DETAILED ACTION
Non-Final Rejection
Notice of Pre-AIA or AIA Status
1. 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
2. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08/13/2025 has been entered.
Status of Claims
3. Claims 2-4, 6-10, 13, 15-17, 23-25 are pending as per filing on 08/13/2025 and are under examination in this office action.
Priority
4. Acknowledgment is made of applicant's claim for priority based on provisional application US 62/769280 filed on 19 November 2018. Receipt of certified copy of related PCT application PCT/US19/61662 with a filing date 15 November 2019 is acknowledged as requirement of certified copies of papers required by 37 CFR 1.55.
Withdrawn Claim Rejections - 35 USC § 112 (a) Written Description
5. The rejection of claim 2 under 35 U.S.C. 112(a) as failing to comply with the written description requirement is withdrawn in view of the claim 2 amendment (claim amendment filed on 08/13/2025) that deleted UL20 gene variant and incorporated a new limitation to comprise UL24 gene variant.
Withdrawn Claim Rejections - 35 USC § 112 (a) Enablement
6. Rejection of claims 17, and 23-25 under 35 U.S.C. 112(a) is withdrawn in view of applicant’s arguments and applicant’s amendment of the claims and in vitro experimental data of the claimed oncolytic HSV infection effects on human breast cancer cell line MDA-MB 231 and MCF7.
Withdrawn Claim Rejections - 35 USC § 112 (d)
7. The rejection of claim 4 under 35 U.S.C. 112(d) is withdrawn in view of amendment of claim 2 to comprise UL24 gene variant. The instant claim 4 depends on claim 2 and thus overcame the rejection.
Claim Rejections - 35 USC § 103
8. In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
9. Claims 2, 6-10, 13, and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over Yao 2008 (US20080008686A1, published 10 Jan 2008) and further in view of O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Avitabile et al 2003 (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Han et al 2012 (published in PNAS, 109 (48) p 19798-19803), Muggeridge et al 2004 (published in Virology 328 (2004) 244 – 253) and Hillen and Berenes (published Ann Review Microbiology, 1994, 48).
Regarding claim 2: Yao discloses oncolytic Herpes Simplex-1 or 2 Viruses (HSV) with regulatable replication controlled using a tetracycline operator/repressor system, inter alia comprising deletion of ICP6, or ICP47 or does not include a functional HSV ICP47 or HSV ICP6 gene within its genome.
Yao teaches claim 2 limitations, a), b), c), and d) as recited below.
Yao (US20080008686A1) discloses the instant claim 2 limitations as given below:
Claim 2 limitation (a) a gene comprising a 5' untranslated region and a HSV -1, or HSV-2, ICP27 gene that is operably linked to an ICP27 promoter comprising a TATA element.
Yao (US20080008686A1) teaches a recombinant DNA molecule that includes a promoter sequence with a TATA element (page number 1, column 2, paragraph [0009] lines 2-3; also see page number 7, column 1, claim 1, b) comprising a 5’ untranslated region (page 4, column 1, paragraph [0032], line 8); the 5’ untranslated region of ICP27 and a HSV-1 or HSV-2, ICP gene that is operably linked to an ICP27 promoter comprising a TATA element (para [0020], lines 1-4). The claim 4 of Yao (US20080008686A1, page number 7, column 1) combined with disclosure of O'Shea and DeHart, supra, teaches an oncolytic HSV-1 or HSV-2 of instant claim 2 with limitation (a).
Claim 2 limitation (b) a tetracycline operator sequence positioned between 6 and 24 nucleotides 3' to said TATA element, wherein the ICP27 gene lies 3' to said tetracycline operator sequence.
Yao (US20080008686A1) teaches a tetracycline operator sequence located between 6 and 24 nucleotides 3' to the said TATA element (para [0020]), wherein the ICP27 gene lies 3' to said operator sequence (para [0032]). Thus, the order of elements for the claim 2 (b) limitation from 5’ to 3’ nucleotide sequence is: TATA element – TetOperator - ICP27 gene.
