DETAILED ACTION
Acknowledgment and entry of the Amendment submitted on 11/14/25 is made.
Claims 1, 2, 5-7, 12, 14, 15, 19, 23-27, 29 and 30 and the Species:
The cell general (single cell/type strain)-claim 2: Lactobacillus.
The origin of the exogenous polypeptide: claim 12: SARS-CoV-2 antigen.
Claims 9 and 31 remain withdrawn from consideration for being drawn to a non-elected invention.
The former 102 and 103 prior art rejections are withdrawn pursuant to the amendment to the claims.
Claim Rejections - 35 USC § 112-2nd paragraph
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 5-7, 12, 14, 15, 19, 23-27, 29 and 30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is vague and indefinite due to the phrase “and any derivatives or fragments thereof.” A fragment reads on as few as one amino acid or many more. It is unclear how the polypeptides ‘vary’ or what structure the ‘derived’ sequence comprises. The term “derivative” does not provide the character or properties from the source that are to be retained in the final product, e.g., paper is derived from wood but is very different from wood. Further, the claim provides no structure and only a vague functional description to identify the proteins of the composition. This is not sufficient to satisfy the Statute's requirement of adequately describing and setting forth the inventive concept. The claim should provide any structural properties, such as the amino acid sequence of the protein or molecular weight, which would allow for one to identify the protein without ambiguity. The mere recitation of a name or general function does not adequately define the claimed protein. The metes and bounds of these claims cannot be understood. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed. Appropriate correction is required.
This rejection was necessitated by the amendment to the claims:
Claim Rejections - 35 USC § 112-Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 2, 5-7, 12, 14, 15, 19, 23-27, 29 and 30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
An engineered L. acidophilus cell with a sequence encoding an immunogenic polypeptide inserted into the SlpA gene along with a sequence encoding the FliC and/or FimH adjuvant peptide, wherein the exogenous polypeptide and at least one adjuvant polypeptide are expressed by the cell; and a vaccine composition comprising said engineered cell wherein the polypeptide is SARS-CoV-2 protein,
does not reasonably provide enablement for:
Any engineered bacterial cell comprising: 1. (i) an exogenous polypeptide; and (ii) at least one adjuvant polypeptide, wherein the at least one adjuvant polypeptide comprises flagellin (FliC) and type 1 fimbrin D-mannose specific adhesin protein (FimH) and any derivatives or fragments thereof; and wherein the exogenous immunogenic polypeptide and the at least one adjuvant polypeptide are expressed by the bacterial cell and a vaccine composition comprising said engineered cell.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The engineered bacterial cell of claim 1 and the vaccine composition of claim 29 may be any Genus/species, the exogenous polypeptide may be from any origin and any source of any structure and the adjuvant polypeptides may be any derivative or fragment from FliC and FimH.
In the instant case, the specification provides a host L. acidophilus cell with a sequence encoding virus peptide inserted into the SlpA gene along with a sequence encoding the FliC and/or FimH adjuvant peptide. The only adjuvant peptides disclosed are the FliC and FimH peptides. The viral peptide antigen shown is the SARS-CoV-2 protein.
The specification and Applicants recite that the invention is a “vaccine platform” comprising genetically engineered microorganisms for use in the production of pharmaceutical vaccine compositions for treating and/or preventing diseases and disorders associated with infections from pathogenic organisms. See paragraph [0033], Detailed Description. Paragraph [0043] provides a laundry list of potential bacterial hosts. However, the examples in the instant specification are only shown (FIG. 1) with various L. acidophilus strains engineered to express the paragraph [0101] immunogenic polypeptides and adjuvant polypeptides within the bacterial surface layer protein (SLP) after genomic insertion. The SlpA gene was cloned and the virus peptide was inserted into a region of the sequence known to preserve SlpA structure and provide surface exposure. The plasmid was transfected into L. acidophilus and selected for homologous recombination using nutrient selective pressure. Example 2 on page 32, teaches that induction of robust immune response is key to preventing infections by pathogens and evaluated the polypeptide adjuvants FliC and FimH.
Accordingly, the specification does not enable a “vaccine platform” comprising any transformed, genetically engineered microorganisms for use in the production of pharmaceutical vaccine compositions for treating and/or preventing diseases and disorders associated with infections from pathogenic organism, but provides for a vaccine platform of L. acidophilus strains engineered to express immunogenic polypeptides and the FliC or FimH adjuvant polypeptides within the bacterial surface layer protein (SLP) after genomic insertion.
