Patent Application
20230181720
The invention relates to single platform vaccines for preventing diseases caused by pathogens and in particular, COVID-19.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), closely related to SARS-CoV, is an enveloped, single-stranded positive RNA virus with a nucleocapsid that belongs to the betacoronavirus genus of the Coronaviridae. Starting in the final months of 2019, the virus caused an ongoing pandemic of COVID-19; the pandemic originated in Wuhan, Hubei Province of China and quickly spread worldwide with millions of confirmed cases and hundreds of thousands of fatalities.
The virus is primarily spread between people during close contact, most often via small droplets produced by coughing, sneezing, and talking. The droplets usually fall to the ground or onto surfaces rather than travelling through air over long distances. The time from exposure to onset of symptoms is typically around five days but may range from two to fourteen days. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. While the majority of cases result in mild symptoms, some progress to acute respiratory distress syndrome (ARDS), multi-organ failure, septic shock, and blood clots.
There are currently no vaccines available to prevent COVID-19. Accordingly, there is a need for vaccines and associated methods designed to protect individuals from COVID-19 infection.
The invention disclosed herein provides a SARS-CoV-2 vaccine vector platform which is useful for preventing the disease COVID-19 caused by SARS-CoV-2 in humans and animals. The invention utilizes a vector termed “LVS ΔcapB”, which is a live attenuated capB mutant of Francisella tularensis Live Vaccine Strain (LVS), itself attenuated by serial passage in the 20th century from Francisella tularensis subsp. holarctica. In this context, LVS has two major attenuating deletions and several minor mutations. The invention is also the use of this vaccine platform to construct and use vaccines against numerous other pathogens caused by bacteria, viruses, parasites, etc.
Embodiments of the invention include an immunogenic composition comprising at least one recombinant attenuated Francisella tularensis subspecies holarctica Live Vaccine Strain (LVS) having a deletion in a capB gene and an antigen expression cassette which comprises a F. tularensis promoter and which expresses at least one antigenic epitope present in a polypeptide expressed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In such compositions, the antigenic polypeptide epitope elicits an immune response in a mammalian host when the immunogenic composition is administered orally (p.o.), intradermally (i.d.), subcutaneously (s.q.), intramuscularly (i.m.), intranasally (i.n.), or by inhalation to the mammalian host.
In typical embodiments of the invention, the at least one antigenic polypeptide epitope present in the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 is present on: a SARS-CoV-2 large surface spike (S) glycoprotein; a SARS-CoV-2 envelope (E) protein: a SARS-CoV-2 membrane (M) glycoprotein: and/or a SARS-CoV-2 nucleocapsid (N) phosphoprotein. Optionally in these compositions, the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 comprises at least two antigenic polypeptide epitopes present in: a SARS-CoV-2 large surface spike (S) glycoprotein: a SARS-CoV-2 envelope (E) protein; a SARS-CoV-2 membrane (M) glycoprotein: and/or a SARS-CoV-2 nucleocapsid (N) phosphoprotein.
In certain embodiments of the invention, the at least one antigenic polypeptide epitope present in the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 is present on SARS-CoV-2 membrane (M) glycoprotein; or SARS-CoV-2 nucleocapsid (N) phosphoprotein. Typically, in these embodiments, the LVS ΔcapB expresses at least two antigenic polypeptide epitopes present on severe acute respiratory syndrome coronavirus 2 including: at least one peptide epitope present in SARS-CoV-2 membrane (M) glycoprotein; at least one peptide epitope present in SARS-CoV-2 nucleocapsid (N) phosphoprotein. In illustrative working embodiments of the invention disclosed herein, the at least two antigenic polypeptide epitopes present on a severe acute respiratory syndrome coronavirus 2 polypeptide are encoded by a sequence found in SEQ ID NO: 1 (e.g., a polynucleotide sequence encoding SARS-CoV-2 membrane (M) glycoprotein coupled via a polypeptide linker to a SARS-CoV-2 nucleocapsid (N) phosphoprotein). In these working embodiments, the antigenic polypeptide is encoded in a codon optimized polynucleotide sequence (i.e., one optimized for expression in Francisella tularensis).