Claim 2 limitation (c) a ribozyme sequence located in said 5' untranslated region of said gene.
Yao (US20080008686A1) teaches a ribozyme sequence located in said 5' untranslated region to reduce the levels of ICP27 expression immediately after HSV infection, a ribozyme sequence was inserted at the 5' untranslated region of ICP27 (page number 4, column 1, para [0032], lines 7-10).
Claim 2 limitation (d) a gene sequence encoding tetracycline repressor operably linked to an HSV immediate-early promoter, wherein the gene sequence is located at the ICP0 locus.
Yao (US20080008686A1) teaches preferable promoters for direct expression of tet repressor in the recombinant DNA molecules are either the HSV immediate-early promoters or the hCMV major immediate-early promoter (page number 2, column 1, para [0010], lines 13-16). The protein products of the immediate-early genes are designated infected cell polypeptides ICP0, etc, and it is the promoters of these genes that are preferably used in directing the expression of tet repressor (tetR) (para [0022], lines 8-12) where in the gene sequence is located at the ICP0 locus. ICP0 null mutant has both copies of ICP0 coding sequence replaced (page 4, column 2, para [0039], lines 7-8).
Claim 2 limitation (e) at least two variant genes compared to wild type, wherein the HSV-1, or HSV-2, at least two variant genes are glycoprotein K (gK) variant and UL24 gene variant, wherein said oncolytic HSV does not encode functional ICPO and functional ICP34.5 protein.
Yao (US20080008686A1) does not teach claim 2 limitation (e).
Avitabile et al 2003 is in the HSV virology art and teaches limitation (e) of claim 2 by disclosing glycoprotein K (gK) variant (e) HSV glycoprotein K (gK) loci within the gK gene that carry syncytial mutations. The best characterized are those initially named syn1 and syn3. The prototype syn1 mutant virus is HSV-1 (MP), which carries the A40V substitution in the first external (or luminal) domain of gK (page number 6836, column 2, paragraph 2, lines 1-5, page 6841 figure 5, upper panel (right), gBDHL+ syn gK) and thus the variant syn gK increases syncytium as compared to wild type gK (Avitabile et al 2003, See, abstract, introduction, results).
Kousoulas 2008 (WO2008141151A2) is in the oncolytic HSV art and teaches a recombinant herpes simplex virus type-1 (HSV-1) has been constructed that carries a deletion of one of the two viral γ1 34.5 genes and other immediate early genes, which render the virus able to selectively replicate in cancer cells but not efficiently replicate in normal cells, and in which specific mutations have been introduced to enable the virus to spread among cancer cells by virus-induced fusion. Specifically, syncytial mutations have been introduced in the genes coding for glycoprotein B and glycoprotein K of the virus, enabling high replication and spread of the virus in cancer cells in the presence of substantially lower amounts of γ1 34.5 protein, which is required for optimum infectious virus produced and virus-induced cell fusion. The oncolytic HSV (HSV-1 or HSV-2) comprises a mutated coding sequence in a gene that encodes a fusogenic protein or a protein that regulates virus- induced cell fusion, wherein presence of said mutated coded sequence increases the ability of the virus to promote virus-induced cell fusion, wherein said gene with a mutated coding sequence is selected from the group consisting of UL27 (gB), UL53 (gK), UL20 or UL24, and wherein the mutated coding sequence is in the UL53 (gK) gene , and HSV comprises mutated coding sequences for at least two different genes that increase the ability of the virus to promote virus-induced cell fusion, mutations in UL24 include deletions, truncations and single amino acid changes (See WO2008141151A2, abstract, claims 1-4, 7-8, 10, para [0034]).
Abdeljelil et al 2013 is in the art and teaches that the UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion, mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. These data supported a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection (See Abdeljelil et al 2013, abstract, entire article, figures and results).
Yao does not teach an oncolytic HSV comprising a recombinant DNA comprising deleted or disrupted or non-functional ICP0 gene or ICP34.5 gene.
