DETAILED ACTION
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 8/28/2025 has been entered.
Claims 1, 5, 7-8, 17-23 are pending in this application. Claims 5, 7, 8 stand withdrawn from consideration as being drawn to a non-elected invention. Claims 21-23 are newly added, and fall within the elected invention.
The invention, elected without traverse, is “method of making a vaccine composition” with the initial claims elected being claims 1-4, and 13. Claims 5-12, and 14 were withdrawn from consideration as being drawn to a non-elected invention, not elected without traverse (2/15/2024). An Additional Election of Species requirement led to the election of the species of claim 4 without traverse, “based on an analysis that one or more of the B. pertussis proteins is cell-surface exposed” (7/31/2024).
Applicant’s amendments to claims 5, 7 and 8, and the request for rejoinder have been carefully considered, however claim 1 is not yet in condition for allowance. When claim 1 is in condition for allowance, this request will be re-evaluated, as the claims now depend from claim 1.
Claims 1 and 17-23 are under examination.
Applicant’s arguments and amendments file 8/28/2025 have been entered, and carefully considered, but are not completely persuasive.
The replacement Drawings filed 8/28/2025 are suitable for examination.
The papers related to the Sequence Listing have been entered.
The amendment to the specification related to the Sequence Listing has been entered.
The IDS filed 8/28/2025 has been entered and considered, and the fee sheet has been accepted.
The OATH filed 8/28/2025 has been entered.
Claim Rejections - 35 USC § 112
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, 17-23 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 directed to a method of making a B. pertussis vaccine composition, through selection of polypeptides, testing for immunogenic effect, synthesizing DNA encoding the selected polypeptide and expressing and purifying the encoded product. Claim 1 has been amended to recite that the “BP” designations indicate a “locus tag”, that the peptides are 100% conserved across B. pertussis strains, selected from surface-exposed regions, and were selected through an in-silico analysis. The claim has been amended to add a step of expressing and purifying the polypeptide encoded by the DNA fragment.
The hallmark of a vaccine is protective immunity. The claims fail to particularly point out and distinctly claim the elements required to achieve that immunity. The metes and bounds of claim 1 are unclear, with respect to the locus tags that allegedly identify proteins of B. pertussis. Claim 1 fails to particularly point out and distinctly claim the peptides to be tested for inclusion in the vaccine, and fails to identify those to be produced. The locus tags (BPXXXX) are laboratory designations, unlinked to any particular protein, with no defined structure, polypeptide sequence, or antigenicity, and laboratory designations, including any associated information, can and do change over time. It is unclear if this locus tag is meant to identify a particular protein of a particular strain, or a particular part of a protein, or the short peptide to be produced. This recitation is similar to claiming a trademarked item, where the trademark indicates the source of the item, but nothing about the item itself. The locus tag is not followed by a SEQ ID NO for the B. pertussis protein, or polypeptide, nor is it clear from the recitation what the actual smaller peptide selected from the unnamed protein of B. pertussis is to encompass. The further limitation that the peptides are 100% conserved across B. pertussis strains fails to remedy this deficiency; if no sequence is provided, by SEQ ID NO: there is nothing to compare against to ensure complete identity. The claim structure also assumes that all the proteins identified by the listed BP tags have a surface-accessible region, presumably in their native environment, however this is not clearly described. If all the B. pertussis proteins, identified by the locus tags do indeed have a surface accessible region in their native environment, the further identification of a surface-exposed region by in-silico analysis seems redundant.
It is noted that in the specification, in the Examples, beginning at [0082] the in-silico analysis occurs first, with an analysis of all surface-exposed proteins (SEP) of one strain of B. pertussis, the Tohama strain. This analysis identifies proteins that have some portion of certain proteins that are surface-exposed in their natural process of infection or persistence. An analysis of the SEP of the various strains was conducted, “to determine homology to other known structurally defined proteins using modeling algorithms… to identify potentially surface exposed regions” of the SEP [0084]. The specification indicates that this is followed by multiple-sequence alignments of various strains of the same proteins, to begin the antigen design process [0085]. From a list of predicted surface exposed regions greater than 25 amino acids long, certain peptides were selected. These peptides were analyzed in-silico for 100% homology across strains, and then “were linked sequentially to generate three individual polypeptides.” [0086]. This is not the same type of method as is now recited in claim 1. Claim 1 creates a single polypeptide, which comprises one or more peptides as described.
