Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
The amended claim set filed 23 April 2026 is acknowledged. Claims 1-22 are currently pending. Of those, claims 2, 5, 8-11, and 19 are currently amended, and claims 21-22 are new. Claims 15-18, and 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 9 Sep 2025. No claims are cancelled. Claims 1-14, 19, and 21-22 will be examined on the merits herein.
References to the specification in this action will use the paragraph numbers from the Pre-Grant Publication US-20230322870-A1 (PTO-892) in order to ensure the record remains clear even if a new specification is filed with different page and line numbers.
Response to Arguments
The Applicants’ arguments filed 23 April 2026 are acknowledged. For clarity, in this action, said arguments will be referred to as “Remarks” and the Non-Final Office Action mailed 25 Nov 2025 will be referred to as “NFOA.”
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 23 April 2026 was filed in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner. A signed copy of the statement is attached with this action.
Claim Interpretation
Regarding the sequences and species election, SEQ ID NO: 4 is SEQ ID NO: 1 comprising K560A (the K at position 560 is replaced with an A) and K686A mutations, SEQ ID NO: 5 is SEQ ID NO: 2 comprising K557A and N687A mutations, and SEQ ID NO: 6 is SEQ ID NO: 3 with K571A and K702A mutations.
Regarding claim 1, the protein names ApxIA, ApxIIA, and ApxIIIA are each interpreted as including fragments named after the proteins, based on dependent claims such as claim 4 that explicitly claim that the proteins may be fragments.
Objection(s) and Rejection(s) Withdrawn
The rejections of claims 2 and 9-11 under 35 U.S.C. 112(d) are withdrawn in view of the claim amendments and arguments.
The rejections of claims 5 and 19 under 35 U.S.C. 112(b) are withdrawn in view of the claim amendments and arguments.
Rejection(s) Maintained
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Rejections - 35 USC § 103
Claims 1-10, 12-14, and 19 remain rejected and claim 21-22 are newly rejected under 35 U.S.C. 103 as being unpatentable over MacInnes et al. (US-6019984-A, filed 1996, published 2000; hereafter MacInnes; PTO-892) in view of Hur and Lee (2013; hereafter Hur; PTO-892), Segers et al. (US-6770275-B1, filed 1997 and published 2004; hereafter Segers; PTO-892), and Oh and Yoo (2017; hereafter Oh; PTO-892).
Regarding claims 1, 12, and 19, MacInnes teaches “a bacterial preparation comprising one or more isolated and purified strain of a microorganism which produces one or more RTX toxins, and which strain has at least one RTX toxin which is substantially cell-associated” (quote from col. 3 ln. 15-19, see also claims 1 and 5), and teaches that the RTX toxins can be produced by selecting strains that produce one or more RTX toxins or by using gene transfer techniques to create the strain (col. 3 ln. 43-52, col. 9 ln. 10-15), and teaches that the bacteria can be modified to include other genes including other toxins that would increase the immunogenicity of the bacterial preparation or induce a response against other antigens (col. 9 ln. 17-19, col. 11 ln. 7-11).
MacInnes teaches that the three RTX toxins in A. pleuropneumoniae are ApxIA, ApxIIA, and ApxIIIA (col. 1 ln. 48-55), but “No A. pleuropneumoniae strains have been identified which produce all three toxins” (col. 1 ln. 60-61). MacInnes teaches that “RTX toxins are also generally characterized by the organization of their operon. The operon generally consists of the following four genes: an activator gene (designated "C"), a structural toxin gene (designated "A"), and two secretion genes (designated "BD").” (col. 6 ln. 26-30).
MacInnes also teaches bacterial preparations comprising all three RTX proteins by teaching that one could use two or more isolated and purified strains of A. pleuropneumoniae which collectively have ApxI, ApxII and ApxIII which are substantially cell-associated (col. 3 ln. 31-34). MacInnes teaches that the toxins can be produced in a “cell associated” manner by several methods, such as treating with a substance that interferes with toxin secretion (col. 10 ln. 22-26) or by making genetic changes to the bacteria such as inactivation or deletion of the B and/or D transport genes (col. 10 ln. 63- col. 11 ln. 5).
Regarding claim 2, the Apx toxins that are naturally present are located at the normal chromosomal locus, and MacInnes teaches plasmid vectors can be used for genetic manipulation (col. 11 ln. 58-61).
Regarding claims 3-7, MacInnes teaches the sequences of ApxIA, ApxIIA, and ApxIIIA in Figures 1 and 2. Alignments against instant SEQ ID NOs: 1-3 are shown below in Alignments 1-3. The proteins all are at least 90% homologous to SEQ ID NOs: 1-3.