O'Shea and DeHart 2017 is in the art and disclose an oncolytic Herpes Simplex Virus (HSV) HSV-1 comprising recombinant DNA wherein the ICP0 and ICP 34.5 genes are entirely deleted or disrupted and does not encode functional ICP0 and ICP34.5 proteins (WO2017189754A1, page number 23, lines 18-22 and page number 17, lines 21-29).
It would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to modify genome of an oncolytic Herpes Simplex Virus (HSV) of Yao (US20080008686A1) to incorporate teachings of O'Shea and DeHart (WO2017189754A1), on the ICP0 and ICP 34.5 genes that are entirely deleted or phenotypically disrupted, and use additional teachings of Yao, Avitabile (variant gK), Kousoulas 2008 (variant gK and variant UL24) and Abdeljelil et al 2013 on UL24 to generate a regulatable and fusogenic oncolytic HSV-1 that is tet operator and tet repressor inducible and regulatable offering control over replication and has enhanced syncytium formation phenotype conferred by variant gK vatiant UL24 resulting in an enhanced fusogenic activity for the oncolytic KTR27-F HSV virus to arrive at the invention of claim 2. An artisan of ordinary skills would have recognized that the modified recombinant oncolytic HSV would provide enhanced fusion and selective killing of cancer or tumor cells. Additionally, the artisan would have recognized that a variant gK gene (conferring fusogenic syn phenotype) and variant UL24 gene resulting in impairment of UL24 function would contribute to the fusogenic phenotype (See, Kousoulas 2008, and Abdeljelil et al 2013). The artisan would have recognized non-functional ICP0 and ICP34.5 would offer safer oncolytic HSV because deletion or disruption of HSV ICP34.5 gene would reduce neurovirulence because ICP34.5 acts as a neurovirulence factor (See, O'Shea and DeHart, WO2017189754A1, page 10, lines 26-30). The ICP0 deletion mutant HSV replicate more efficiently in cancer cells and has impaired replication in normal cells (See, O'Shea and DeHart, WO2017189754A1, page 32, lines 29-33; page 33 lines 1-7). Another motivation to make the modifications taught by Yao, Avitabile et al, Kousoulas 2018, and Abdeljelil et al 2013 in the claimed oncolytic HSV would be teachings of Han et al that in cell cultures, wild type HSV-1 rarely induces syncytia; however, mutations in genes encoding gB, gK, or UL24 can give rise to this property (See, Han et al, page 19798, col 1, para 1) and clinical isolates of HSV-1 and HSV-2 are rarely syncytial (See, page 245, col 2, para 2, Muggeridge et al, 2004, Virology 328 (2004) 244 – 253). There would be motivation for commercial success and interest as well. One would have been motivated to do so and there would have been a reasonable expectation of success given the disclosures of the combined prior art teachings as recited above.
Regarding claims 6-10 (dependent on claim 2): The combined teachings of Yao, O'Shea and DeHart, Avitabile, Kousoulas 2018, and Abdeljelil et al 2013, Han, Muggeridge and Hillen and Berenes teaches claim 2 as recited supra.
Yao (US20080008686A1) teaches the limitations of claim 6-10 as follow:
Regarding claim 6, Yao teaches the tetracycline operator sequence comprises two Op2 repressor binding sites. The promoter is under the control of a tetracycline operator sequence that has two op2 repressor binding sites joined together by between two and twenty linking nucleotides. The positioning of the operator sequence is important to achieve effective control over the promoter (page number 1, column 2, para [0009], lines 3-9)
Regarding claim 7, Yao teaches an HSV-1, KTR-27, in which the essential ICP27 gene is under the control of the tetO-bearing ICP27 promoter (page 4, para [0032], lines 1-6 and para [0041], lines 1-3).
Regarding claim 8, Yao in an example of embodiment on HSV-1 teaches preferable promoter for direct expression of immediate-early gene or for direct expression of tet repressor in the recombinant DNA molecule are the HSV immediate-early promoter (page 2, column 1, para [0010]).