Further in claim 1, the claim fails to particularly point out and distinctly claim what aspect of the B. pertussis protein, identified by the locus tag, undergoes the “in-silico structural analysis” and fails to point out what the analysis determines. The recitation of “in-silico structural analysis” implies a three-dimensional type of analysis, but the point of the analysis is undefined. It is not clear whether this is for possible antigenicity, for multiple-sequence alignment, for scoring compared to some reference, for docking with a known protective antibody et al. The type of analysis performed on the B. pertussis full length protein, or on a surface-accessible part of a B. pertussis protein, will change the identified shorter peptide sequence, depending on the point of the analysis. It is unclear whether the structural analysis is intended to be performed individually on each portion of each B. pertussis protein (unlinked to any other element), or whether it is intended to be performed on the combined polypeptide structure of multiple shorter peptides from each protein (and any linker or other structural element).
The specification, beginning at [0099] recites a further set of steps to identify putative vaccine peptides targeting B. pertussis. This includes a similar set as those previously set forth, but also include an analysis of relative gene abundance in whole RNA transcriptomic studies, leading to the selection of the designed polypeptides BPPoly100 and BPPoly 101.
“[00100] In our initial studies, putative vaccine peptides targeting B. pertussis were selected based on the following criteria:
1) Identification of the species conserved core of surface exposed proteins (SEPs) using the available B. pertussis genomes. These include secreted and surface exposed proteins embedded in the outer membrane as well as proteins located in the periplasmic space as the latter are variably expressed both on the surface and in the periplasm;
2) sequence conservation, based on analysis of multi-sequence alignments of each protein;
3) Surface exposure of the core proteins, based on in-silico modelling to determine the 3-dimensional structure and the potentially surface exposed residues.
Using these criteria, a pool of approximately 150 peptides that are > 20 amino acid residues in length have been identified for B. pertussis. From these a single Bacteria Vaccine Polypeptide (BVP) was previously designed with a random assortment of peptides. This BVP, Bp Poly 1 was shown to be significantly protective in the mouse lung model (data not shown).
[00101] To further refine the selection of peptides, we investigated the relative abundance of gene specific mRNA in whole RNA transcriptomic studies to determine whether high level transcription, which generally correlates with the quantity of protein produced in bacteria, may be a useful criterion to identify protective targets… To investigate this criteria, we designed a polypeptide (Bp Poly 100) using peptides with the above criteria and derived from genes with low level transcription. We also designed a polypeptide (Bp Poly 101) using peptides with the above criteria and derived from genes with high level transcription. Each polypeptide was purified and their protective capacities were compared in the mouse model of pertussis...
[00103] To test whether transcription level may be useful for selecting protective peptides, Bp Poly 100 and Bp Poly 101 were designed using a final step of prioritization of the vaccine peptide selection based on transcriptomic data indicated by quantitative mRNA… Using our defined core of SEP genes, the individual relative abundance (RA) of each was determined, based on the data of de Gouw et al' Proteins in commercially available B. pertussis vaccines were excluded.
The peptides from proteins with the lowest RA of mRNA (values range 29-374) were incorporated into Bp Poly 100.
The peptides from proteins with the highest RA of mRNA (values range 11,819-47,656) were incorporated into Bp 101 (See Figures 6 and 7)…”
“[00112] The data show that Bp Poly 101, consisting of peptides from proteins encoded by genes with high level transcription, demonstrated significantly better protection at each time point than either the control group (adjuvant alone) or Hi Poly 100, consisting of peptides from proteins encoded by genes with low-level transcription.”
The wording of claim 1, appears to be circular in nature. Claim 1 already sets forth 7 locus tags, that allegedly identify surface-exposed peptides of a B. pertussis protein, to use in challenge experiments, and then create DNA fragments encoding a polypeptide of the up to seven peptides. It is unclear, beyond the identified BP- locus tags, what peptides are intended to be produced.
As such, the claims remain indefinite, as to what peptides are encompassed by the claim which provide protective immunity. It appears the claims lack certain necessary and sufficient limitations to create a polypeptide which upon administration will provide protective immunity.
The Examiner points out, with respect to (withdrawn, currently amended) claim 5, the amendment changes the dependency to depend from claim 1, but recites a new method, and appears to be broadening in scope, as the only result required is an immunogenic response which is not commensurate in scope with a protective immune response.
With respect to claims 17-18, and 21-22, the same issues as written above apply. Claim 17 indicates that at least two peptides are selected which is not commensurate with the polypeptide that demonstrated protective immunity. Claim 18 adds a repeated peptide, claim 21 adds linker sequences, and claim 22 only requires one or more of SEQ ID NO 11-17. The particular peptides are not particularly pointed out, nor are they clearly described such that one of skill would be apprised as to what peptides to introduce, and what additional peptides are considered to fall within the scope of the claim. They are not clearly any of the peptides represented in the sequence listing (other than claim 22).
Claims 19-20 fail to particularly point out and distinctly claim what additional peptides are to be introduced to the polypeptide, which would still provide protective immunity. The particular peptides, be they 7 or 30, are not particularly pointed out, nor are they clearly described such that one of skill would be apprised as to what peptides to introduce, and what additional peptides are considered to fall within the scope of the claim. They are not clearly any of the peptides represented in the sequence listing,
Applicant’s Arguments:
Applicant’s arguments have been carefully considered, and address some of the issues from the previous rejection, but some indefiniteness remains, or was added by amendment.