The claims recite any fragments or derivatives from FimH or FliC, but the specification provides no guidance as which amino acids may be changed without causing a detrimental effect to the polypeptides and with the added adjuvant function requirement. It is unpredictable as to which amino acids could be removed and which could be added. While it is known that many amino acid substitutions are possible in any given protein, the position within the protein’s sequence where amino acid substitutions can be made with a reasonable expectation of success are limited. Other positions are critical to the protein’s structure/function relationship, e.g., such as various positions or regions directly involved in binding, catalysis in providing the correct three-dimensional spatial orientation of binding and catalytic sites. These regions can tolerate only very little or no substitutions. Selective point mutation to one key residue could eliminate the function of the polypeptide. It could eliminate its functional properties. If the range of decreased binding ability after single point mutation of a protein antigen varies, one could expect point mutations in the protein antigen to cause varying degrees of loss of function. A protein having multiple point mutations, or accumulated point mutations at key residues could create a new antigen that is precipitously or progressively unrecognizable. As stated above, Applicants have not shown the particular substitution and the result it produces. Applicants have provided no guidance to enable one of ordinary skill in the art how to determine, without undue experimentation, the effects of different amino substitutions and the nature and extent of the changes that can be made. It is expensive and time consuming to make amino acid substitutions at more than one position, in a particular region of the protein, in view of the many fold possibilities for change in structure and the uncertainty as to what utility will be possessed. Amino acids owe their ‘significance’ to their inclusion in a pattern which is directly involved in recognition by, and binding to, the receptor and the significance of the particular amino acids and sequences for different amino acids cannot be predicted a priori, but must be determined from case to case by painstaking experimental study. The instant claims allow for substitutions with amino acids of vastly different properties and they do not recite the specific changes in the claims.
Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001 clearly states: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 148 USPQ 689, 696 (1966) (stating, in context of the utility requirement, that "a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.") Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.”
Claim Rejections - 35 USC § 112-Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 2, 4-7, 12, 14, 15, 19, 20, 23-27, 29 and 30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Instant claim 1 recites:
(Currently Amended) (Any) engineered bacterial cell comprising: 1. (i) an exogenous polypeptide; and (ii) at least one adjuvant polypeptide, wherein the at least one adjuvant polypeptide comprises flagellin (FliC) and type 1 fimbrin D-mannose specific adhesin protein (FimH) and any derivatives or fragments thereof; and wherein the exogenous immunogenic polypeptide and the at least one adjuvant polypeptide are expressed by the bacterial cell.
Note: the bacterial cell may be any Genus/species (not even limited to a lactic acid bacteria), the exogenous polypeptide may be from any origin and any source of any structure and is not even required to be an immunogenic polypeptide or virulence factor, etc.
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. The courts have stated:
"To fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that "the inventor invented the claimed invention." Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997); In re Gostelli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989) ("[T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed."). Thus, an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations, not that which makes it obvious," and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention." Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966." Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials.' Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Consequently, it is the Office’s position that (1) the independent claim constitutes a "broad generic claim” based on generically claimed bacteria and/or generically claimed nucleic acid sequences encoding generically claimed exogenous polypeptides and generically claimed adjuvant polypeptides (2) the claimed genus has substantial variation because of the numerous options and combinations of options permitted.
The specification does not provide substantive evidence for possession of this large and variable genus, encompassing a massive number of polypeptides/nucleic acid vectors (e.g. portions, subportions, and/or truncated portions of otherwise known or described structures from any sources of any structures) claimed only by broad categorizations.
MPEP §2163 states that for a generic claim the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. If the genus has a substantial variance (as in the instant case), the disclosure must describe a sufficient variety of species to reflect the variation within that genus. Although the MPEP does not define what constitutes a sufficient number of representative species, the courts have indicated what does not constitute a representative number to adequately describe a broad genus. The courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus (e.g. see In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618). Further, MPEP §2163 states that the disclosure of only one or two species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure "indicates that the patentee has invented species sufficient to constitute the gen[us]. "See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) "[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated."). "A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when ... the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed." In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).
In the instant case, the specification provides a host L. acidophilus cell with a sequence encoding virus peptide inserted into the SlpA gene along with a sequence encoding the FliC and/or FimH adjuvant peptide. The only adjuvant peptides disclosed are the FliC and FimH peptides. The viral peptide antigen shown is the SARS-CoV-2 protein.
However, the specification does not adequately describe a representative number and a representative variety of the numerous other options encompassed by the generically claimed bacteria, nucleic acid sequences, SARS-Cov-2 antigens, promoters, secretions signals, adjuvant peptides, and/or colorization factors. Further, the specification does not adequately describe a nexus between the fully described plasmids and DNA sequences, specifically adapted for the elected Lactobacillus delivery vectors, and the breadth and diversity of the other options alone or in mixed-and-matched combinations as encompassed by the claims. The specification does not describe portions of the otherwise known structures that are sufficient to maintain their claimed functional properties. Consequently, based on this lack of information within the specification, there is evidence that a representative number and a representative variety of the numerous options with both these structural attributes (i.e. arbitrary combinations of genetically modified bacteria, DNA and/or RNA sequences, SARS-CoV-2 antigens, promoters, secretion signals, adjuvant peptides, and/or colonization factors) and the claimed functional properties (e.g. colonization of at least one tissue under non-lethal conditions, and/or antigenicity, and/or adjuvanticity, and/or gut colonization) have not yet been identified. Therefore, it is the Office’s position that even one of skill in the art would not conclude that Applicant was in possession of the entire genus.