Regarding claim 9, Yao teaches the HSV immediate-early promoter, preferably either an ICP0 promoter or ICP4 promoter. (Page 3, column 2, para [0025], lines 4-5; claim 9, page 7).
Regarding claim 10, Yao’s (US20080008686A1) claim 11 through its dependency on claim 1 (page 7, column 1, claim 11 and claim 1) teaches the recombinant DNA is part of the HSV-1 genome or HSV-2 genome (page 2, column 1, para [0010], lines 1-4).
It would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to further modify the combined prior art teachings as applied to claim 2 above and recited above with additional teachings of Yao to arrive at the inventions of claims 6-10. The motivation would be to develop a tightly regulated tetracycline inducible oncolytic HSV with HSV immediate early promoter for enhanced transcription efficacy and oncolytic properties leading to commercial success. One of the ordinary skills in the art would have a reasonable expectation of success given the disclosures of the combined prior art teachings applied as recited above.
10. Claims 3, 13, and 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Yao 2008 (US20080008686A1, published 10 Jan 2008), O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Avitabile et al 2003 (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Han et al 2012 (published in PNAS, 109 (48) p 19798-19803), Muggeridge et al 2004 (published in Virology 328 (2004) 244 – 253), Hillen and Berenes (published Ann Review Microbiology, 1994, 48) as applied to claim 2 above, and further in view of Dolter et al 1994 (Published December 1994, Journal of Virology, 68 (12) pages 8277-8281).
Claim 3 is dependent on claim 2 with further added limitation that, the oncolytic HSV of claim 2, wherein the variant gene is a gK variant gene that encodes an amino acid substitution selected from the group consisting of: an Ala to Val amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2; an Ala to "x" amino acid substitution corresponding to amino acid 40 of SEQ ID NO: 2, wherein "x" is any amino acid; an Asp to Asn amino acid substitution corresponding to amino acid 99 of SEQ ID NO: 2; a Leu to Pro amino acid substitution corresponding to amino acid 304 of SEQ ID NO: 2; and an Arg to Leu amino acid substitution corresponding to amino acid 310 of SEQ ID NO: 2.
The combined teachings of Yao, O’Shea and DeHart, and Avitabile et al, Kousoulas 2008 and, Abdeljelil et al 2013 as applied to claim 2 do not teach an oncolytic HSV of claim 2 with the variant gK gene that encodes Ala40Vak mutation for enhanced cell syncytium formation through increased fusogenic property of gK protein.
Dolter et al discloses syncytial mutations in the Herpes Simplex Virus -1 (HSV-1) gK gene corresponding to SEQ ID NO: 2, the amino acid position 40 as Ala40Val mutation (page 8279, figure 2, amino acid sequence of gK, and table 2, showing gK variants with syn20b, MPc, syn33b, syn102, syn105, syn33), the resulting oncolytic HSV virus with variant gK Alanine40Valine mutation confers enhanced fusogenic property for improved oncolysis of cells. The amino acid sequence is available in Uniport database with ID Q69472_HHV1. The SEQ ID NO:2 of the instant application and gK amino acid sequence reported by Dolter et al and entered in Uniport database on 01 Nov 1996, integrated into UniProtKB/TrEMBL shows 100 % homology and Alanine at position 40 in bold face letter as shown below and further in view Dolter et al (page 8279, table 2, syn mutations in gK) is read as oncolytic HSV virus with variant gK Ala40Val.
Qy: SEQ ID NO:2 from instant application.
Db: SEQ ID Q69472_HHV1 from Uniport data base.