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, 17-23 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 the creation of a DNA fragment encoding “Bp Poly101” for use in creation of a vaccine against B. pertussis, does not reasonably provide enablement for the creation of DNA fragments encoding only one peptide or any other peptide fusion less than or different from Bp Poly101. 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 the invention commensurate in scope with these claims.
In In re Wands (8 USPQ2d 1400 (CAFC 1988)) the CAFC considered the issue of enablement in molecular biology. The CAFC summarized eight factors to be considered in a determination of "undue experimentation". These factors include: (a) the quantity of experimentation necessary; (b) the amount of direction or guidance presented; (c) the presence or absence of working examples; (d) the nature of the invention; (e) the state of the prior art; (f) the relative skill of those in the art; (g) the predictability of the art; and (h) the breadth of the claims.
In considering the factors for the instant claims:
a) In order to practice the claimed invention one of skill in the art must analyze B. pertussis protein sequences from one or more strains, select at least one peptide from a set of tagged proteins meeting the conditions (including in-silico analysis), test the peptides for the immunogenic effect through a protection assay in a mouse model of pertussis, create a DNA fragment that encodes that peptide in a fusion polypeptide, express and purify the polypeptide, which upon administration provides protective immunity from infection with B. pertussis. For the reasons discussed below, there would be an unpredictable amount of experimentation required to practice the claimed invention.
b) The specification provides generic guidance for selecting specific polypeptides which are a) conserved across B. pertussis strains; 2) not similar to other structurally defined proteins using modeling algorithms, and 3) are cell-surface exposed in Example 1. Each of these analyses are computer-implemented using known algorithms, such as BLAST, CHIMERA, and multiple sequence alignments.
c) The specification provides working examples of a DNA construct which encodes a fusion polypeptide comprising sub-peptides of B. pertussis proteins, which are BpPoly1, and BpPoly3 as set forth at paragraph [0087-0088] and Figure 4. Two Bp polynucleotide fragments encoding two Bp fusion polypeptides are created. DNA fragments encoding “one or more” of the identified peptides are not clearly produced. Individual peptides from the tagged proteins are not individually tested for eliciting protective immunity. Only the fusion polypeptides as set forth in [0088], in the particular order listed, appear to have been made, then purified and subsequently tested in the required mouse model to determine whether they induce protective immunity against challenge with B. pertussis.
“[0088] As used herein, the polypeptide "BpPolyl" includes 21 unique peptides from 13 B. pertussis proteins and has a theoretical molecular weight of 99-kDa.
Also as used herein, the polypeptide "BpPoly3" contains 30 unique peptides derived from 12 proteins and is 99-kDa in molecular weight.”
These sentences do not identify the “unique” peptides included for each polypeptide, nor the particular order of the peptides. Induction of an immune response is strongly related to the three-dimensional structure of the entire polypeptide, including linkers, purification motifs, and the specific order in which the peptides are fused.
The specification provides routine procedures for purifying fusion proteins, and testing them in immunizing mice, at [0089-0095]. Various titers of anti-Bp polypeptide antisera were obtained from the immunized mice. These tests of the BpPoly1 and BpPoly3 polypeptides was not disclosed or shown as to whether they elicit protective immunity, merely that they were immunogenic.
The hallmark of a vaccine is protective immunity; merely being immunogenic is not commensurate in scope with protection from future infection with B. pertussis. Challenge experiments with B. pertussis were not clearly disclosed with BpPoly1 or BpPoly3. [00101] states “BpPoly1 was shown to be significantly protective in the mouse lung model (data not shown).”
As set forth in the claims, and specification, the locus tags (BPXXXX) are laboratory designations, unlinked to any particular protein, with no defined structure, polypeptide sequence, or antigenicity, and laboratory designations, including any associated information, can and do change over time. The locus tag is not followed by a SEQ ID NO for the B. pertussis protein, or polypeptide, nor do the claims recite what the actual smaller peptide selected from the unnamed protein of B. pertussis is to encompass. The further limitation that the peptides are 100% conserved across B. pertussis strains fails to remedy this deficiency; if no sequence is provided, by SEQ ID NO: there is nothing to compare against to ensure complete identity. The claim structure also assumes that all the proteins identified by the listed BP tags have a surface-accessible region, presumably in their native environment, however this is not clearly described.