With regards to the state of the art at the time the invention was filed, vaccine development for SARS-CoV-19, including the use of genetically engineered bacteria to deliver nucleic acid constructs, was under development, and thus necessarily unpredictable as evidenced by, for example, Diamond et al. 2020 (The Challenges of Vaccine Development against a New Virus during a Pandemic; Cell Host & Microbe; 27: 699-703) and Wang et al. 2020 (An Evidence Based Perspective on mRNA-SARS-CoV-2 Vaccine Development; Medical Science Monitor 26: 2924700). For example, Diamond teaches SARS-CoV-2 is a positive sense single stranded RNA virus first isolated in December of 2019 (e.g. see page 699, right most column) and although several vaccine candidates against the virus are in development, no vaccine against human coronaviruses are currently approved and available (e.g. see Table 1). Diamond teaches the development of an effective viral vaccine is a challenge typically guided by decades of basic research on viral biology and host response to infection, but this traditional path is not feasible for a rapidly-emerging, highly-virulent virus for which a dire need for a vaccine exists (e.g. page 702, third column). Diamond teaches several questions remain regarding the key elements of a protective immune response including: Will neutralizing antibodies correlate with protection? Do protective responses require antibody targeting of specific epitopes? Is there a threshold neutralizing antibody response that protects against SARS-CoV-2? Do assays commonly used to measure neutralization faithfully predict protective activity in vivo? What is the role of non-neutralizing anti-S antibodies that bind the surface of infected cells? What is the contribution of mucosal immunity in the respiratory tract for protection against infection or dissemination? Are humoral responses to other viral open reading frames important for immunity, especially those with immune antagonist activity that are displayed on the cell surface or secreted extracellularly? Diamond teaches that while some of this information can be gleaned from pre-clinical and early vaccine trials with SARS-CoV and MERS-CoV, even skilled artisans are not certain that these correlates of protection will transfer to the pandemic SARS-CoV-2 (e.g. see page 702, first column). Further, Diamond teaches another major challenge is the absence of high-throughput small animal disease models to facilitate vaccine candidate down-selection and the detailed study of protective immunity because it appears that conventional laboratory strains of mice are not susceptible to SARS-CoV-2 infection (e.g. page 702, first column). Diamond teaches against the backdrop of the many unknowns about coronavirus immunity there is some risk associated with expected vaccine development during a pandemic and that this uncertainty must be balanced by the enormous cost of inaction (e.g. page 703, first column). Further, Wang also teaches there remain significant challenges in the development of vaccines as rapidly as possible to control COVID-19 (e.g. see abstract; and page 6 “Perspectives”). Wang teaches finding the most suitable target site for SARS-CoV-2 vaccine development is extremely important and the current key target is the spike glycoprotein called spike protein and/or S protein (e.g. page 3, right column; and Figure 1). Wang teaches that even with hopes of an mRNA-based vaccine (i.e. a nucleic acid sequence encoding a spike protein antigen) the stoichiometric relationship between antigen and the immune response occurs in many different ways and thus it is difficult to ensure that any new vaccine targeted to the S protein could be used long term, or in the new future, because there is the possibility that the vaccine will not be effective based of differences in the S-protein sequences (e.g. page 5, left column; and page 6, right column).
Thus, the state of the art provides evidence that there is unpredictability in the results obtained from any species other than those specifically enumerated, and accordingly, the evidence indicates ordinary artisans could not reliably predict the operability in the invention of any species other than the one disclosed. Therefore, the state of the art does not provide adequate written description support for which options and combinations of options would predictably retain their functional activities; thus the only way to determine if any given combination of generically claimed, and arbitrarily combined, structures actually works, is empirical testing of each and every combination.
Consequently, neither the specification nor the state of the art provides sufficient written description to support the genus encompassed by the claims. Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that "applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the 'written description' inquiry, whatever is now claimed." (See page 1117.) The specification does not "clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed." (See Vas-Cath at page 1116.). Given the above analysis of the factors as a whole, which the courts have determined are critical in determining whether Applicant is in possession of, or the specification supports, the claimed invention, Applicant has not satisfied the requirements as set forth under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph.