Regarding claims 8-9 and 22, MacInnes teaches that the bacteria can be A. pleuropneumoniae serotype 1 (col. 8 ln. 43-57) and teaches the use of this serotype in the Examples (col. 16 ln. 16).
Regarding claim 10, the inactivation of deletion to the B and/or D transport genes modifies an additional gene.
Regarding claims 12-14, the bacteria can be used as vaccines, either by being inactivated or in a live attenuated form (col. 13 ln. 54-59, col. 14 ln. 7-24). The vaccine may have an immunologically acceptable carrier or diluent and may comprise adjuvants (col. 14 ln. 65 – col. 15 ln. 10).
Regarding claim 19, the A. pleuropneumoniae attenuated strain further comprises the rest of the genome which encodes additional A. pleuropneumoniae antigens. For example, MacInnes teaches that outer membrane proteins (OMPs) are known in the art as protective antigens for a vaccine (col. 2 ln. 45-48).
MacInnes does not explicitly teach a single microorganism comprising all three Apx toxins, as in claim 1 and dependent claims. MacInnes does not teach inactive ApxIA, ApxIIA, and ApxIIIA with common antigenic cross-reactivity with wild-type proteins, as in claim 3, or specifically with the sequences of SEQ ID NOs: 1-3, as in claim 7. MacInnes does not teach making substitutions or deletions to ApxIA at K560 and K686, ApxIIA at K557 and N687, or ApxIIIA at K571 and K702, as in claims 4-6.
Regarding claim 1, Hur teaches “the Actinobacillus pleuropneumoniae antigens ApxIA, ApxIIA, ApxIIIA and OmpA were expressed in an attenuated strain of Salmonella (ΔlonΔcpxRΔasd)” (Abstract). Specifically, Hur teaches gene was inserted into a plasmid and separately electroporated into the attenuated strain (pg. 88 col. 1 par. 4), and mice were immunized using “approximately 1×105 CFU/ml in 10 μl of an equal-volume mixture of the four constructs” (pg. 88 col. 1 par. 4). Regarding claim 3, the genes are wild-type ApxIA, ApxIIA, and ApxIIIA because no mutations are disclosed. Regarding claims 12-13, the bacteria in the vaccine are necessarily present in a carrier or diluent in order to be in a liquid form (note the use of μl as a measurement), and the OmpA expressing strain can be considered an adjuvant. The use of colony-forming units (CFU) as a unit of measurement requires that the vaccine is live because dead bacteria do not form colonies. Regarding claim 19, OmpA is an additional swine pathogen antigen (pg. 87 col. 1 par. 2). Regarding claim 21, the genes are on a plasmid (i.e. extra-chromosomal).
Regarding claims 1-3, Segers confirms that the toxin gene (A) requires an activator gene (C) for activation (col. 2 ln. 21-30) and teaches that live attenuated vaccines have advantages over non-live vaccine types because the live vaccine is self-replicating so it can be administered in lower doses, and it mimics the natural infection in triggering more than only B-cell immunity (col. 2 ln. 57 to col. 3 ln. 55). To achieve this goal, Segers teaches using bacteria that produce the RTX-A toxin in a non-activated form, and reduces this to practice in A. pleuropneumoniae by deleting the ApxIC gene (Example 2) or both the ApxIC and ApxIIC genes (Example 3) (i.e. an additional gene is modified, see claim 10). In the double deletion, the strain still produced ApxIA and ApxIIA (col. 12 ln. 39-44), but the deletions led to strong or complete losses of hemolytic activity (col. 12 ln. 1-7). Segers teaches that the double deletion mutant “MBHPP147” (defined in the title of Table 2) “can be used as a live vaccine which confers significant homologous and heterologous protection.” (col. 14 ln. 6-8). Segers also teaches: “Alternatively, it is possible to modify the target-site of the RTX Activator protein, i.e. the acylation-site at the RTX-toxin. If this site is modified to the extent that acylation is decreased or absent, this also results in the production of an RTX-toxin in a non-activated form. The acylation site can easily be mutated using recombinant DNA techniques. Mutation can e.g. be obtained by deletion of a restriction fragment that comprises the acylation site, or by site-directed mutagenesis of the acylation site.” (col. 5 ln. 7-14). Regarding claim 2, the ApxIA and ApxIIA are located at the normal chromosomal locus. Regarding claims 3 and 7, Segers teaches that the bacteria produce ApxIA and ApxIIA that is inactive (because the proteins are not activated). There is no evidence of record that the inactive ApxIA and ApxIIA taught by Segers is not capable of cross-reacting with the wild-type proteins having SEQ ID NOs: 1-3 as in claim 7. Regarding claim 8-9, the microorganism is the Actinobacillus strain A. pleuropneumoniae that express endogenous ApxIA and ApxIIA and is derived by modifying a serotype 1 strain (Example 3). Regarding claims 12-13, the bacteria can be used in a live attenuated vaccine (col. 6 ln. 44-46, Example 3). Segers teaches that the vaccine also comprises a pharmaceutically acceptable carrier or diluent such as water, culture fluid, or physiological salt (col. 6 ln. 58-65) and optionally an adjuvant may be added to the vaccine (col. 7 ln. 5-6). Regarding claim 19, the A. pleuropneumoniae attenuated strain further comprises the rest of the genome which encodes additional A. pleuropneumoniae antigens (col. 4 ln. 28-30). For example, Segers teaches that hemolysins, cytotoxins, and outer membrane proteins could be used as antigens in a vaccine (col. 2 ln. 61-63). Segers also teaches that the vaccine may be modified to additionally carry genetic information encoding one or more additional antigens from other bacteria (col. 7 ln. 36-43).