The specification provides, in the Examples, beginning at [0082] that the in-silico analysis occurs first, with an analysis of all surface-exposed proteins (SEP) of one strain of B. pertussis, the Tohama strain. This analysis identifies proteins that have some portion of certain proteins that are surface-exposed in their natural process of infection or persistence. An analysis of the SEP of the various strains was conducted, “to determine homology to other known structurally defined proteins using modeling algorithms… to identify potentially surface exposed regions” of the SEP [0084]. Antigens from known B. pertussis vaccines are excluded. The specification indicates that this is followed by multiple-sequence alignments of various strains of the same proteins, to begin the antigen design process [0085]. From a list of predicted surface exposed regions greater than 25 amino acids long, certain shorter peptides were selected. These peptides were analyzed in-silico for 100% homology across strains, and then “were linked sequentially to generate three individual polypeptides.” [0086]. This is not the same type of method as is now recited in claim 1. Claim 1 creates a single polypeptide, which comprises one or more peptides as described.
Further in claim 1, The type of analysis performed on the B. pertussis full length protein, or on a surface-accessible part of a B. pertussis protein, will change the identified shorter peptide sequence, depending on the point of the analysis. The specification does not provide guidance as to whether the structural analysis is intended to be performed individually on each portion of each B. pertussis protein (unlinked to any other element), or whether it is intended to be performed on the combined polypeptide structure of multiple shorter peptides from each protein in a particular order (and any linker or other structural element).
As set forth above, the specification, beginning at [0099] recites a further set of steps to identify putative vaccine peptides targeting B. pertussis. This includes a similar set as those previously set forth, but also include an analysis of relative gene abundance in whole RNA transcriptomic studies, leading to the selection of the designed polypeptides BPPoly100 and BPPoly 101.
“[00100] In our initial studies, putative vaccine peptides targeting B. pertussis were selected based on the following criteria:
1) Identification of the species conserved core of surface exposed proteins (SEPs) using the available B. pertussis genomes. These include secreted and surface exposed proteins embedded in the outer membrane as well as proteins located in the periplasmic space as the latter are variably expressed both on the surface and in the periplasm;
2) sequence conservation, based on analysis of multi-sequence alignments of each protein;
3) Surface exposure of the core proteins, based on in-silico modelling to determine the 3-dimensional structure and the potentially surface exposed residues.
Using these criteria, a pool of approximately 150 peptides that are > 20 amino acid residues in length have been identified for B. pertussis. From these a single Bacteria Vaccine Polypeptide (BVP) was previously designed with a random assortment of peptides. This BVP, Bp Poly 1 was shown to be significantly protective in the mouse lung model (data not shown).”
The specification fails to clearly identify the 150 peptides from which the peptides are selected for Bp Poly 1. The randomized order of peptides is not identified. The indication that Bp Poly 1 was “significantly protective” is vague with respect to whether Bp Poly 1 actually provided the required protective immunity against a challenge with B. pertussis to be identified as a vaccine.
The specification continues:
“[00101] To further refine the selection of peptides, we investigated the relative abundance of gene specific mRNA in whole RNA transcriptomic studies to determine whether high level transcription, which generally correlates with the quantity of protein produced in bacteria, may be a useful criterion to identify protective targets… To investigate this criteria, we designed a polypeptide (Bp Poly 100) using peptides with the above criteria and derived from genes with low level transcription. We also designed a polypeptide (Bp Poly 101) using peptides with the above criteria and derived from genes with high level transcription. Each polypeptide was purified and their protective capacities were compared in the mouse model of pertussis...
[00103] To test whether transcription level may be useful for selecting protective peptides, Bp Poly 100 and Bp Poly 101 were designed using a final step of prioritization of the vaccine peptide selection based on transcriptomic data indicated by quantitative mRNA… Using our defined core of SEP genes, the individual relative abundance (RA) of each was determined, based on the data of de Gouw et al' Proteins in commercially available B. pertussis vaccines were excluded.
The peptides from proteins with the lowest RA of mRNA (values range 29-374) were incorporated into Bp Poly 100.
The peptides from proteins with the highest RA of mRNA (values range 11,819-47,656) were incorporated into Bp 101 (See Figures 6 and 7)…”
“[00112] The data show that Bp Poly 101, consisting of peptides from proteins encoded by genes with high level transcription, demonstrated significantly better protection at each time point than either the control group (adjuvant alone) or Hi Poly 100, consisting of peptides from proteins encoded by genes with low-level transcription.”