Response to Applicants’ arguments:
Applicants argue:
Applicant submits that the subject matter of the present claims relates to a vaccine platform technology designed to be used with any exogenous, immunogenic polypeptide, of which coronavirus antigens are exemplary embodiments. The vaccine platform technology encompassed by the currently pending claims can also be used with any bacterial host cell, of which L. acidophilus is one exemplary embodiment. Furthermore, one of ordinary skill in the art would readily acknowledge that other immunogenic antigens and bacterial host cells can be used with the "at least one adjuvant polypeptide compris[ing] flagellin (FliC) and type 1 fimbrin D- mannose specific adhesin protein (FimH) and any derivatives or fragments thereof" to generate "a platform that is generally regarded as safe (GRAS) by the FDA; a platform that is relatively inexpensive to manufacture; a platform that is useful to target novel viral antigens to induce both mucosal and systemic immune responses while avoiding ADE; a platform that combines adjuvant strategies shown to enhance mucosal immune responses; and a platform that does not require a cold chain or special training to administer." Application, paragraph [0065].
These arguments have been fully and carefully considered but are not deemed persuasive. The bacterial cell may be any Genus/species (not even limited to a lactic acid bacteria), the exogenous polypeptide may be from any origin and any source of any structure and is not even required to be an immunogenic polypeptide or virulence factor, etc. The instant specification has only provided results and working examples for a single vaccine platform, e.g., An engineered L. acidophilus cell with a sequence encoding an immunogenic polypeptide inserted into the SlpA gene along with a sequence encoding the FliC and/or FimH adjuvant peptide, wherein the exogenous polypeptide and at least one adjuvant polypeptide are expressed by the cell; and a vaccine composition comprising said engineered cell wherein the polypeptide is SARS-CoV-2 protein.
The instant claims encompass a genus of variant species, an adequate written description of the claimed invention must include sufficient description of at least a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics sufficient to show that Applicant was in possession of the claimed genus. However, factual evidence of an actual reduction to practice has not been disclosed by Applicant in the specification; nor has Applicant shown the invention was "ready for patenting" by disclosure of drawings or structural chemical formulas that show that the invention was complete; nor has Applicant described distinguishing identifying characteristics sufficient to show that Applicant were in possession of the claimed invention at the time the application was filed. The Guidelines further state, "[f]or inventions in an unpredictable art, adequatewritten description of a genus which embraces widely variant species cannot beachieved by disclosing only one species within the genus'" (Id. at 1106);accordingly, it follows that an adequate written description of a genus cannot beachieved in the absence of a disclosure of at least one species within the genus. Otherwise, one has only a research plan, leaving it to others to explore the unknown contours of the claimed genus. See Ariad, 598 F.3d at 1353 (The written description requirement guards against claims that "merely recite a description of the problem to be solved while claiming all solutions to it and . . . cover any compound later actually invented and determined to fall within the claim's functional boundaries."). Abbvie Deutschland GmbH & Co. v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 U.S.P.Q.2d 1780, 1790, 2014 BL 183329, 12 (Fed. Cir. 2014).
The following rejections were necessitated by the amendment to the claims:
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1, 2, 5-7, 15, 19, 23-27, 29 and 30 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kajikawa et al (PLoS One. 2015 Oct 28;10(10): 1-15) in view of Makvandi et al (Microbial Pathogenesis. May 2018. Vol 118: 87-90) and Paul-Ehrlich-Insititut (https://www.pei.de/EN/newsroom/press-releases/year/2017/12-mode-of-action-of-vaccine-adjuvant-flagellin-in-fusion-proteins-has-been-clarified.html#:~:text=The%20benefit%20of%20flagellin%20is%20that%20the,various%20different%20antigens%20using%20genetic%20engineering%20technologies December 2017).
Kajikawa teaches that surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, Kajkawa constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides. See abstract. The reference teaches that the probiotic strain Lactobacillus acidophilus NCFM is particularly promising as an oral vaccine vector because: (1) it is acid and bile tolerant; (2) it expresses mucus-binding proteins and associates with the intestinal mucosa; and (3) it binds to dendritic cells (DCs) through DC-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and other pattern recognition receptors described above. The reference recites that proof of principle has been demonstrated by Mohamadzadeh et al., who constructed recombinant L. acidophilus producing the Bacillus anthracis protective antigen and succeeded in inducing protective immunity in a murine model.