Regarding claims 4-6, Oh teaches that the acylation modification sites for ApxIA are at K560 and K686, the acylation modification sites for ApxIIA are at K557 and N687, and the acylation modification sites for ApxIIIA are at K571 and K702 (Figure 2B).
One of ordinary skill in the art at the time of filing would consider it prima facie obvious to improve the MacInnes A. pleuropneumoniae bacteria comprising cell-wall associated ApxIA and ApxIIA by expressing recombinant ApxIIIA on a plasmid as taught in Hur as the specific example of the other toxin that MacInnes teaches can be expressed, thereby arriving at the invention for claims 1-2, 7-10, 12-14, 19, and 21-22, because both MacInnes and Hur teach a vaccine comprising all three Apx toxins, and because MacInnes teaches that additional antigens can be expressed in general while Hur provides the specific methodological details to recombinantly express ApxIIIA. Therefore, combining the three Apx toxins in a single strain would be desirable to improve the immune response relative to a two-toxin vaccine and to result in a single-strain vaccine that is easier to prepare and administer by removing the step of separately growing and mixing multiple different strains. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination.”
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that applying a known technique to a known device, method or product ready for improvement is obvious because a particular known technique is recognized as part of the ordinary capabilities of one skilled in the art. In the instant case, MacInnes contains a “base” product of a live attenuated vaccine strain comprising ApxIA and ApxIIA; and Hu contains a similar product wherein the technique of recombinantly expressing ApxIIIA is taught as advantageous in order to produce a vaccine strain that can raise an immune response against ApxIIIA. Thus, one of ordinary skill in the art would have recognized that applying the known technique taught by Hu would have yielded predictable results (i.e. the same advantages) and an improved system.
As on optional additional modification, one of ordinary skill in the art at the time of filing would consider it prima facie obvious to improve the Apx toxins being expressed in the MacInnes strain(s) by inactivating the toxins by deleting or substituting the acylation sites as taught by Segers and using the specific acylation site amino acids taught by Oh, thereby further arriving at the invention for claims 3-7, because a live attenuated vaccine strain producing an inactive toxin would still be effective, as shown by Segers, while being safer because the toxins cannot perform their toxic activities. Therefore, following the strategy of Segers to delete or substitute the acylated amino acids disclosed in Oh would be desirable to make a safer live attenuated strain with inactive Apx toxin proteins. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination.” The resulting proteins would be inactive proteins which have common antigenic cross-reactivity with SEQ ID NOs: 1-3 or at least the variant wild-type sequences taught in MacInnes in the absence of any evidence to the contrary and in view of the minor modification being made (substitution or deletion of a single amino acid).
The modifications to express ApxIIIA in the live attenuated strain of MacInnes and to delete or substitute the amino acids could be performed with a reasonable expectation of success because the required genetic modification techniques were well known at the time of filing. Note that MacInnes and Segers both explicitly teach that the bacteria can be modified to express new genes, even though they were both filed more than 20 years before the effective filing date, and that MacInnes specifically teaches the wild-type sequences of the Apx toxins, and that Oh specifically teaches the locations for modification.
Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
The response to arguments follows the alignments.
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Alignment 1: Alignment of instant SEQ ID NO: 1 and SEQ ID NO: 2 of MacInnes. The sequences have 97.9% identity.