Related embodiments of the invention method of making an immunogenic composition, such methods comprising introducing a polynucleotide encoding at least one antigenic epitope present in a polypeptide expressed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into a recombinant attenuated Francisella tularensis subspecies holarctica Live Vaccine Strain (LVS). In these methods, the LVS has a deletion in a capB gene; and the antigenic polypeptide epitope encoded by the polynucleotide elicits an immune response to SARS-CoV-2 in a mammalian host when the immunogenic composition is administered orally (p.o.), intradermally (i.d.), subcutaneously (s.q.), intramuscularly (i.m.), intranasally (i.n.) or by inhalation to the mammalian host. Embodiments of the invention include making compositions of matter that further comprise additional agents such as a pharmaceutical excipient selected for a specific route of administration, for example oral or intranasal administration. In certain embodiments, the at least one antigenic polypeptide epitope present in the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 is present on SARS-CoV-2 membrane (M) glycoprotein; or SARS-CoV-2 nucleocapsid (N) phosphoprotein. Typically, in these embodiments, the LVS ΔcapB expresses at least two antigenic polypeptide epitopes including: at least one peptide epitope present in SARS-CoV-2 membrane (M) glycoprotein; at least one peptide epitope present in SARS-CoV-2 nucleocapsid (N) phosphoprotein. In illustrative working embodiments of the invention that are disclosed herein, the at least two antigenic polypeptide epitopes present on a severe acute respiratory syndrome coronavirus 2 polypeptide arc encoded by SEQ ID NO: 1.
Other embodiments of the invention include the use of an immunogenic composition disclosed herein for inducing immunity to SARS-CoV-2. Such embodiments of the invention include methods of generating an immune response in a mammal comprising administering the immunogenic composition disclosed herein (e.g., a LVS ΔcapB transformed with a polynucleotide encoding a SARS-CoV-2 M and N fusion protein such as the polynucleotide of SEQ ID NO: 1) to the mammal so that an immune response is generated to the antigenic polypeptide epitope present in a severe acute respiratory syndrome coronavirus 2 polypeptide. In certain embodiments of the invention, the immunogenic composition is administered orally. In other embodiments of the invention, the immunogenic composition is administered intranasally.
Embodiments of the vaccine platform disclosed herein can be modified to accommodate mutated antigens of SARS-CoV-2 and future SARS-CoV-like viruses should such strains arise and be sufficiently different from SARS-CoV-2 that persons or animals vaccinated with an earlier vaccine version are no longer immune. The vaccine platform can be used to construct vaccines against other viruses including but not limited to SARS, MERS, and other coronaviruses: Influenza A and B: Hepatitis A. Hepatitis B, Hepatitis C, Hepatitis E; Ebolavirus; Lassa; Nipah; Rift Valley Fever; Zika; Chikungunya; Cocksackie A16; Enterovirus 68, Enterovirus 71; Marburg; HIV; Dengue; Rabies: Arenaviruses including Guanarito, Junin, Lassa, Lujo, Machupo, Sabia, Dandemong, lymphocytic choriomeningitis; Bunyaviruses including Andes, Bwamba, Crimean-Congo Hemorrhagic Fever, Oropouche, Rift Valley, Severe Fever with Thrombocytopenia, Syndrome (SFTS); Flaviviruses including Japanese encephalitis, Usutu, West Nile; Togaviruses including Bamah Forest, O'nyong-nyong, Ross River, Semliki Forest, Venezuelan Equine Encephalitis; Filviruses including Bundibugyo Ebola, Lake Victoria Marburg, Sudan Ebola: Herpesviruses: Polyomaviruses: Poxviruses, Cytomegalovirus, Epstein-Barr, etc. The vaccine platform can be used to construct vaccines against bacteria including but not limited to Burkholderia, pseudomallei, Burkholderia mallei, Francisella tularensis, Bacillus anthracis, Yersinia pestis, Mycobacterium tuberculosis, Mycobacterium leprae, Legionella pneumophila, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Listeria monocytogenes, Brucella species, etc. The vaccine platform can be used to construct vaccines against rickettsia including but not limited to Rickettsia prowazekii, R. typhi, R. rickettsia, R. tsutsugamushi, Coxiella burnetii, etc. The vaccine platform can be used to construct vaccines against protozoa including but not limited to Leishmania species, Trypanosoma cruzi, Toxoplasma gondii, etc. The vaccine platform can be used to construct vaccines against fungi including but not limited to Histoplasma capsulatum, Coccidioides immitis or Coccidioides posadasii, etc.