For construction of recombinant L. acidophilus as a vaccine candidate, there are three strategies for the subcellular distribution of antigens: cytoplasmic accumulation, secretion, and cell surface display. In this study, Kajikawa inserted a linear epitope from the membrane proximal external region (MPER) of HIV-1 into the highly expressed bacterial surface layer protein (SlpA) of L. acidophilus, as a prototype oral mucosal vaccine platform, and assessed immunogenicity in a mouse model. n a preliminary experiment, L. acidophilus NCK2208 was only weakly immunogenic with no antibody response to MPER. To improve the mucosal immunogenicity of NCK2208, matured murine IL-1β was employed since IL-1 and IL-1 family proteins are known to act as mucosal adjuvants. We previously showed that biologically active IL-1β can be produced and secreted by another recombinant Lactobacillus strain. In the first round of i.g. immunization with the recombinant strain and reference strains, both MPER-specific Abs and the specific IgA-producing cells were detected exclusively in the group receiving the IL-1β-secreting strain. On the other hand, SlpA-specific responses did not rely on the cytokine. These results implied that the induction of MPER-specific but not SlpA-specific Abs was adjuvant-dependent. However, in the second trial where mice received four additional boosts, both L. acidophilus strains eventually elicited MPER-specific Ab responses regardless of IL-1β co-expression. This suggests that IL-1β was not essential for, but possibly expedited the specific immune responses. Additional studies are needed to confirm the adjuvant effect of IL-1β and better define the mechanism of action.
While Kajikawa teaches an engineered L. acidophilus cell comprising an exogenous polypeptide and at least one adjuvant polypeptide inserted into the S-layer protein, e.g., as a prototype oral mucosal vaccine platform, they do not particularly exemplify the use of FliC and/or FimH as the adjuvant polypeptide or the use of additional exogenous polypeptides.
Makvandi et al teach that flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system. Methods: The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR. Results: The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine. Conclusions: The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.
The reference from the Paul-Ehrlich-Institut teaches the benefit of flagellin is that the gene sequence encoding for flagellin can be merged with gene sequences of various different antigens using genetic engineering technologies. This produces so-called fusion proteins, in which the antigen against which an immune response is to be induced and flagellin are closely connected with each other in a single molecule. Although multiple animal models and clinical studies have shown before that the administration of such fusion proteins efficiently induces immune responses against the fused antigen, it has up to now largely been unknown how such fusion proteins exactly induce these immune responses.
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made that an appropriate adjuvant polypeptide to be co-expressed with a therapeutic/vaccine exogenous polypeptide in the successful vaccine platform taught by Kajikawa is flagellin polypeptide. Makvandi et al teaches that these adjuvant polypeptides can be expressed in prokaryotic host cell systems and the Paul-Ehrlich Insitut teaches they can be merged with various different antigens and expressed as fusion proteins. The use of second or additional exogenous polypeptides as recite din instant claims 26 and 27 would be an obvious design choice and one of ordinary skill in the art would be motivated to include additional antigens which reduce the number of immunizations and allow for a broader immunological response.
Claim(s) 12 and 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kajikawa et al (PLoS one. October 2015. 10(10): 1-15) in view of Makvandi et al (Microbial Pathogenesis. May 2018. Vol 118: 87-90) and Paul-Ehrlich-Insititut (https://www.pei.de/EN/newsroom/press-releases/year/2017/12-mode-of-action-of-vaccine-adjuvant-flagellin-in-fusion-proteins-has-been-clarified.html#:~:text=The%20benefit%20of%20flagellin%20is%20that%20the,various%20different%20antigens%20using%20genetic%20engineering%20technologies December 2017), as applied to claims 1, 2, 5-7, 15, 19, 23-27, 29 and 30 above, and further in view of Dimitrov et al. 2006 (US 2006/0240515).
The teachings of Kajikawa, Makvandi and the Paul-Ehrlich-Institut are set forth above. Although they teach an engineered L. acidophilus cell comprising an viral polypeptide and at least one adjuvant polypeptide inserted into the S-layer protein, e.g., as a prototype oral mucosal vaccine platform, and FliC and/or FimH as adjuvant polypeptide, they do not particularly exemplify wherein the exogenous polypeptide is a SARS-CoV-2 antigen, and particularly one form a nucleocapsid protein, spike protein, envelope protein or membrane protein.
