals were observed by fluorescence microscopy (Carl Zeiss®).
Soc-GFP protein was produced as described previouslyl9. Briefly, the recombinant Soc-GFP proteins were purified according to the basic protocol described as follows. The BL21 (DE3) RIPL cells harboring the recombinant clones were induced with 1 mM IPTG for 2 h at 30° C. The cells were harvested by centrifugation (4,000×g for 15 min at 4° C.) and resuspended in 50 mL of HisTrap binding buffer (50 mM Tris-HCl, pH 8.0, 20 mM imidazole, 300 mM NaCl). The cells were lysed using French-press (Aminco®) and the soluble fraction containing the His-tagged fusion protein was isolated by centrifugation at 34,000×g for 20 min. The supernatant was loaded onto a HisTrap column (GE® Healthcare) and washed with 50 mM imidazole containing buffer, and the protein was eluted with 20-500 mM linear imidazole gradient. The peak fractions were concentrated and purified by size exclusion chromatography using Hi-Load 16/60 Superdex®-200 (prep-grade) gel filtration column (GE® Healthcare) in a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The peak fractions were concentrated and stored at ˜80° C.
In vitro display of Soc-GFP on the T4- SpyCatcher or T4-S trimer phage was assessed by the co-sedimentation, similar to the procedures of S trimer display on T4 phage. The T4-SpyCatcher-GFP or T4-S trimer-GFP phages were resuspended in Opti-MEM medium (Thermo Fisher®) and then added to ACE2-transfected HEK293T cells. After 6 hrs incubation, the unbound phages were removed by rinsing three times in PBS for 5 mins each. The GFP signals were observed by fluorescence microscopy (Carl Zeiss®).
We followed the recommendation of the National Institutes of Health about mouse study (the Guide for the Care and Use of Laboratory Animals). All mouse experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of America (Washington, DC) (Office of Laboratory Animal Welfare assurance number A4431-01) and the University of Texas Medical Branch (Galveston, Tex.) (Office of Laboratory Animal Welfare assurance number A3314-01). The SARS-CoV-2 virus challenge study was conducted in the animal biosafety level 3 (ABSL3) suite, and the principal investigators have registered with the CDC to work with the virus.
Six- to eight-week-old female BALB/c mice (The Jackson Laboratory (JAX®)) were randomly grouped (5 mice per group) and allowed to acclimate for 14 days. Three times intramuscular immunizations were administrated into their hind legs with phage vaccine candidates. A total of 6 ×1011 phages was injected on days 0 (prime), 21 (boost 1), and 42 (boost 2). Negative control mice received the same volume of PBS buffer (Naive) or the same amount of T4 control phage. A group of mice immunized with S trimer (20 μg) adjuvanted with Aluminum Alhydrogel was included as the positive control. Blood was drawn from each animal on days 0 (prebleed), 14, 35, and 56, and the isolated sera were stored at −80° C.
All experiments were performed at Envigo®/Cocalico Biologicals® (Reamstown, PA) in accordance with institutional guidelines. Adult New Zealand White rabbits were immunized i.m. in the flank region with 3×1011 PFU T4 phages/dose in 0.2 mL saline (n =4 for each group). Pre-immune test-bleeds were first obtained via venipuncture of the marginal vein of the ear on Day 1. Animals were immunized on Days 1, and 15 (Prime+One-boost regimen). Immune sera were obtained on Day 25.
ELISA plates (Evergreen Scientific®) were coated with 100 μl per well of 1 μg/ml of SARS-CoV-2 S-ecto protein (Sino Biological®), SARS-CoV-2 RBD-untagged protein (Sino Biological®), SARS- CoV-2 NP protein (Sino Biological®), or SARS-CoV-2 E protein (1-75 aa) (Thermo Fisher®) in coating buffer [0.05 M sodium carbonate-sodium bicarbonate (pH 9.6)]. After overnight incubation at 4° C., the plates were washed two times with PBS buffer and blocked for 2 hr at 37° C. with 200 pl per well PBS-5% BSA buffer. Serum samples were diluted with a 5-fold dilution series beginning with an initial 100-fold dilution in PBS-1% BSA. One hundred microliters diluted serum samples were added to each well, and the plates were incubated at 37° C. for 1 hour. After washing five times with PBST (PBS+0.05% Tween-20), the secondary antibody was added at 1:10,000 dilution in PBS-1% BSA buffer (100 μl per well) using either goat-anti-mouse IgG-HRP, goat-anti-mouse IgG1-HRP, goat-anti-mouse IgG2a-HRP (Thermo Fisher®), or goat-anti-rabbit IgG-HRP (Abcam®). After incubation for 1 hour at 37° C. and five times washes with PBS-T buffer, plates were developed using the TMB (3,3′,5,5′-tetramethylbenzidine) Microwell Peroxidase Substrate System (KPL®). After 5-10 min, the enzymatic reaction was stopped by adding TMB BlueSTOP (KPL®) solution. The absorbance was read within 30 min at 650 nm on a VersaMax spectrophotometer. The endpoint titer was defined as the highest reciprocal dilution of serum to give an absorbance more than 2-fold of the mean background of the assay.