Alignment 2: Alignment of instant SEQ ID NO: 2 and SEQ ID NO: 8 of MacInnes
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. The sequences have 100% identity.
Alignment 3: Alignment of instant SEQ ID NO: 3 and SEQ ID NO: 11 of MacInnes
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. The sequences
Response to Arguments
Applicant argues (Remarks pg. 11) that “the rejections appear to be based on an impermissible hindsight reconstruction”.
This argument has been carefully considered but is not found persuasive. In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). The rejection is based on the teachings of the art and modifications that would have been obvious to one of ordinary skill in the art at the time of filing based on that art, as described in the rejection above. The rejection does not rely on knowledge that was not publicly available at the time of filing, such as the instant specification.
Applicant argues (Remarks pg. 11-12) that none of the cited references discloses or suggests an Actinobacillus pleuropneumoniae (APP) strain expressing all three RTX toxins, ApxIA, ApxIIA, and ApxIIIA, in a single microorganism.
This argument has been carefully considered but is not found persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The examiner agrees that none of the cited references discloses the subject matter of claim 1 on their own; therefore, this is an obviousness rejection rather than an anticipation rejection.
Applicant argues (Remarks pg. 12) that because none of the cited references discloses or suggests the subject matter, “Therefore, the Examiner's conclusion relies on selectively combining isolated disclosures from unrelated references without any teaching or motivation in the art to do so.”
This argument has been carefully considered but is not found persuasive. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the motivation is found in the knowledge generally available to one of ordinary skill in the art, specifically the other rationales from the courts that were cited in the obviousness argument. As stated above:
“both MacInnes and Hur teach a vaccine comprising all three Apx toxins, and because MacInnes teaches that additional antigens can be expressed in general while Hur provides the specific methodological details to recombinantly express ApxIIIA. Therefore, combining the three Apx toxins in a single strain would be desirable to improve the immune response relative to a two-toxin vaccine and to result in a single-strain vaccine that is easier to prepare and administer by removing the step of separately growing and mixing multiple different strains. See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination.”
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that applying a known technique to a known device, method or product ready for improvement is obvious because a particular known technique is recognized as part of the ordinary capabilities of one skilled in the art. In the instant case, MacInnes contains a “base” product of a live attenuated vaccine strain comprising ApxIA and ApxIIA; and Hu contains a similar product wherein the technique of recombinantly expressing ApxIIIA is taught as advantageous in order to produce a vaccine strain that can raise an immune response against ApxIIIA. Thus, one of ordinary skill in the art would have recognized that applying the known technique taught by Hu would have yielded predictable results (i.e. the same advantages) and an improved system. “
Applicant argues (Remarks pg. 12-13) that no naturally occurring strain of APP exists which expresses all three of ApxIA, ApxIIA and ApxIIIA, even after analysis of hundreds of isolates. “This by itself is strongly indicative that a person with ordinary skill in the art would not have a reasonable expectation of success. There are 732 strains of APP with unique genome sequences, and not even one of these (as evidenced by Angen et al.) encodes all of ApxI, ApxII, and ApxIII. APP is a bacterium that is naturally transformable, and it is well-established in the art that there is a high degree of intra-species genetic exchange through mechanisms involving uptake signal sequences (USSs)…. In all these tens, if not hundreds, of millions of years, not a single APP strain with all three of ApxI, ApxII, and ApxIII has arisen despite the established high levels of intra-species genetic exchange.
In other words, despite the high genetic diversity of APP, its natural competence, frequent recombination, and the common occurrence of co-infections in pigs, no APP strain according to the invention has ever arisen. Self-evidently, given these factors, if this combination were readily achievable, it would reasonably be expected to arise in nature. Therefore, it complete absence strongly indicates that the claimed configuration is not straightforward, and that fundamental and insurmountable biological problems preclude such a strain. As such, a person with ordinary skill in the art would not have a reasonable expectation that such a strain could be produced.”
Applicant further argues (Remarks pg. 13) that “First, as noted above, despite a high level of genetic exchange between APP strains through USS and competence, over a period of millions of years, an APP expressing all three of ApxI, ApxII and ApxIII has never arisen. This alone suggests that there are insurmountable biological problems inherent within APP that preclude the production of such a modified APP.”
This argument has been carefully considered but is not found persuasive. See MPEP 716.01(c): “Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.” (emphasis added). The argument cannot substitute for evidence that there is not a reasonable expectation of success. If evidence were provided that there are “fundamental and insurmountable biological problems [that] preclude such a strain”, using standard genetic modification techniques known to the art at the time of filing, this evidence would be considered.