As noted above, in certain embodiments of the invention, combinations of vaccines expressing different SARS-CoV-2 antigens can be administered together. The vaccine platform has consistently resulted in a strong antibody response and a strong cell-mediated immune response to recombinant pathogen antigens expressed by the vaccine. The vaccine composition is administered to humans or animals by injection intradermally or by another route, e.g., subcutaneously, intramuscularly, orally, intranasally, or by inhalation. Each vaccine composition can be administered intradermally (i.d.) or by another route, e.g., subcutaneously (s.q.), intramuscularly (i.m.), intranasally (i.n.), inhaled, or even orally (p.o.) to a mammalian host. The vaccine can be administered as part of a homologous or heterologous prime-boost vaccination strategy. In certain implementations, the host is administered a single dose of a first vaccine and one or more doses of a homologous or heterologous booster vaccine.
This single platform simplifies manufacture, regulatory approval, clinical evaluation, and vaccine administration, and would be more acceptable to people than multiple individual vaccines, and be less costly. Currently, no single bacterial platform vaccine against SARS-CoV-2 is available. Regarding manufacture, vaccines constructed from the same vectors can be manufactured under the same conditions. That is, the manufacture of the LVS ΔcapB vector will be the same regardless of which antigen it is expressing or overexpressing. Similarly, manufacture of the L. monocytogenes vector will be the same regardless of which antigen it is expressing.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention.
All publications mentioned herein are incorporated by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. This application is related to U.S. patent application Ser. No. 16/319,812, filed on Jan. 22, 2019, entitled “SAFE POTENT SINGLE PLATFORM VACCINE AGAINST TIER 1 SELECT AGENTS AND OTHER PATHOGENS” the contents of which are incorporated herein by reference.
The current pandemic of COVID-19 has sickened over a hundred and fifty million people, killed over 3 million, and wreaked havoc on the world's economy. There is a tremendous need for a safe and effective COVID-19 vaccine to end the current devastating pandemic. An effective COVID-19 vaccine can end this pandemic quickly.
The invention disclosed herein utilizes a vaccine vector platform termed “LVS ΔcapB”, which is a live attenuated capB mutant of Francisella tularensis Live Vaccine Strain (LVS), itself attenuated by serial passage in the 20th century from Francisella tularensis, subsp. holarctica (see, e.g., Jia et al., Infect Immun. 78:4341-4355. (Epub 2010 07-19). PMID 20643859. PMCID: PMC2950357. doi: 10.1128/IAI.00192-10; Salomonsson et al., Infect. Immun. 77:3424-343: and Rohmer et al., Infect. Immun. 74:6895-6906: the contents of which are incorporated herein by reference).
In this context, embodiments of the invention include immunogenic (vaccine) compositions that comprise an attenuated recombinant Francisella tularensis subspecies holarctica Live Vaccine Strain (LVS) that does not express CapB protein (e.g., LVS ΔcapB), wherein this LVS further expresses one or more antigens present on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Embodiments of the invention also include methods of immunizing a susceptible host against a pathogen comprising administering to the host a vaccine that comprises an attenuated recombinant Live Vaccine Strain lacking a polynucleotide encoding CapB (LVS ΔcapB), wherein the LVS ΔcapB expresses one or more antigens expressed by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polypeptide.
One major advantage of the immunogenic vaccine compositions disclosed herein is the capacity to manufacture vaccines cheaply and quickly. The head of GAVI (the Vaccine Alliance) has pointed out how important it is that vaccines being developed for COVID-19 be available to all of the world's population and not just the privileged. The capacity to manufacture huge quantities of vaccine quickly and cheaply would allow that eventuality. Live, attenuated bacterial vaccines, such as LVS ΔcapB vectored vaccine against COVID-19 are much less expensive to manufacture, as they can be grown readily in inexpensive broth and require no purification. Vaccine cost is of critical importance in developing countries.