An ELISA to analyze the binding of rRBD, S-ecto-6P-spytag trimer, T4 displayed RBD/S-trimer to human ACE2 protein was performed similarly to the above described. Briefly, 100 ng protein or 1×1010 phages were coated on plates overnight at 4° C. After blocking with PBS-5% BSA buffer, recombinant human ACE2-mouse Fc protein (Sino Biological®) with a series of dilution was added and incubated for 1 hr at 37° C. Plates were then incubated with the secondary goat-anti-mouse IgG-HRP antibody and developed with TMB substrate. Reactions were stopped and the absorbance was measured at 650 nm on a VersaMax spectrophotometer.
Challenge of the Mice with Adapted Live SARS-CoV-2 Virus
Immunized mice were challenged with the MA SARS-CoV-2 MA10 strain, a gift from R. Baric at the University of North Carolina, by the intranasal route as previously described. Briefly, mice were inoculated with 60 ul of SARS-CoV-2 MA10 at a dose of about 105 median tissue culture infectious dose. The animals were weighed every day over the indicated period of time for monitoring the onset of morbidity (weight loss and other signs of illness) and mortality, as the end points for evaluating the vaccine efficacy.
Neutralizing antibody titers in mouse immune sera were quantified by Vero E6 cell-based microneutralization assay using SARS-CoV-2 US-WA-1/2020 strain as previously described50. Briefly, serially 1:3 downward diluted mouse sera that were decomplemented at 56° C. for 60 min in a 60-ul volume were incubated for 1 hour at RT in duplicate wells of 96-well microtiter plates that contained 120 infectious SARS-CoV-2 viruses in 60 ul in each well. After incubation under BSL-3 conditions, 100 ul of the mixtures in individual wells was transferred to Vero E6 cell monolayer grown in 96-well microtiter plates containing 100 ul of MEM and 2% fetal bovine serum medium in each well and was cultured for 72 hours at 37° C. before assessing the presence or absence of cytopathic effect (CPE). Neutralizing antibody titers of the tested specimens were calculated as the reciprocal of the highest dilution of sera that completely inhibited virus-induced CPE in at least 50% of the wells and expressed as 50% neutralizing titer.
Neutralizing antibody titers in rabbit immune sera were quantified using an automated, liquid handler-assisted, high-throughput, microfocus neutralization/high-content imaging methods developed at ViroVax. Briefly, rabbit sera (paired preimmune and immune) were first decomplemented at 56° C. for 60 min and were then serially diluted in 384-well plates, in duplicate, using a BioTek Precision 2000 liquid handler, along with two reference sera. Twenty-microliter aliquots of SARS-CoV-2 US-WA-1/2020 were added to all test wells and positive control wells to yield a final MOI of 10 under BSL-3 conditions. Vero (American Type Culture Collection®, CCL-81) cells were maintained in a high-glucose DMEM supplemented with 10% fetal bovine serum (HyClone® Laboratories, South Logan, Utah) and 1% penicillin/streptomycin at 37° C. with 5% CO2. After preincubating the plates for 1 hour, 20 ul of Vero cells (106/ml), containing propidium iodihdde (PI) (50 μg/ml), was added to all wells using the liquid handler. Plates were then loaded in an IncuCyte® S3 high-content imaging system (Essen BioScience/Sartorius®, Ann Arbor, Mich). Longitudinal image acquisition and processing for virus-induced CPE and cell death (PI uptake) were performed every 6 hours, until cell death profiles had crested and stabilized (3.5 days). Neutralizing antibody titers which were expressed as median inhibitory concentration (IC50) or IC90, were obtained from four-parameter logistic curve fits of cell death profiles using OriginPro 9 (OriginLab® Corp., Northampton, Mass.).
All the data were presented as means ±SEM except indicated. Statistical analyses were performed by Graph Pad® Prism 9.0 software using one-way or two-way Analysis of variance (ANOVA) according to the data. Tukey's multiple comparisons post-test was used to compare individual groups. Significant differences between two groups were indicated by *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ns, no significance. P-values of <0.05 were considered significant.
It is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.
All documents, patents, journal articles and other materials cited in the present application are incorporated herein by reference.
The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
The following references are referred to above and are incorporated herein by reference:
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The foregoing applications, and all documents cited therein or during their prosecution (“appin cited documents”) and all documents cited or referenced in the appin cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, products specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
While the present disclosure has been disclosed with references to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present disclosure, as defined in the appended claims. Accordingly, it is intended that the present disclosure is not limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.
This application claims benefit of priority of U.S. Provisional Patent Application No. 63/126,047 entitled, “UNIVERSAL BACTERIOPHAGE T4 NANOPARTICLE PLATFORM TO DESIGN MULTIPLEX SARS-COV-2 VACCINE CANDIDATES BY CRISPR ENGINEERING” filed Dec. 16, 2020. The entire contents and disclosures of these patent applications are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63126047 | Dec 2020 | US |