The data of Angen showing that A. pleuropneumoniae strains do not naturally express all three Apx toxins does not provide evidence about the expectation of success because the claims do not require that the bacteria be produced by natural transformation, and claim 1 does not require the use of any particular species of bacteria. In contrast to applicant’s argument that there is not a reasonable expectation to express the genes, Hur teaches successful introduction of all three genes ApxIA, ApxIIA, and ApxIIIA into the same species of bacteria using genetic engineering techniques. Introducing genes into genetically tractable bacteria is routine at the time of filing, and results in microorganisms that cannot be produced naturally. Therefore, the argument is not persuasive because the only evidence submitted is limited to naturally occurring strains, but the arguments do not provide any evidence to overcome the art’s teaching that genetic modifications can be made with a reasonable expectation of success using well-characterized techniques.
Additionally, it is noted that the instant specification teaches that “The introduction, replacement or modification of a nucleic acid encoding an ApxIA, ApxIIA and/or ApxIIIA polypeptide may be carried out by any appropriate technique” and gives several examples of art-known genetic modification techniques [0138], so the inventors did not do not consider the method for making the genetic modification unpredictable enough that the specification needs to explain it in detail. Also, Also, Example 2 does not disclose any modifications that needed to be made to overcome alleged “fundamental and insurmountable biological problems”. Applicant is warned that if evidence were submitted to show that the genetic modification(s) to add three Apx genes to a bacteria cannot be performed with a reasonable expectation of success, the teachings of the specification would be re-evaluated in view of the newly submitted evidence to determine whether the specification teaches how to make and use the full scope of what is claimed without undue experimentation.
Applicant argues (Remarks pg. 12-13) that “Hur describes the production of S. typhimurium which separately express each of ApxIA, ApxIIA, and ApxIIIA, as well as OmpA. As an initial point, no Apx toxin has ever been reported to be expressed using plasmids. Further, in Hur and Lee, no export genes are co-expressed with the individual Apx toxins. S. typhimurium does not naturally express such export proteins. Therefore, the induvial Apx will remain intracellular within the bacterial cells. As such, any immune response generated in Hur would be different from that of the present invention, which accurately mimics the natural expression of the Apx toxins. For this reason alone, a person with ordinary skill in the art would not have a reasonable expectation of succeeding in arriving at present invention by combining the teaching of MacInnes and Hur.”
Applicant further argues (Remarks pg. 13-14) that “Second, RTX toxins rely on dedicated secretion systems, and the simultaneous expression of three large Apx toxins (ApxI, ApxII and ApxIII are approximately 100-120 kDa each) imposes significant constraints. It was known in the art that ApxI and ApxIII may be co- expressed. However, based on the known state of the art in relation to RTX toxins, one of ordinary skill in the art would have expected that the addition of an ApxII expressing construct would disrupt expression of the other toxins, because the intrinsic limitations of the secretion machinery preclude the efficient expression and secretion of all three of ApxI, ApxII and ApxIII. … the cited art, including Hur, provides no guidance on how to overcome these constraints, and thus, does not support a reasonable expectation that all three toxins could be stably expressed together.”
This argument has been carefully considered but is not found persuasive. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., expression of the proteins, export of the proteins, generating an immune response that mimics the natural expression, expression of full proteins and secretion machinery) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Claim 1 requires only that the microorganism comprise the three nucleic acid sequences, or fragments thereof (see claim 4 and claim interpretation from NFOA par. 15).
Applicant argues (Remarks pg. 14) that “Third, the combination of multiple large toxin operons, each with distinct regulatory requirements, presents substantial challenges in terms of genetic stability. Prior art methods based on serial single cross-over events or strain-specific systems, such as those acknowledged in the present application as unsuitable, do not provide a reliable pathway to generate stable triple mutants. In contrast, the inventors have developed a specific two-step natural transformation method using linear DNA templates to achieve precise, unmarked chromosomal integration of each modified toxin gene. This approach is neither taught nor suggested in the cited art and was critical to successfully generating the claimed microorganism.”
This argument has been carefully considered but is not found persuasive. See MPEP 716.01(c): “Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.” (emphasis added). The argument cannot substitute for evidence that there is not a reasonable expectation of success when making the claimed modification. There is no evidence of record that the “specific two-step natural transformation method using linear DNA templates” is required for success and that other genetic modification techniques cannot be used with a reasonable expectation of success. If evidence were provided that the inventor’s approach yielded unexpected results and/or that the art-known genetic modification techniques are not operable to produce the bacteria comprising all three genes, then that evidence would be considered.