Another major advantage of the immunogenic vaccine compositions disclosed herein is that the vector is a more attenuated derivative of a vaccine already safely administered to people. Hence it is anticipated to be extremely safe. Another likely advantage of the immunogenic vaccine compositions disclosed herein is that as a live attenuated vaccine, it is much more likely to induce long-lasting protection than a protein/adjuvant vaccine, DNA/RNA vaccine, or non-replicating virus-vectored vaccine. Another major advantage of the immunogenic vaccine compositions disclosed herein is that the single vector platform that we are using is easily expandable to other infectious diseases. In fact, we have already employed the single platform to generate potent vaccine candidates against other pathogens. Finally, the immunogenic vaccine compositions disclosed herein is easily altered in response to mutations in the SARS-CoV-2 virus that may render initial vaccines against it no longer effective.
As there are currently no licensed vaccines against COVID-19 comprising a replicating bacterial vector, this vaccine meets a major unmet need. Previous human trials have demonstrated reasonable safety of the double-deletional parent vector (LVS). The even more attenuated but still highly immunogenic triple-deletional platform vector (LVS ΔcapB) derived from the parent is >10,000 fold less virulent in a mouse model (as measured by intranasal LD50; all animals survived highest dose tested). Because the vaccine is based upon a bacterial vector, it can be inexpensively manufactured in broth culture—no purification is necessary as in the case of viral-vectored vaccines.
Advantages of the invention disclosure herein include that there is no need for animal products, in contrast to viral-vectored vaccines grown in cell culture. In addition, there is no need for adjuvant; and the vaccine can be readily altered to accommodate mutations in the SARS-CoV-2 virus. In addition, single vector platform simplifies manufacture, regulatory approval, clinical evaluation, and vaccine administration, and would be more acceptable to people than multiple individual vaccines, and be less costly. Regarding manufacture, vaccines constructed from the same vectors can be manufactured under the same conditions. That is, the manufacture of the LVS ΔcapB vector will be the same regardless of which antigen it is expressing or overexpressing.
The invention disclosed herein has a number of embodiments. Embodiments of the invention include an immunogenic composition comprising at least one recombinant attenuated Francisella tularensis subspecies holaretica Live Vaccine Strain (LVS) having a deletion in a capB gene and an antigen expression cassette which comprises a F. tularensis promoter and which expresses at least one antigenic epitope present in a polypeptide expressed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In such compositions, the antigenic polypeptide epitope elicits an immune response in a mammalian host when the immunogenic composition is administered by at least one route of administration selected from orally (p.o.), intradermally (i.d.), subcutaneously (s.q.), intramuscularly (i.m.), intranasally (i.n.), or by inhalation to the mammalian host.