Q69472_HHV1
Query Match 100.0%; Score 1782; Length 338;
Best Local Similarity 100.0%;
Matches 338; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MLAVRSLQHLSTVVLITAYGLVLVWYTVFGASPLHRCIYAVRPTGTNNDTALVWMKMNQT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MLAVRSLQHLSTVVLITAYGLVLVWYTVFGASPLHRCIYAVRPTGTNNDTALVWMKMNQT 60
Qy 61 LLFLGAPTHPPNGGWRNHAHICYANLIAGRVVPFQVPPDATNRRIMNVHEAVNCLETLWY 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LLFLGAPTHPPNGGWRNHAHICYANLIAGRVVPFQVPPDATNRRIMNVHEAVNCLETLWY 120
Qy 121 TRVRLVVVGWFLYLAFVALHQRRCMFGVVSPAHKMVAPATYLLNYAGRIVSSVFLQYPYT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 TRVRLVVVGWFLYLAFVALHQRRCMFGVVSPAHKMVAPATYLLNYAGRIVSSVFLQYPYT 180
Qy 181 KITRLLCELSVQRQNLVQLFETDPVTFLYHRPAIGVIVGCELMLRFVAVGLIVGTAFISR 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KITRLLCELSVQRQNLVQLFETDPVTFLYHRPAIGVIVGCELMLRFVAVGLIVGTAFISR 240
Qy 241 GACAITYPLFLTITTWCFVSTIGLTELYCILRRGPAPKNADKAAAPGRSKGLSGVCGRCC 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GACAITYPLFLTITTWCFVSTIGLTELYCILRRGPAPKNADKAAAPGRSKGLSGVCGRCC 300
Qy 301 SIILSGIAMRLCYIAVVAGVVLVALHYEQEIQRRLFDV 338
||||||||||||||||||||||||||||||||||||||
Db 301 SIILSGIAMRLCYIAVVAGVVLVALHYEQEIQRRLFDV 338
Therefore, it would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to modify an oncolytic HSV generated by combined teachings of Yao, O'Shea and DeHart, and Avitabile et al, Kousoulas 2008 and, Abdeljelil et al 2013 to further incorporate, the teachings of Dolter et al. The motivation to incorporate Dolter et al teachings on a variant gK with Alanine40Valine mutation at position 40 corresponding to SEQ ID NO:2 would be that the resulting oncolytic HSV virus would confer with enhanced cell fusogenic property to the variant gK comprising Alanine40Valine substitution and would lead to commercial success. One of the ordinary skills in the art would have a reasonable expectation of success given the teachings of Yao, O'Shea and DeHart, Avitabile et al, Kousoulas 2008 and, Abdeljelil et al 2013 on generating a recombinant oncolytic HSV in the art.
Claims 13, and 15-17: The combined teachings of Yao, O'Shea and DeHart, Avitabile, Kousoulas 2008 and, Abdeljelil et al 2013, Han, Muggeridge and Hillen and Berenes teaches claim 2 as recited supra.
Regarding claim 13: O'Shea and DeHart further teaches added limitation of instant claim 13 by disclosing an oncolytic HSV, inter alia, further comprises a heterologous gene which encodes an immunostimulatory molecule, interleukin 12 (IL-12) that can increase the efficacy of oncolytic HSV to induce an anti-tumor specific immunity (See, claims 11 and 12).
It would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to modify the invention of Yao to incorporate teaching of O'Shea and DeHart comprise heterologous immunostimulatory molecule IL-12 encoding gene in the oncolytic HSV to arrive at the invention claim 13. One of the ordinary skills would have been motivated to modify the invention of Yao because interleukin 12 (IL-12) can increase the efficacy of oncolytic HSV to induce an anti-tumor specific immunity (See, O'Shea and DeHart, WO2017189754A1, claims 11 and 12). There would have been a reasonable expectation of success given the disclosures of O'Shea and DeHart as recited supra.
Regarding claims 15: The instant claim 15 is directed to a composition comprising an oncolytic HSV according to claim 2 of the instant application. The composition comprising an oncolytic HSV as claimed in instant claim 15 is same to that of a recombinant oncolytic HSV as per claim 2. Therefore, combined teachings of O'Shea and DeHart, Yao, Avitabile, Han, Muggeridge and Hillen and Berenes teaches claim 2 as recited supra renders the claim 15 obvious. (See, WO2017189754A1, O’Shea and DeHart, page 21, lines 1-2 where O’Shea and DeHart describe compositions comprising the recombinant HSV).