These two additional, differing fusion proteins, represented peptides from proteins encoded by genes with low levels of transcription or high levels of transcription: BpPoly100 and BpPoly101. As set forth above, Example 2, beginning at [0100] describes the steps performed to select the peptides, including a step of mRNA transcription abundance analysis not required for the pending claims. The sequences of these peptides are not clearly provided, beyond the listing in Fig 5 and Fig 6. The specification fails to provide any other specific peptide sequences from B. pertussis that are suitable for inclusion in the polypeptide beyond those identified in the sequence listing. The specification fails to provide information as to whether short antigenic peptides are further selected from the SEQ ID NO identified in Figs 5 and 6, or whether all the peptides in Fig 5 are fused end to end in the given order to create BpPoly100, or whether all the peptides in Fig 6 are fused end to end in the given order to create BpPoly101. Neither the individual selected peptide sequences nor the total fusion polypeptide sequence (including linkers, or N-terminal region) are clearly identified. These additional fusion polypeptides were tested in a mouse model of pertussis, and only the BpPoly101 was shown to elicit protective immunity from challenge with B. pertussis.
d) The invention is broadly drawn to methods of making a vaccine composition, which comprises one or more unspecified peptides from one or more proteins of B. pertussis (selected from seven listed proteins), encoded by a DNA fragment, and seven proteins are listed by laboratory designation only. The selected sub-peptides which make up the fusion polypeptide are not clearly set forth, nor is there a clear single polypeptide sequence making up the polypeptide of Fig 6 and BpPoly101 set forth. The method comprises testing the one or more selected unspecified peptides from one or more proteins of B. pertussis in a mouse model of pertussis, and then creating a DNA fragment encoding the peptide(s), expressing the polypeptide, and isolating it. The claims are unlimited as to the strain of the bacteria, the peptides have few requirements to meet, the actual peptide sequences are not claimed, the order of the peptides is undefined, and the actual polypeptide sequence of the full polypeptide is not clearly claimed.
e) g) The area of Bioinformatics, and particularly Immunoinformatics is complex, and issues with immunogenicity and peptide sequences are considered to be unpredictable in the prior art. The elicitation of protective immunity requires the right three-dimensional structure of the antigens presented. Antigen recognition by the immune system, and the generation of a response to that antigen depends on that three-dimensional context. Nearly any antigen will elicit some sort of immune response upon administration to an immune-competent host. Whether the antigen(s) elicits a protective response depends on a multiplicity of factors beyond the simple amino acid sequence. A protective response is the production of antibodies, and activated immune cells which prevent future infection with that pathogen. Selecting subunits of bacterial proteins for use in a subunit or acellular vaccine for any given species of bacteria is a process of trial and error, even in the age of sophisticated statistical analysis and computer programs. ‘
As previously stated, the hallmark of a vaccine is that it elicits protective immunity in the host against challenge with the pathogen. To be a successful vaccine, the vaccine must be non-toxic, it should be broadly cross-reactive to all species of the bacteria, and it must elicit protective immunity against challenge with the bacteria. While these tenets are broadly understood in immunology, when it comes down to actually creating and producing the vaccine, one of skill in the art often finds great difficulties. As acknowledged in the specification at [0007] various reasons exist to the increased incidence of pertussis and Whooping Cough, including genetic vaccine escape, and reduced vaccine effectiveness.
[0007] “Multiple factors have contributed to the increased incidence of pertussis, including heightened awareness, improved diagnostic methods, and waning immunity after implementing acellular pertussis vaccines. Genetic vaccine escape of B. pertussis may also have contributed since B. pertussis strains isolated in the United States no longer uniformly express pertactin and FHA [Marieke, Schmidtke]. Because of reduced vaccine effectiveness and the resultant reduction in herd immunity, the current recommendations for pertussis immunization include immunization of every pregnant women during every pregnancy to temporarily provide protection to newborns (CDC, Pregnancy and Whooping Cough).”
Pollard et al. (2021) discuss the principles of vaccination, and issues with creating vaccines for certain pathogens, including acellular vaccines for B. pertussis.