In typical embodiments of the invention, the at least one antigenic polypeptide epitope present in the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 is present on: a SARS-CoV-2 large surface spike (S) glycoprotein; a SARS-CoV-2 envelope (E) protein: a SARS-CoV-2 membrane (M) glycoprotein: and/or a SARS-CoV-2 nucleocapsid (N) phosphoprotein. Optionally in these compositions, the polypeptide expressed by severe acute respiratory syndrome coronavirus 2 comprises at least two antigenic polypeptide epitopes present in: a SARS-CoV-2 large surface spike (S) glycoprotein: a SARS-CoV-2 envelope (E) protein; a SARS-CoV-2 membrane (M) glycoprotein: and/or a SARS-CoV-2 nucleocapsid (N) phosphoprotein (e.g. an epitope present on an S1 subunit of the SARS-CoV-2 large surface spike (S) glycoprotein and an epitope present on a S2 subunit of the SARS-CoV-2 large surface spike (S) glycoprotein). In certain embodiments of the invention, the antigenic polypeptide epitope is encoded in a codon optimized polynucleotide sequence. Optionally, the at least one antigenic epitope present in a polypeptide expressed by severe acute respiratory syndrome coronavirus 2 is encoded in a polynucleotide of SEQ ID NO: 1-SEQ ID NO: 9 (e.g. a polynucleotide segment in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 that is at least 25, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or 8000 nucleotides in length and/or is not more than 25, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or 8000 nucleotides in length). Embodiments of the invention include Francisella tularensis subspecies holarctica Live Vaccine Strain immunogenic compositions that are designed to express multiple SARS-CoV-2 proteins from different genetic elements in this microorganism. For example, as shown in
In certain embodiments of the invention, the LVS is engineered to express at least two antigenic polypeptide epitopes present on severe acute respiratory syndrome coronavirus 2 including: at least one peptide epitope present in SARS-CoV-2 membrane (M) glycoprotein; at least one peptide epitope present in SARS-CoV-2 nucleocapsid (N) phosphoprotein. In certain embodiments of the invention, the LVS is transformed with a polynucleotide encoding polypeptide epitopes found on SARS-CoV-2 membrane (M)glycoprotein, with such polynucleotide sequences being coupled to a polynucleotide encoding a polypeptide linker, with this (encoded) linker also being coupled to a polynucleotide encoding polypeptide epitopes found on a SARS-CoV-2 nucleocapsid (N) phosphoprotein. In such embodiments, the genetically engineered LVS ΔcapB thereby expresses a MN fusion protein that is presented to immune cells. In illustrative working embodiments of the invention disclosed herein, the at least two antigenic polypeptide epitopes present on a severe acute respiratory syndrome coronavirus 2 polypeptide are encoded by a sequence found in SEQ ID NO: 1 (which is a polynucleotide sequence encoding a fusion protein comprising SARS-CoV-2 membrane (M) glycoprotein coupled in frame via an encoded polypeptide linker to a SARS-CoV-2 nucleocapsid (N) phosphoprotein). In certain embodiments, the antigenic polypeptides can be encoded in a codon optimized polynucleotide sequence.
Embodiments of the invention include concurrent administration of one vaccine embodiment of the invention along with one or more other vaccine embodiments using the same vector. Furthermore, a single vector platform vaccine also has the advantage that different vaccines comprising the same vector but expressing different antigens can be safely and effectively administered at the same time. That is, individual LVS ΔcapB vaccines expressing Burkholderia pseudomallei (Bp) antigens. Francisella tularensis subsp. tularensis (Ft) antigens, Bacillus anthracis (Ba) antigens, Yersinia pestis (Yp) antigens, SARS-CoV-2 antigens, and the antigens of other pathogens, can be administered together.
As discussed in detail below, nine COVID-19 immunogenic vaccine compositions have been constructed and demonstrated to express the relevant SARS-CoV-2 proteins singly and in combination. Embodiments of the invention include an immunogenic composition comprising a recombinant attenuated Francisella tularensis subspecies holarctica Live Vaccine Strain (LVS) having a deletion in a capB gene and which comprises a heterologous promoter that expresses a fusion protein comprising an antigenic polypeptide epitope present in a SARS-CoV-2 virus polypeptide. It is desirable to include large segments of SARS-CoV-2 virus polypeptides in this invention in order to present a large number of immunoreactive epitopes to the mammalian immune system. Optionally the LVS expresses two or more antigenic polypeptide epitopes present in a SARS-CoV-2 virus polypeptide. In this context, illustrative embodiments of the invention include vaccine combinations or combinations of proteins in a single vaccine. Such illustrative combinations include (SARS-CoV-2 proteins bolded):
1. rLVS ΔcapB/SCoV2 (SΔTM)+rLVS ΔcapB/SCoV2 (MN)
2. rLVS ΔcapB/SCoV2 (S1)+rLVS ΔcapB/SCoV2 (MN)
3. rLVS ΔcapB/SCoV2 (S)+rLVS ΔcapB/SCoV2 (MN)
4. rLVS ΔcapB/SCoV2 (S2)+rLVS ΔcapB/SCoV2 (MN)
5. rLVS ΔcapB/SCoV2 (S2E)+rLVS ΔcapB/SCoV2 (MN)
6. rLVS ΔcapB/SCoV2 (S1)+rLVS ΔcapB/S2 (S2)
7. rLVS ΔcapB/SCoV2 (S1)+rLVS ΔcapB/SCoV2 (S2E)
Another embodiment of the invention is a method of generating an immune response in a mammal comprising administering one or more of immunogenic compositions disclosed herein to the mammal so that an immune response is generated to the one or more antigenic polypeptide epitopes present in a SARS-CoV-2 virus polypeptide. In one such embodiment, the method comprises administering an LVS immunogenic composition disclosed herein in a primary vaccination; and administering the same immunogenic composition of LVS immunogenic composition disclosed herein in a subsequent homologous booster vaccination. Typically, the method consists essentially of administering the immunogenic composition of an LVS immunogenic composition disclosed herein in a primary vaccination; and administering the immunogenic composition of LVS immunogenic composition disclosed herein in a subsequent homologous booster vaccination. Optionally, the method comprises administering the immunogenic composition to the mammal less than 4 times.