In contrast to applicant’s argument, it is noted that the instant specification teaches that “The introduction, replacement or modification of a nucleic acid encoding an ApxIA, ApxIIA and/or ApxIIIA polypeptide may be carried out by any appropriate technique” and gives several examples of art-known genetic modification techniques [0138], so the inventors did not do not consider the method for making the genetic modification unpredictable enough that the specification needs to explain it in detail. Also, Example 2 does not disclose any modifications that needed to be made to overcome alleged “fundamental and insurmountable biological problems”. Applicant is warned that if evidence were submitted to show that the genetic modification(s) to add three Apx genes to a bacteria cannot be performed with a reasonable expectation of success, the teachings of the specification would be re-evaluated in view of the newly submitted evidence to determine whether the specification teaches how to make and use the full scope of what is claimed without undue experimentation.
Applicant argues (Remarks pg. 14-15) that “The Examiner further relies on Segers and Oh to suggest that modification of acylation sites would have been obvious. However, Segers refers only to inactivation via deletion of the activator gene apxC and provides no sequence-level guidance or suggestion of residue-specific modifications within the Apx toxins themselves. Oh identifies acylated residues but neither teaches that modifying these residues would yield inactive yet antigenically functional toxins, nor addresses the feasibility of implementing such modifications across all three toxins in a chromosomal APP context. In fact, Oh is entirely theoretical, providing no experimental evidence to validate its hypothesis. Accordingly, the claimed mutations, and particularly the insertion of these mutations in all three of ApxI, ApxII and ApxIII as expressed in a single microorganism, is not routine, but instead represent a non-trivial and nonobvious and advantageous technical development.”
This argument has been carefully considered but is not found persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Segers teaches to modify the acylation site of the protein, as described in the rejection above, which is a known and defined site on the protein at the time of filing, as taught by Oh. Applicant appears to be arguing that looking up known facts (the Apx acylation site’s location in the protein) is beyond the level of ordinary skill at the time of filing, even when the reference (Segers) specifically identifies the acylation location as the site that should be mutated. However, this is incorrect. MPEP 2141 states: “The person of ordinary skill in the art is a hypothetical person who is presumed to have known the relevant art at the relevant time.” The person of ordinary skill would be aware of the teachings of Oh and that the same acylation site is discussed in both references. It is routine experimentation to choose the amino acids corresponding to the proteins’ known acylation sites from Oh in order to modify the acylation site as taught in Segers.
Also, see MPEP 716.01(c): “Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.” (emphasis added). The argument cannot substitute for evidence that the choice of the acylation site residues actually results in unexpected results.
Applicant argues (Remarks pg. 15) that “a person with ordinary skill in the art would have been deterred by concerns relating to toxicity and metabolic burden. ApxI and ApxIII are known in the art to be highly cytotoxic, and simultaneous expression of all three toxins would reasonably be expected to impair bacterial viability or impose significant metabolic stress. The cited art provides no indication that these challenges could be overcome, further undermining any assertion of a reasonable expectation of success.
Hur does not remedy these deficiencies. The work reported in Hur is conducted in a heterologous host, does not involve secretion through native RTX systems, and does not combine multiple toxins within a single microorganism.”
This argument has been carefully considered but is not found persuasive. See MPEP 716.01(c): “Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.” (emphasis added). The argument cannot substitute for evidence that there is not a reasonable expectation of success. There has been no evidence presented that Apx genes are toxic to bacteria. Instead, the specification states that ApxI and ApxII proteins are cytotoxic to host species such as pigs [0002-0003].
Additionally, in response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., expression of the proteins, “heterologous host”, secretion) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Claim 1 requires only that the microorganism comprise the three nucleic acid sequences, or fragments thereof (see claim 4 and claim interpretation from NFOA par. 15).
Additionally, in response to applicant's arguments against the references individually (“Hurr… does not combine multiple toxins within a single microorganism”), one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues (Remarks pg. 15-16) that “In addition, the claimed invention provides significant and unexpected advantages over the cited art. A single APP strain expressing ApxI, ApxII, and ApxIII offers broader antigenic coverage across serotypes than naturally occurring strains, which express only subsets of these toxins. Simultaneous presentation of all three toxins enables a more comprehensive immune response compared to single-toxin or multi-strain approaches. In addition, the use of a single engineered strain simplifies manufacturing, improves consistency, and facilitates formulation relative to existing multi-strain vaccines. Importantly, targeted acylation-site modifications render the toxins non-toxic while preserving antigenicity, thereby improving safety without compromising efficacy. None of the cited references teaches or suggests that would have led a person with ordinary skill in the art to predict these significant and unexpected advantages, which arise only through the inventors' inventive contribution.”