Regarding claims 16: The claim 16 is directed to a composition comprising an oncolytic HSV according to claim 15 with an added limitation that recite "pharmaceutically acceptable carrier”. O'Shea and DeHart discloses compositions comprising the recombinant HSV may comprise a pharmaceutically acceptable carrier (see page 21, lines 1-12). Therefore, combined teachings of O'Shea and DeHart, Yao, Avitabile, Sarfo, Han, Muggeridge and Hillen and Berenes teaches claim 15 as recited supra renders the claim 15 obvious.
Regarding claims 17: The invention in instant claim 17 is directed to a method for treating cancer, the method comprising administering the oncolytic HSV of claim 2 to a subject having cancer, wherein the cancer is the breast cancer (claim 17 limitation).
O'Shea and DeHart (WO2017189754A1) teach claim 17 recombinant HSV of is administered to a subject with cancer (13 and 23 of O'Shea and DeHart (WO2017189754A1, claim 13, page number 46, line numbers 12-14 and page number 47, lines 15-16). The instant claim 17 teaching is supported by methods of treatment using oncolytic HSV in the specification of O'Shea and DeHart (WO2017189754A1, page number 23, methods of treatment using oncolytic HSV, lines 18-21 and page number 17, lines 21-27). O'Shea and DeHart discloses treatment of cancer-a soft tissue sarcoma. The specification further includes embodiments on treatment of solid tumors including carcinomas of breast (breast cancer), colon, rectum, lung, oropharynx, hypopharynx using the oncolytic HSV as supra (claim 34, claims on page 48, line 23-24; and methods of treatment using oncolytic HSV in the specification of page number 23, lines 18-21 and page number 24, lines 15-17).
It would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the above recited teachings of O'Shea and DeHart (WO2017189754A1) into Yao’s invention of tetracycline regulatable oncolytic HVS to arrive at the instant claims 17. The motivation would be to formulate the oncolytic HSV of the claim 2 in a form of pharmaceutical composition comprising carrier for treatment of cancer, solid tumor, breast cancer to perform animal experiments of tumor model or human cancer treatment trial (O'Shea and DeHart, WO2017189754A1). There would have been a reasonable expectation of success given the disclosures of O'Shea and DeHart and disclosures in the art as recited supra. Formulation of pharmaceutical composition comprising a carrier is a routine in the art and treatment of cancer in experimental animal models is performed as per standard protocol irrespective of the outcome or end results and is routine in the art of oncolytic HSV virotherapy of breast cancer.
11. Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Yao (US20080008686A1, published 10 Jan 2008) and Avitabile et al (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Hiroaki and Hideaki (US20210386807A1, published 16 December 2021, with an earlier priority filed application JP2018-203553 of 30 October 2018), as applied to claim 2 above, and further in view of Bertrand et al 2010 (Journal of Virology, 2010, vol 84 (1) p. 109–118), Pearson and Bertrand 2014 (US8771698B2, 07/08/2014), and Carmichael et al 2018 (PLoS Pathog 14(5): e1007054).
Claim 4 is dependent on claim 2 with an added limitation, the oncolytic HSV of claim 2, wherein the variant gene is a UL24 gene that encodes a Ser to Asn amino acid substitution corresponding to amino acid 113 of SEQ ID NO: 3.
Regarding claim 4, combined teachings of O'Shea and DeHart, Yao and Avitabile et al, Kousoulas 2008 and Abdeljelil et al 2013 as applied to claim 2 does not teach an oncolytic HSV of claim 4 with UL24 gene that encodes a Ser113Asn mutation/substitution corresponding to SEQ ID NO:3 of instant application.