In another embodiment of the invention, the method comprises administering an LVS composition as disclosed herein in a primary vaccination; and administering a second heterologous immunogenic composition comprising the antigenic polypeptide epitope present in a SARS-CoV-2 virus in a subsequent booster vaccination. Optionally, the second immunogenic composition comprises an attenuated strain of Listeria monocytogenes expressing the antigenic polypeptide epitope. In certain embodiments, the method comprises administering LVS immunogenic composition disclosed herein and a second immunogenic composition to the mammal less than a total of four times. Optionally for example, the method comprises administering a single dose of a first LVS immunogenic composition disclosed herein, and one or more doses of a second immunogenic composition disclosed herein.
Studies illustrating aspects and properties of the invention are published in Jia et al., NPJ Vaccines. 2021 Mar. 30; 6(1):47. doi: 10.1038/s41541-021-00321-8, the contents of which are incorporated by reference.
Construction and Characterization of Recombinant LVS ΔcapB Expressing SARS-CoV-2 Antigens
The complete genome sequence of SARS-CoV-2 and the polypeptides encoded by this genome are known in the art. See, e.g. “Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal”, Sah et al., Microbiology Resource Announcements March 2020.9 (11) e00169-20; DOI: 10.1128/MRA.00169-20, the contents of which are incorporated by reference; and SARS-CoV-2 sequenced genomes are available at GenBank (e.g. MN988668 and NC_045512, the contents of which are incorporated by reference). See also Zhou P, Yang X L, Wang X G, Hu B. Zhang L, Zhang W, Si H R, Zhu Y, Li B, Huang C L, Chen H D, Chen J, Luo Y. Guo H, Jiang R D, Liu M Q, Chen Y, Shen X R, Wang X, Zheng X S, Zhao K, Chen Q J, Deng F, Liu L L, Yan B. Zhan F X, Wang Y Y, Xiao G F, Shi Z L. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798):270-3. Epub 2020/02/06. doi: 10.1038/s41586-020-2012-7. PubMed PMID: 32015507. See also Wu et al, Nature volume 579, pages 265-269 (2020) and Genebank MT152824 (US), the contents of which are incorporated by reference, for the complete genomic sequence which was used herein for gene optimization.
Similar to other coronaviruses, including SARS-CoV and MERS-CoV. SARS-CoV-2 encodes 4 structural proteins: a large surface spike (S) glycoprotein (1273 aa) (1, 3); an envelope (E) protein (75 aa); a membrane (M) glycoprotein (222 aa); and a nucleocapsid (N) phosphoprotein (419 aa) (
Construction and Verification of rLVS ΔcapB Prime Vaccines Expressing Immunogenic SARS-CoV-2 Antigens.
1A. Construct rLVS ΔcapB Vaccines Expressing SARS-CoV-2 Antigens (rLVS ΔcapB/SCoV2).