This argument has been carefully considered but is not found persuasive. See MPEP 716.01(c): “Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.” (emphasis added). The argument cannot substitute for evidence that the alleged benefits actually occur and are not expected in view of the art.
New Rejection(s)
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 4 and 7 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. The parent claims 1 and 3 each recite the genes ApxIA, ApxIIA, and ApxIIIA. Child claims 4 and 7 recite amino acid sequences for these genes that include fragments comprising at least 30% of the amino acids, which is a broader recitation.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements. In the interest of compact prosecution, the recitation of the gene names is broadly interpreted as including fragments of the ApxIA, ApxIIA, and ApxIIIA genes.
Claim Rejections - 35 USC § 112(b)
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 5, 9-11, 13-14, and 19 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.
Regarding claims 5, 9-11, 13-14, and 19, these claims recite limitations that are “preferably” or “most preferably” present. These are relative terms that present ambiguity. It is unclear if the limitations are required or not.
Claim 10 recites the limitation "at least one additional gene is modified". There is insufficient antecedent basis for this limitation in the claim because neither independent claim 1 nor new claim 22 recites that at least one gene has been modified previously, so that an additional gene can be modified in claim 10. Also, the claim is indefinite because it is unclear whether the term “additional” modifies only the term gene (i.e. any gene other than ApxIA, ApxIIA, ApxIIIA that were disclosed in parent claims) or whether it refers to additional genes that have been modified, which leads to a difference in scope because there is no requirement that all of the genes in claim 1 have been modified.
Claim 11 recites the limitation "the at least one additional gene which is modified". There is insufficient antecedent basis for this limitation in the claim because neither independent claim 1 nor new claim 22 recites that at least one additional gene is modified.
Claim Rejections - 35 USC § 103
Claims 1-14, 19, and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over MacInnes et al. (US-6019984-A, filed 1996, published 2000; hereafter MacInnes; PTO-892 mailed 25 Nov 2025) in view of Hur and Lee (2013; hereafter Hur; PTO-892 mailed 25 Nov 2025), Segers et al. (US-6770275-B1, filed 1997 and published 2004; hereafter Segers; PTO-892 mailed 25 Nov 2025), and Oh and Yoo (2017; hereafter Oh; PTO-892 mailed 25 Nov 2025) as applied to claims 1-10, 12-14, 19, and 21-22 above, and further in view of Segers (EP-0875574-A2; hereafter Segers2; PTO-892).
The combination of MacInnes, Hur, Segers, and Oh teach all elements of claims 1 and 22, which are the parent claims of claim 11. Briefly, the combination teaches that it would be obvious to improve a live attenuated vaccine strain of A. pleuropneumoniae bacteria comprising cell-wall associated ApxIA and ApxIIA by expressing recombinant ApxIIIA as taught in Hur as the specific example of the other toxin that MacInnes teaches can be expressed, and that optionally it would also be obvious to improve the Apx toxins being expressed in the MacInnes strain(s) by inactivating the toxins by deleting or substituting the acylation sites as taught by Segers and using the specific acylation site amino acids taught by Oh.
The combination does not teach at least one additional gene which is modified is (i) apxIVA; (ii) sxy; or (iii) apxIVA and sxy, as in claim 11.
Segers2 teaches a “live attenuated bacteria of the genus Actinobacillus pleuropneumoniae that have a mutation in an apxIV gene such that no functional ApxlV toxin can be produced” (Abstract). Segers2 teaches that the apxIV gene is named apxIVA (Figure 4). Segers2 teaches that apxIV deletion mutants are viable but are less virulent compared to their apxIV-possessing parent strains without impairing the immunogenic properties of the strains (pg. 3 ln. 47-48, pg. 3 ln. 58- pg. 4 ln. 1). Segers2 teaches that “animals vaccinated with a live attenuated Actinobacillus pleuropneumoniae strain according to the invention will not have antibodies against ApxIV toxin in their serum. In a comparative test, e.g. an ELISA test, such sera will therefore react with all immunogenic Actinobacillus pleuropneumoniae-proteins such as e.g. Apxl, II and/or III, but not with ApxIV. Sera from pigs infected with an Actinobacillus pleuropneumoniae field strain however will react with all immunogenic Actinobacillus pleuropneumoniae proteins, including ApxIV. Therefore, the live attenuated Actinobacillus pleuropneumoniae according to the present invention turns out to be a very suitable marker vaccine, i.e. a vaccine strain that can be discriminated from a field strain.” (pg. 7 ln. 45-51).