“A vaccine is a biological product that can be used to safely induce an immune response that confers protection against infection and/or disease on subsequent exposure to a pathogen. To achieve this, the vaccine must contain antigens that are either derived from the pathogen or produced synthetically to represent components of the pathogen. The essential component of most vaccines is one or more protein antigens that induce immune responses that provide protection... Protection conferred by a vaccine is measured in clinical trials that relate immune responses to the vaccine antigen to clinical end points (such as prevention of infection, a reduction in disease severity or a decreased rate of hospitalization).” P83
“The adaptive immune response is mediated by B cells that produce antibodies (humoral immunity) and by T cells (cellular immunity). All vaccines in routine use.., are thought to mainly confer protection through the induction of antibodies (Fig. 3).” P85
“Another important feature of vaccine-induced protection is the induction of immune memory. Vaccines are usually developed to prevent clinical manifestations of infection. However, some vaccines, in addition to preventing the disease, may also protect against asymptomatic infection or colonization, thereby reducing the acquisition of a pathogen and thus its onward transmission, establishing herd immunity.” P88
“The waning of antibody levels varies depending on the age of the vaccine recipient (being very rapid in infants as a result of the lack of bone marrow niches for B cell survival), the nature of the antigen and the number of booster doses administered. For example, the virus-like particles used in the HPV vaccine induce antibody responses that can persist for decades, whereas relatively short-term antibody responses are induced by pertussis vaccines; and the inactivated measles vaccine induces shorter-lived antibody responses than the live attenuated measles vaccine.” P89
Pollard continues to discuss pertussis: “… the pertussis vaccine, where the focus of vaccine programmes is the prevention of disease in infancy; this is achieved both by direct vaccination of infants as well as by the vaccination of other age groups, including adolescents and pregnant women in some programmes, to reduce transmission to infants and provide protection by antibody transfer across the placenta. Notably, in high-income settings, many countries (starting in the 1990s) have switched to using the acellular pertussis vaccine, which is less reactogenic than (and therefore was thought to be preferable to) the older whole-cell pertussis vaccine that is still used in most low-income countries. It is now apparent that acellular pertussis vaccine induces a shorter duration of protection against clinical pertussis and may be less effective against bacterial transmission than is the whole-cell pertussis vaccine47. Many high-income countries have observed a rise in pertussis cases since the introduction of the acellular vaccine, a phenomenon that is not observed in low-income nations using the whole-cell vaccine48.” P89-90 Fig 1F,
“Whether vaccines prevent infection or, rather, the development of disease after infection with a pathogen is often difficult to establish, but improved understanding of this distinction could have important implications for vaccine design.” P91
“The level of protection afforded by vaccination is affected by many genetic and environmental factors, including age, maternal antibody levels, prior antigen exposure, vaccine schedule and vaccine dose.” P91-92
As previously set forth, the prior art shows that the selection of epitopes or small peptides from bacterial proteins, to be used in subunit or fusion protein vaccines is not routine, and that significant inventive skill and decision making is required, to create a workflow or pipeline of bacterial protein sequence analysis, structural analysis, conservation, and expression pattern, and the selected fusion polypeptide must be tested in a relevant challenge model to determine that the composition provides protective immunity against the relevant bacteria.
Plotkin (2014) (PTO-1449) discusses the many challenges in creating vaccines against B. pertussis, and provides certain options for improving current acellular vaccines. Plotkin notes “there is ample evidence that the alleles responsible for producing the main vaccine antigens have changed from the 1960’s…. [and] it is not clear that simply changing alleles will improve the situation.” One strategy suggested by Plotkin is to add additional peptides from B. pertussis to the acellular vaccine Tdap, such as the adenylate cyclase toxin. Another suggestion is to increase the amount of peptide in the vaccine, as opposed to adding more epitopes.
Gabutti (2015) (PTO-1449) is a review of the vaccine picture for pertussis approximately around the time of filing. Gabutti notes that B. pertussis has a complex antigen structure, in which certain proteins are known to be immunogenic, however protection from challenge with the bacteria is not lifelong and wanes over a period ranging from 4-10 years. (p109).
“With regard to infections caused by B. pertussis, antibody levels against a single antigen or a combination of antigens that can certainly be associated with clinical protection are not currently known.” (p111)
“Clinical studies have not provided certain and definitive evidence on the protective role of antibodies against PT, FHA, PRN or FIM and on the existence of a serologic correlate of protection against pertussis.”
Gabutti further discusses the levels of response against various proteins of B. pertussis and the likelihood of later infection in this section.
“Taken together, these observations snow that in order to evaluate a vaccine it is necessary to resort to clinical efficacy data. Studies aimed at evaluating antibody kinetics, which show a relatively rapid decline of the antibody level are not reliable, since it is not possible to establish with certainty a cut-off value below which the subject is susceptible to the infection.”
Burns et al. (2014) (PTO-1449) discusses issues with a resurgence in pertussis and possible solutions at length. The issues include the short-lived immunity obtained by immunization, suboptimal balance of the immune response, the need for additional vaccine antigens for optimal protection, insufficient or incorrect balance of antigens, antigen mismatch with current circulating strains, etc. One of skill in the art of vaccine creation must balance all of these issues in the selection of epitopes or short peptide sequences from proteins of pertussis, and the selected peptides or peptide fusion must elicit protective immunity against challenge.
One of skill in the art would not expect a single antigen, epitope or peptide sequence from a single protein of B. pertussis to provide protective immunity against challenge with B. pertussis for all the reasons cited above. Pertussis is a complex microorganism, with multiple proteins that may be immunogenic, or have immunogenic regions. No one protein has been shown to be sufficient to elicit protective immunity. No single epitope from a single protein has been shown to elicit protective immunity.
As discussed above, it is not only the selection of the peptides, it is the ultimate order and balance of the peptides, and the final three-dimensional structure of the fusion polypeptide which all require significant inventive skill and decision making.
Hozori et al (2023) is a recent example of applying bioinformatics protocols to B. pertussis in the attempt to create an acellular, multi-epitope vaccine against pertussis. Hozori is an example of the use of specific, trained modeling programs and in-silico analysis in the selection of subunits of polypeptides. Hozori applies a pipeline of well-defined programs, each identified by name and with the relevant parameters provided, as well as the point of each program.