We previously have successfully constructed rLVS ΔcapB vaccines expressing shuttle plasmid-encoded Ft, Ba, Yp, and Bp antigens and demonstrated potent protection by the rLVS ΔcapB vaccines against lethal respiratory challenge with the relevant pathogens. We now have used a similar approach to construct vaccines against SARS-CoV-2. For expression of the S protein (protein id QIH55221.1), a gene encoding full-length SARS-CoV-2 S (Genebank MT152824) with two stabilizing proline substitutions at the S2 fusion machinery (K986P and V987P) (1, 5) was codon-optimized for expression in LVS ΔcapB and synthesized by Atum.com. Similarly, genes encoding SARS-CoV-2 E, M, N proteins were also codon-optimized and synthesized by Atum.com. The synthesized genes encoding the full-length S protein (145 kDa), the fusion proteins of S2-E (72 kDa), and the fusion protein of MN (71 kDa) linked by flexible linker (GGSG) were cloned separately into a pFNL-derived expression shuttle plasmid downstream of the pbfr promoter by the Electra Cloning System (ATUM) and traditional molecular cloning methods (6). Subsequently we performed a deletional mutagenesis of the codon-optimized gene for full-length S protein to generate pFNL-derived expression shuttle plasmids for SΔTM. S1 and S2 subunits. We shall also construct a pFNL-derived shuttle plasmid carrying expression cassettes for both S1 and S2 subunits driven by the Francisella omp and bfr promoter, respectively, as indicated in
As expected, the fusion protein of MN with or without N-terminal tags were abundantly expressed by the LVS ΔcapB vector and recognized by the guinea pig polyclonal antibody to SARS CoV (NR-10361, BEI Resources). Surprisingly, the full-length Spike protein (145 kDa) was also abundantly expressed by the LVS ΔcapB vector and recognized by the guinea pig polyclonal antibody to SARS CoV (NR-10361, BEI Resources). This is the largest protein we have successfully expressed from the LVS ΔcapB vector. The SΔTM, S1, and S2 were also expressed by the LVS ΔcapB vector as demonstrated by Western blotting analysis by using the monoclonal antibody to the N-terminal tag (FLAG) and by using the polyclonal antibody to SARS CoV.
1B. Characterize rLVS ΔcapB Vaccines In Vitro, Including Protein Expression and Growth Kinetics in Broth and in Macrophages, and Genetic Stability of the Integrated Antigen Expression Cassette.
1B1. Protein Expression by rLVS ΔcapB/SCoV2 Vaccine Grown on Agar Plates.
Heterologous protein expression by rLVS ΔcapB/SCoV2 vaccines on Chocolate agar plates were analyzed by Western blotting using polyclonal antibody to SARS-CoV or monoclonal antibodies to the N-terminal tags of the SCoV2 protein, as described by us previously (7-9).
In studies of embodiments of the invention disclosed herein, a major unexpected finding was that only the vaccines expressing the Membrane (M) and Nucleocapsid (N) proteins (e.g. the MN fusion protein of SEQ ID NO: 1) were protective (either when administered alone or with vaccines expressing other proteins), whereas all of the vaccines expressing only the S protein (or a part of the S protein i.e. SΔTM, S1, or S2) or the S2 protein fused to the Envelope (E) protein (S2E) were not protective. It was also unexpected that the MN fusion protein expressing vaccines worked just as well when administered by the intranasal route as by the intradermal route. Specifically, we used the LVS ΔcapB vector platform to construct six COVID-19 vaccines expressing one or more of all four structural proteins of SARS-CoV-2 and tested the vaccines for efficacy, administered intradermally (ID) or intranasally (IN), against a high dose SARS-CoV-2 respiratory challenge in hamsters. These studies showed that the LVS ΔcapB vaccine expressing COVID-19 MN proteins, but not the vaccines expressing the S protein or its subunits in various configurations, is highly protective against severe COVID-19-like disease including weight loss and lung pathology, and also that protection is highly correlated with serum anti-N antibody levels. See
This concludes the description of embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.
This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 63/026,480, filed on May 18, 2020, and U.S. Provisional Patent Application Ser. No. 63/182,111, filed on Apr. 30, 2021, which applications are incorporated by reference herein.
This invention was made with Government support under grant number AI141390, awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/32203 | 5/13/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63182111 | Apr 2021 | US | |
| 63026480 | May 2020 | US |