Hiroaki and Hideaki (US20210386807A1) disclose an oncolytic HSV with UL24 gene SEQ ID NO: 5 with 100% identity to instant claim 4 UL24 gene SEQ ID NO:3, however, does not teach S113N substitution/mutation. Hiroaki and Hideaki teach an oncolytic HSV with syn mutations of the UL24 protein as shown in amino acid positions of the UL24 protein, by using the amino acid numbers of SEQ ID NO: 5. Examples of the syn mutation of the UL24 protein may include T62, R63 and V64 mutations such as T62G/R63V/V64S (See, para [0070], para [0065]- [0068]). The “syn mutation (syncytial mutation)” means a mutation introduced mainly into the gB gene (or gB protein), gK gene (or gK protein), UL20 gene (or UL20 protein: envelope protein UL20) and/or UL24 gene (or UL24 protein: nuclear protein UL24) of HSV. According to this mutation, the membrane fusion activity of the HSV is promoted. The amino acid sequence of the gB protein of the KOS strain, the amino acid sequence of the gK protein of the KOS strain, the amino acid sequence of the UL20 protein of the KOS strain, and the amino acid sequence of the UL24 protein of the KOS strain are shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively (See, para [0065]).
Qy: SEQ ID NO:3 of instant application.
Db: SEQ ID NO 5 of US20210386807A1 by Hiroaki and Hideaki.
ORGANISM: human herpesvirus 1
Query Match 100.0%; Score 1359; Length 269;
Best Local Similarity 100.0%;
Matches 269; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAARTRSLVERRRVLMAGVRSHTRFYKALAKEVREFHATKICGTLLTLLSGSLQGRSVFE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAARTRSLVERRRVLMAGVRSHTRFYKALAKEVREFHATKICGTLLTLLSGSLQGRSVFE 60
Qy 61 ATRVTLICEVDLGPRRPDCICVFEFANDKTLGGVCVIIELKTCKYISSGDTASKREQRAT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ATRVTLICEVDLGPRRPDCICVFEFANDKTLGGVCVIIELKTCKYISSGDTASKREQRAT 120
Qy 121 GMKQLRHSLKLLQSLAPPGDKIVYLCPVLVFVAQRTLRVSRVTRLVPQKVSGNITAVVRM 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GMKQLRHSLKLLQSLAPPGDKIVYLCPVLVFVAQRTLRVSRVTRLVPQKVSGNITAVVRM 180
Qy 181 LQSLSTYTVPMEPRTQRARRRRGGAARGSASRPKRSHSGARDPPEPAARQVPPADQTPAS 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 LQSLSTYTVPMEPRTQRARRRRGGAARGSASRPKRSHSGARDPPEPAARQVPPADQTPAS 240
Qy 241 TEGGGVLKRIAALFCVPVATKTKPRAASE 269
|||||||||||||||||||||||||||||
Db 241 TEGGGVLKRIAALFCVPVATKTKPRAASE 269
Pearson and Bertrand 2014 (US8771698B2) teach modulation of UL24 interactions with protein targets and uses thereof for inhibition of herpesvirus infection. The UL24-associated syncytial phenotype suggests that UL24 is involved in membrane fusion events during infection. Thus, UL24 has an impact on the subcellular distribution of viral glycoproteins involved in membrane fusion, particularly at late times in infection (See, Example 8, SEQ ID NO: 30). The UL24 the affects co-localization of HSV-1 glycoproteins gB and gD with F-Actin. The quantification confirmed that the co-localization association of gB and gD with F-actin at late times in infection was affected by UL24, and thus that UL24 plays a role in the association of viral glycoproteins involved in fusion events with microfilaments (See, Example 9, SEQ ID NO: 30).
Bertrand et al 2010 teaches conserved residues in the UL24 protein of HSV-1 are important for dispersal of the nucleolar protein nucleolin from nucleolar foci throughout the nuclei of infected cells. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39 degrees C. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli (See, abstract).