Dimitrov teaches whole-cell, microorganism-based vaccines, e.g., engineered bacterial cells, for the treatment of severe acute respiratory syndrome (SARS) comprising nucleic acid constructs encoding SARS-CoV spike proteins, including the use of operably linked prokaryotic promoters (e.g. [0011-19, 0079-87]; and Dimitrov claims 1, 20-21, and 32-33). Dimitrov provides an example of expression and secretion of the spike protein from E. coli cells (e.g. [0032, 0076]; Figure 2 and Example 7. Dimitrov teaches the microorganisms used for vaccines (i.e. necessarily adapted for administration to a subject) express the spike protein and include Listeria and Salmonella (e.g. [0024, 0099]; and Dimitrov claim 33; meeting limitations found in claims 1 and 17). Dimitrov teaches the spike proteins may be use alone or coupled to carrier proteins (i.e. peptide adjuvants), including formulated as a fusion protein (e.g. [0058, 0069, 0090, 0094]. [0090] An immune composition of the invention can include an adjuvant and a nucleic acid, polypeptide, peptide fragment, a peptidomimetic, a coupled protein, an immunopeptide of the invention, or any combination thereof. [0093] The invention also provides vaccines that include a nucleic acid, polypeptide, a peptide fragment, a peptidomimetic, a coupled protein, an immunopeptide of the invention, a nucleic or any combination thereof. Such vaccines can be formulated as described herein or as known in the vaccine arts. [0094] The invention also provides nucleic acid-based vaccines that express a polypeptide, a peptide fragment, or a coupled protein of the invention. [0099] The invention also provides microbe-based vaccines. Generally, these vaccines relate to microbes that have been transformed with a nucleic acid construct that provides for the expression of a polypeptide, a peptide fragment, or a coupled protein of the invention. For example, Listeria monocytogenes may be used as a vector to elicit T-cell immunity. This is because it infects antigen-presenting cells and also because infection originates at the mucosa. According, Listeria may be transformed with a nucleic acid construct that provides for the expression of a polypeptide, a peptide fragment, or a coupled protein that elicits an immune response against the spike protein from the coronavirus that causes severe acute respiratory syndrome. Highly attenuated forms of Listeria may be constructed according to methods reported in the art. Lieberman and Frankel, Vaccine, 20:2007 (2002). Salmonella may also be used as a vector to elicit a cytotoxic T lymphocyte (CTL) response against the coronavirus that causes severe acute respiratory syndrome. Accordingly, Dimitrov includes engineered bacterial cells wherein the adjuvant is expressed with the exogenous polypeptide antigen.
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to use any of the Covid viral proteins taught by Dimitrov in the Lactobacillus acidophilus vaccine platform taught by Kajikawa et al because Kajikawa teaches numerous advantages over E. coli. For example, the L.acidophilus: 1) it is acid and bile tolerant; (2) it expresses mucus-binding proteins and associates with the intestinal mucosa; and (3) it binds to dendritic cells (DCs) through DC-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and other pattern recognition receptors. Additionally, Kajikawa teaches that the SLAP is a highly expressed bacterial surface layer protein (SlpA) which would be advantageous for expression of the exogenous peptides. The use of Kajikawa’s vaccine platform and the polypeptide adjuvants taught by Makvandi and Paul-Erlich institute to express the covid antigens taught by Dimitrov would have been prima facie obvious to one of ordinary skill in the art as it would be expected to be an improved vaccine platform as Kajikawa teaches L.acidophilus: 1) it is acid and bile tolerant; (2) it expresses mucus-binding proteins and associates with the intestinal mucosa; and (3) it binds to dendritic cells (DCs) through DC-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and other pattern recognition receptors. Additionally, Kajikawa teaches that the SLAP is a highly expressed bacterial surface layer protein (SlpA) which would be advantageous for expression of the exogenous peptides
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Pertinent art, not relied upon:
Exp Mol Med
. 2017 Sep 1;49(9):e373. doi: 10.1038/emm.2017.172
Bacterial flagellin—a potent immunomodulatory agent
Irshad A Hajam 1
Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent
The elicitation of immune responses at mucosal surfaces through the use of flagellin has the potential to eradicate or at least prevent the bad outcome of diseases. During the past two decades, flagellin has been extensively tested as a mucosal adjuvant against epitope-based influenza vaccines,19, 20, 70, 71 West Nile virus (WNV),72 Escherichia coli,73 Yersinia pestis,45, 74 Clostridium tetani,75 C. jejuni,76 Streptococcus77 and Plasmodium falciparum.78, 79
Green et al
RESEARCH
Microbial Cell Factories volume 18, Article number: 10 (2019)
Open access
Published: 18 January 2019
Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein. We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.