Segers2 teaches “Live attenuated bacteria according to the invention can be obtained in several ways. One possible way of obtaining such bacteria is by means of classical methods such as the treatment of wild-type Actinobacillus pleuropneumoniae bacteria with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment. Strains that do not produce a functional ApxIV toxin do not or to a lesser extend induce anti-ApxIV toxin antibodies, and therefore can easily be selected in animal tests. The necessary antiserum can be obtained as described below in Example 3. Another possibility is to deliberately introduce, using recombinant DNA-technology, a well-defined mutation in the gene encoding the ApxIV toxin. Such a mutation may be an insertion, a deletion, a replacement of one nucleotide by another one or a combination thereof, with the only proviso that the mutated gene no longer encodes a functional ApxIV toxin. It can easily be seen, that especially mutations introducing a stop-codon in the open reading frame, or mutations causing a frame-shift in the open reading frame are very suitable to obtain a strain which no longer encodes afunctional ApxIV toxin. Such mutations can e.g. be made by means of in vitro site directed mutagenesis using the Transformer® kit sold by Clontech. Many other standard recombinant DNA techniques such as digestion of the gene with a restriction enzyme, followed by endonuclease treatment and re-ligation, are equally applicable.” (pg. 4 ln. 40-53).
Segers2 teaches “Given the large amount of vaccines given nowadays to pigs, it is clear that combined administration of several vaccines would be desirable, if only for reasons of decreased vaccination costs. It is therefore very attractive to use live attenuated vaccine strains as a recombinant carrier for heterologous genes, encoding antigens selected from other pathogenic micro-organisms or viruses. Administration of such a recombinant carrier has the advantage that after administration of such a carrier, immunity is induced against two or more diseases at the same time. The live attenuated bacteria according to the present invention provide a very suitable carrier for heterologous genes, since the gene encoding the ApxIV toxin can be used as an insertion site for such heterologous genes. The use of the apxIV gene as an insertion site has the advantage that at the same time the apxIV gene is inactivated, and the newly introduced heterologous gene can be expressed in accordance with the homologous Actinobacillus pleuropneumoniae genes. The construction of such recombinant carriers can be done routinely, using standard molecular biology techniques such as homologous recombination.” (pg. 5 ln. 3-13).
One of ordinary skill in the art at the time of filing would consider it prima facie obvious to improve the live attenuated bacteria/vaccine from the combination of MacInnes, Hur, and optionally the combination also including Segers, and Oh by using an A. pleuropneumoniae strain with an apxIVA deletion as taught in Segers2, thereby arriving at the claimed invention, because Segers2 teaches that this strain has several benefits for a live attenuated vaccine strain including that it allows the user to differentiate vaccinated versus infected animals based on their immune response, and that the deletion of the apxIVA gene generates an advantageous site for the insertion of heterologous genes in order to reduce vaccine costs. Therefore, the combination would be desirable because to obtain these benefits for the combination’s live attenuated vaccine strain that comprises a heterologous gene (the ApxIIIA gene). See MPEP 2144(II): “The strongest rationale for combining references is a recognition, expressly or impliedly in the prior art … that some advantage or expected beneficial result would have been produced by their combination.” This modification can be made with a reasonable expectation of success because Segers2 explicitly states that the generation of apxIVA mutants and the insertion of heterologous genes can be done with routine experimentation using standard molecular biology techniques.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that applying a known technique to a known device, method or product ready for improvement is obvious because a particular known technique is recognized as part of the ordinary capabilities of one skilled in the art. In the instant case, the combination of MacInnes and Hu, and optionally the combination with Segers and Oh contains a “base” product of a live attenuated A. pleuropneumoniae vaccine strain comprising ApxIA, ApxIIA, and ApxIIIA; and Segers2 contains a similar product wherein the technique of deleting apxIVA is taught as advantageous in order to produce a vaccine strain that allows the user to differentiate vaccinated versus infected animals based on their immune response and that generates an advantageous site for the insertion of heterologous genes. Thus, one of ordinary skill in the art would have recognized that applying the known technique taught by Segers2 would have yielded predictable results (i.e. the same advantages) and an improved system. Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMELIA N DICKENS whose telephone number is (571)272-0381. The examiner can normally be reached M-F 8:30-4:30 (EDT/EST).
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/AMELIA NICOLE DICKENS/Examiner, Art Unit 1645
/SAMIRA J JEAN-LOUIS/Supervisory Patent Examiner, Art Unit 1642
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