“Fortunately, bioinformatics methods offer a cost-effective and time-efficient approach to achieve this goal. In this study, we selected the most promising translatable proteins with peptide signals and high immunogenicity, extracted suitable epitopes from these proteins, assembled a candidate vaccine, and performed comprehensive validation at each stage. The result is a candidate pertussis multi-epitope subunit vaccine that holds promise for further immunization efforts.” P2
Hozori identifies open reading frames present in B. pertussis genome data, identifies extracellular and outer membrane proteins and protein sequence data, identifies any post-translational modifications for those proteins, and then analyzes the selected proteins with a pipeline including prediction of allergenicity, assessment of homology to other human and animal proteins, prediction of antigenicity, prediction of toxicity, prediction of topology, prediction of antigenic regions using specific 3-dimensional modeling programs, selection of the most suitable epitopes for eliciting protective immunity, and for production in an acellular vaccine formulation. P2-3. Hozori tests and evaluated the immunogenicity of the epitopes separately and in the multi-epitope form after production of the multi-epitope fusion protein. Hozori emphasizes that the entire multi-epitope candidate, including all elements such as linkers, purification tags, or adjuvants must be further analyzed.
“The multi-epitope vaccine candidate was designed by joining T cell and B cell epitopes with appropriate adjuvant and linkers. The candidate vaccine sequence was formed using candidate epitopes, Adjuvant (Gen Bank: AKI95666.1) [37], linkers, HIV-Tat peptide (Gen Bank: AEK79606.1), and 6xHis tag… Linkers or so-called “spacers” are important elements in the design and construction of protein vaccines. They play important roles in terms of structural stability, domain-to-domain interactions, and vaccine functionality [8]. HIV-Tat is a short, positively charged amino acid that acts as a cellular diffuser and enhances the immune response against CPP-fusion [33]. A 6x His-Tag was used to purify the vaccine at the end of the sequence [38].” P3
The multi-epitope vaccine was further analyzed in-silico, to evaluate antigenicity, solubility and allergenicity of the full fusion protein, and included a physiochemical evaluation of the vaccine construct. Physiological stability is one of the analyses. P3 Secondary and tertiary structure of the multi-epitope vaccine was analyzed in-silico using several programs, databases, and scoring metrics. Protein stability was engineered by identifying structural disulfides important for epitope presentation. Codon optimization was performed, and the optimal DNA sequence encoding the multi-epitope vaccine was generated.
Hozori et al continue the analysis by performing molecular docking experiments (a 3D analysis) which identify a rigid body docking, clustering of lowest energy structures, and structural improvements through energy minimization. P4. A 3D model of the best performing fusion protein was selected using PyMOL. Hozori perform molecular dynamics simulations with the selected multi-epitope protein, using GROMACS5.1.4, modified with a variety of molecular dynamics algorithms and parameters.
“In any in-silico study, molecular dynamics analysis is critical to determine the stability of the protein-protein complex. Protein stability can be determined by comparing the dynamics of native proteins under normal conditions [32]. The molecular dynamics of the vaccine-TLR4 complex were simulated by normal mode analysis (NMA) through GROMACS5.1.4 software. This server is also used to determine various dynamic parameters such as protein structure in an aqueous environment, Leonard Jones and electrostatic potential between ligand and protein, RMSD, RMSF, the radius of gyration of protein, the surface of protein accessible to solvent and secondary structure of the protein in water and after ligand binding. was used [49].”
Hozori further used C-IMMSIM to predict how the host would respond to the proposed multi-epitope vaccine. (p4).
“The C-IMMSIM server (https://kraken.iac.rm.cnr.it/C-IMMSIM/ index.php) was used to predict the vaccine’s immune response. This prediction simulates the three main elements of the functioning mammalian system (bone marrow, thymus, and lymph nodes). This server is an agent-based model that uses position-specific scoring matrices (PSSM) for peptide prediction derived from artificial intelligence methods [50]. Immune simulation input parameters were determined as default.”
Sections 3-4 of Hozori discusses each step, and the results obtained. The list of antigens is given in tables 4 and 5, and Fig 1 is a diagram of the vaccine construct.
“Immunoinformatics solutions offer cost-effective and less harmful alternatives to traditional vaccine production, reducing the need for extensive laboratory experiments. These techniques have become more accessible and affordable over the years. In recent decades, substantial progress has been made in the design of in-silico drugs. In line with this, our primary objective in this study was to propose a candidate pertussis subunit multi-epitope vaccine with improved immunogenicity. This study, has tried to provide a vaccine with high immunogenicity by placing the stages of vaccine immunogenicity in different parts of selecting the appropriate protein as well as the appropriate epitopes and obsessing over choosing the best sequence, which in articles similar to these stages or only for the selected protein It was applied either only for selected epitopes and not for both steps. Also, the consistent and complete validation steps and the reactions between the ligand and the protein in the molecular dynamics simulator that was carried out in this study, in our opinion, can be the potential difference between this research and other similar studies.