Applied Microbio. and Biotech. Vol. 99, pates 2967-2977, March 2015
Lactic acid bacteria—20 years exploring their potential as live vectors for mucosal vaccination:
Over the past two decades, intensive genetic and molecular research carried out on LAB, mainly Lactococcus lactis and some species of the Lactobacillus genus, has revealed new, potential biomedical LAB applications, including the use of LAB as adjuvants, immunostimulators, or therapeutic drug delivery systems, or as factories to produce therapeutic molecules. LAB enable immunization via the mucosal route, which increases effectiveness against pathogens that use the mucosa as the major route of entry into the human body. In this review, we concentrate on the encouraging application of Lactococcus and Lactobacillus genera for the development of live mucosal vaccines. First, we present the progress that has recently been made in the field of developing tools for LAB genetic manipulations, which has resulted in the successful expression of many bacterial, parasitic, and viral antigens in LAB strains. Next, we discuss the factors influencing the efficacy of the constructed vaccine prototypes that have been tested in various animal models. Apart from the research focused on an application of live LABs as carriers of foreign antigens, a lot of work has been recently done on the potential usage of nonliving, nonrecombinant L. lactis designated as Gram-positive enhancer matrix (GEM), as a delivery system for mucosal vaccination. The advantages and disadvantages of both strategies are also presented. Genetic engineering tools used for cloning heterologous genes in Lactococcus lactis
So far, L. lactis remains the model microorganism in LAB research. Rapid research progress on the use of LAB for treatment and prophylaxis occurred at the turn of the twenty-first century. In these studies, plasmid-cured strains of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363, whose genomes were sequenced in 1999/2001 and 2007, respectively, are most commonly used (Bolotin et al. 2001; Wegmann et al. 2007; Linares et al. 2010). Currently, databases provide genetic information on 33 strains of the species L. lactis (http://www.ncbi.nlm.nih.gov/genome/genomes/156, November 2014). The first vectors for cloning foreign genes in LAB were developed about 30 years ago, on the basis of the replication systems of the two cryptic, broad host-range plasmids of L. lactis: pWV01 and pSH71 (de Vos 1987; Kok et al. 1984). To date, many derivatives of these plasmids have been created (Shareck et al. 2004). Another widely used replicon is pAMbeta-1, isolated from Enterococcus faecalis. On the basis of its replication system, the low-copy plasmid pIL252 and the high-copy plasmid pIL253 were constructed (Simon and Chopin 1988; Shareck et al. 2004). A number of different systems for the expression of genes encoding heterologous proteins in the cells of L. lactis were developed using both constitutive and inducible promoters. The most commonly used system for expression of heterologous proteins is the NICE system, utilizing the nisin promoter (Mierau and Kleerebezem 2005). Many of the developed expression systems have inducible promoters whose expression depends on environmental conditions. These include, for example, the P170 promoter, active at low pH and subject to self-induction by accumulated lactic acid when the culture enters the stationary phase of growth (Madsen et al. 1999), or the zit operon promoter that is regulated by ZitR protein and is activated by low concentration of zinc ions (the so-called zinc hunger) (Llull and Poquet 2004). The recently described Zirex system enables induction of genes regulated by the concentration of zinc ions and by the use of the pneumococci regulatory protein SczA (Mu et al. 2013). In studies aimed at examining the potential application of LAB in immunoprophylaxis, promoters induced under conditions prevailing in the immunized body are particularly useful. One such expression system is stress inducible controlled expression system (SICE). The SICE system uses a promoter of the L. lactis groESL operon, whose expression is induced under stress conditions (Benbouziane et al. 2013). LAB as carriers of heterologous bacterial, parasitic, and viral antigens
LAB are characterized by their high physiological and genetic diversity. Thus, the abilities of different strains to persist and multiply in an immunized organism differ substantially. Moreover, dissimilarities in the composition of their cell walls results in significant differences in the stimulated immune response. Some cell-wall components such as peptidoglycan, lipoproteins, or lipoteichoic acids are recognized by eukaryotic Toll-like (TLR) or nucleotide oligomerization domain (NOD) receptors involved in the anti-inflammatory immune response. Strain-specific effects of LAB result from the induction of diverse immune regulatory pathways (Zeuthen et al. 2008; Macho Fernandez et al. 2011). Therefore, the immunization schedule developed for vaccines that have been generated on the basis of one member of the LAB group as an antigen carrier is often unsuitable for immunization of different host targets. The data obtained by different research groups are not consistent, and the difficulties comparing them result from the use of different antigens, vectors, and immunization schedules. Parameters affecting the immune response induced by prototypes of LAB vaccines will be discussed in a later part of this subsection.
: Engineering infectious cDNAs of coronavirus as
bacterial artificial chromosomes
AUTHOR(S): Almazan, Fernando; Galan, Carmen; Enjuanes, Luis
CORPORATE SOURCE: Department of Molecular and Cell Biology, Centro
Nacional de Biotecnologia, CSIC, Madrid, Spain
SOURCE: Methods in Molecular Biology (Totowa, NJ, United
States) (2008), 454(SARS- and Other Coronaviruses),
275-291
CODEN: MMBIED; ISSN: 1064-3745
DIGITAL OBJECT ID: 10.1007/978-1-59745-181-9_20
PUBLISHER: Humana Press Inc.
DOCUMENT TYPE: Journal
LANGUAGE: English
ED Entered STN: 16 Oct 2008
AB The construction of coronavirus (CoV) infectious clones had been hampered
by the large size of the viral genome (around 30 kb) and the instability
of plasmids carrying CoV replicase sequences in Escherichia coli.
Several approaches have been developed to overcome these problems. Here
we describe the engineering of CoV full-length cDNA clones using bacterial
artificial chromosomes (BACs). In this system the viral RNA is
expressed in the cell nucleus under the control of the cytomegalovirus
promoter and further amplified in the cytoplasm by the viral replicase.
The BAC-based strategy is an efficient system that allows easy
manipulation of CoV genomes to study fundamental viral processes and also
to develop genetically defined vaccines. The procedure is illustrated by
the cloning of the genome of SARS coronavirus, Urbani strain.
Hou et al:
New recombinant Lactobacillus casei exhibits porcine
rotavirus VP7 protein on its surface used as e.g.
anti-porcine rotavirus, comprises gene sequence in which
porcine rotavirus VP7 protein inserted into genome of
Lactobacillus casei
PATENT NO KIND APPLICATION DATE
------------------------------------------------------------------
CN 110042076 A CN 2019-10287460 20190411
PRIORITY APPLN. INFO: CN 2019-10287460 20190411
INT. PATENT CLASSIF.:
IPC ORIGINAL: A61K0039-15 [I,A]; A61P0031-14 [I,A]; C12N0001-21 [I,A];
C12N0015-74 [I,A]; C12R0001-225 [N,A]
CPC: A61K0039-15; A61P0031-14; C07K0014-005; C12N0015-74;
C12N2720-12322
BASIC ABSTRACT:
CN 110042076 A UPAB 20190917
NOVELTY - A recombinant Lactobacillus casei exhibits a porcine
rotavirus VP7 protein on its surface comprises a gene sequence in which a
porcine rotavirus VP7 protein is inserted into the genome of L.casei , is
new.
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the
following: a method for constructing the recombinant L.casei , involving
(a) amplifying the gene fragment of the porcine rotavirus VP7 protein, (b)
constructing the target fragment into the shuttle plasmid PLA,
transforming into Escherichia coli , and screening the positive
recombinant plasmid pLA-VP7, and (c) transforming the positive recombinant
plasmid pLAVP7 into L.casei , and screening the positive recombinant
L.casei pLA-VP7/ L.paracasei .
ACTIVITY - Virucide.
USE - The recombinant L.casei is useful as anti-porcine rotavirus
(claimed), and for preparing oral rotavirus genetically engineered lactic
acid bacteria. The invention claims a porcine rotavirus VP7 protein surface display of
recombinant lactobacillus casei, are inserted gene sequence of porcine
rotavirus VP7 protein gene group in the Lactobacillus paracasei.
construction method and application of invention further provides the
recombinant Lactobacillus casei class. The invention obtains porcine
rotavirus VP7 full-length gene sequence, constructing recombinant
Lactobacillus paracasei, selecting oral way immune mouse and piglet,
and to evaluate the immune effect of the recombinant bacteria, the result
shows enhances the immune effect of antigen epitopes, for preparing oral
research of rotavirus gene engineering lactic acid bacteria
provides feasible basis.
Sievers et al. 2020 (Clinical Trials Identifier: NCT04334980; bacTRL-Spike-1; Symvivo Corporation; first posted 04/06/20—after first effective filing date of the instant application; https://www.clinicaltrials.gov /ct2/show/NCT04334980) as evidenced by Symvivo’s bacTRL product information.
Sievers teaches compositions for use as a vaccine in human clinical trials (i.e. adapted for administration to a human) comprising Bifidobacterium longum bacteria genetically modified to comprise plasmids containing synthetic DNA encoding a spike protein of SARS-CoV-2 (i.e. called bacTRL; e.g. see page “Brief Summary” on page 2; and Table starting on page 3. As evidenced by Symvivo’s product information, their bacTRL platform binds directly to intestinal epithelial cells (i.e. colonization of at least one tissue and necessarily requires a gut colonization factor) and encodes a hybrid transport protein for the coordinated secretion and transfection of the entire pDNA-protein complex which subsequently results in the robust expression of the transgenes to elicit an immune response against the Covid-19 spike protein (i.e. necessarily requires promoters and secretion signals; see Figure 1).
Bermudes et al (US Patent No. 10973908; priority to 5/14/20—after the effective filing date of the instant application 3/30/20) A live genetically engineered bacterium, comprising a genetically engineered construct comprising a nucleic acid sequence encoding at least one portion of a SARS-CoV-2 antigen, the live genetically engineered bacterium being adapted for administration to a human or animal and colonization of at least one tissue under non-lethal conditions. The antigen is preferably the SARS-CoV-2 spike protein. The nucleic acid sequence preferably includes an associated promoter.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JENNIFER E GRASER/Primary Examiner, Art Unit 1645 1/27/26