Epitope-based vaccines aim to activate the immune system by pinpointing specific B-cell and T-cell epitopes. The effectiveness of subunit vaccines greatly depends on the careful selection of appropriate epitopes that can trigger both humoral and cell-mediated immune responses. Linkers compatible with these epitopes are employed to merge them with adjuvants, ensuring the vaccine’s structural integrity, interdomain interactions, and overall functionality. Furthermore, epitope-centric vaccines are regarded as a secure choice since they eliminate potential allergens, toxins, and undesirable components.” (p10-11)
“Using the candidate CTL and B-cell epitopes, along with adjuvants, linkers, TAT HIV peptide, and a 6x His-Tag, we constructed the candidate vaccine sequence. Linkers were crucial in ensuring the structural stability, interdomain interactions, and functionality of the vaccine [8].” P11
“We performed three-dimensional modeling to gain precise insights into the spatial configuration of key protein elements, which can aid in studying interactions with ligands, dynamics, and function [32]. Energy minimization was carried out to reduce the binding energy (BE) of the entire system, thus enhancing the stability of the complexes bound to the TLR4 protein. Codon optimization was performed to maximize the expression level of our recombinant protein-based vaccine in the E. coli K12 strain, ensuring high GC content (54.30%) and codon adaptation index (CAI). Disulfide engineering was applied to increase the vaccine’s thermal stability. Safety simulations demonstrated compatibility with acceptable safety responses. The immune response was observed to improve after repeated exposure to the antigen, suggesting practical synthesis of immunoglobulins (Ig), which play a critical role in humoral immune response. Macrophages and dendritic cells also displayed adequate activity. Finally, the optimal validation results affirmed the excellent quality and safety of the 18-epitope vaccine candidate.
The multi-epitope vaccine developed in this research distinguishes itself by utilizing the complete B. pertussis proteome (accession no: NC_018518.1 in NCBI) to identify target epitopes. These epitopes are derived from highly virulent proteins, giving them a significant advantage over existing vaccines and traditional methods. This vaccine comprises B-cell and CTL epitopes sourced from exceptionally antigenic proteins, which stimulate both humoral and cellular immunity in the host. Importantly, it excludes pathogen proteins that resemble human proteins, thereby reducing the risk of autoimmune diseases. Such vaccines have the potential to establish enduring immunity in the host.” P12
Even with the thorough in-silico analysis of the engineered multi-epitope vaccine candidate, Hozori points out that significant in vivo testing would be required, including challenge experiments, to demonstrate the elicitation of protective immunity in the desired subjects.
“It’s important to stress that this predictive in-silico work needs confirmation through in vivo experiments to fully validate these approaches. The multi-epitope vaccine presented in this study, incorporating B-cell and CTL epitopes with an appropriate adjuvant, holds promise as a pertussis vaccine. While this study represents an integrated computational approach, further laboratory research is essential to verify its safety and efficacy.” P12
Knowledge that any single epitope may aid in eliciting protective immunity does not speak to any other different epitope or peptide sequence. Knowledge that the full BpPoly101 having certain linkers, and tags, elicits protective immunity does not speak to whether any individual peptide making up BpPoly101 would also elicit protective immunity on its own. Knowledge that BpPoly101 elicits protective immunity does not speak to any sub-combinations of peptides from BpPoly101 that would elicit protective immunity. Only the full length BpPoly101 has been shown to possess that ability.
f) The skill of those in the art of immunology, immunoinformatics and bioinformatics is high.
h) The claims are broad because they are drawn to methods of a vaccine composition comprising one or more undefined antigens or peptide sequences from seven listed pertussis protein tags, where the method of creating the vaccine includes selection of one or more sub-peptides from the listed proteins, testing the peptides in an animal model of pertussis, creating a DNA fragment which encodes the one or more peptides, expressing and purifying the polypeptide. The claims do not set forth the overall structure of the sub-units, or particularly identify the sequences in each portion. The specification does not provide evidence that any one peptide has the ability to induce protective immunity against B. pertussis, nor that any sub-combination of peptides has that ability. The only polypeptide construct shown to induce protective immunity in the mouse model is the Bp Poly 101 construct, and that is the only construct enabled for this function: a vaccine.
The skilled practitioner would first turn to the instant specification for guidance to practice methods of making a vaccine composition comprising sub-peptides of proteins of B. pertussis. However, the instant specification does not provide specific guidance to practice these embodiments. As such, the skilled practitioner would turn to the prior