Carmichael et al (2018) is in the art and teaches the HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B. Carmichael et al teaches Strikingly, many of the accessory proteins needed for cell-to-cell spread are also necessary for the syncytial (Syn) phenotype exhibited by certain HSV-1 mutants. These mutants inappropriately cause cell fusion, resulting in large multinucleated cells. Most syncytial mutations cause changes in the cytoplasmic tail of gB or the amino-terminal segment of gK, but alterations in UL20 and UL24 can also produce the Syn phenotype. Syn variants arise from alterations to any of four different proteins (gB, gK, UL20, or UL24), the Syn variants of UL20 and UL24 (UL24syn (G121A) have intrinsically weaker phenotypes, the UL20syn and UL24syn variants are poorly fusogenic (Fig 1A, p. 4 para 1, p. 11, p. 20 para 2, p.24 legend for figure S5 and fig S5).
The claimed UL24 gene S113N substitution or mutation (instant claim 4) has occurred spontaneously during the in vitro passage of KTR27 HSV in U2Os cells to generate the claimed KTR27-F (fusogenic) oncolytic HSV mutant virus and whether the S113N substitution in the UL24 gene contributes to the fusogenic activity of KTR27-F remains to be determined (See, the instant application specification para [00114]). As per the teachings of Bertrand et al 2010, the location of the naturally occurred UL24 gene S113N substitution/mutation (naturally generated variant UL24 gene) is within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif as disclosed by Bertrand et al 2010 and Carmichael et al (2018) (See, Bertrand et al 2010 and Carmichael et al (2018) entire article) and therefore would be expected to have impairment in the function of variant UL24 comprising S113N mutation as disclosed by Bertrand et al 2010 and Pearson and Bertrand 2014, and contribute to enhanced fusogenic phenotype of the KTR27-F HSV variant.
Therefore, it would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to modify an oncolytic HSV generated by combined teachings of O'Shea and DeHart, Yao and Avitabile et al to further incorporate, as per teachings of Hiroaki and Hideaki, Bertrand et al 2010 and Pearson and Bertrand 2014 and Carmichael et al 2018
a variant UL24 gene, and the variant UL24 can comprise naturally occurred S113N substitution corresponding to SEQ ID NO:3 so that the resulting oncolytic HSV virus has increased syncytium formation (fusogenic) phenotype by comprising variant gK and variant UL24 and would be expected of commercial success. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the invention as claimed in claim 4. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, G).
12. Claims 23-25 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Yao 2008 (US20080008686A1, published 10 Jan 2008), O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Avitabile et al 2003 (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Han et al 2012 (published in PNAS, 109 (48) p 19798-19803), Muggeridge et al 2004 (published in Virology 328 (2004) 244 – 253), Hillen and Berenes (published Ann Review Microbiology, 1994, 48) as applied to claim 2 above and further in view of Yao et al 2010 (published in J Virol. 2010 Jun 2;84(16):8163–8171).
Regarding claim 23-25: The inventions of claims 23-25 are directed to the method of claim 17, further comprising administering an agent that regulates a tetracycline operator-containing promoter (claim 23); the method of claim 23, wherein the agent is doxycycline or tetracycline (claim 24); the method of claim 23, wherein the agent is administered locally or systemically (claim 25).
The combined teachings of Yao, O'Shea and DeHart, Avitabile, Kousoulas 2008, Abdeljelil et al 2013, Han, Muggeridge and Hillen and Berenes teaches claim 2 as recited supra.
Yao 2008 teaches a tetracycline repressor regulated oncolytic HSV-1 virus for treatment of cancer patients with solid tumors (See, US20080008686A1, abstract, claims 1-10). Yao further teaches administering locally the oncolytic HSV to the solid tumor for a period sufficient to allow infection of tumor cells and administering tetracycline either systematically or locally to the tumor in a patient; wherein said tumor is a melanoma or a tumor of the breast (breast cancer). (See, US20080008686A1, claims 19, and claim 23; para [0015]).
Yao et al 2010 provides working examples of application of the tetracycline operator comprising regulatable oncolytic HSV type 1 recombinant virus to demonstrate efficacy of the virus in breast cancer cell line (See, page 8168, figure 6) and treatment of solid tumor in mice (See, page 8168, figure 7).
It would have been obvious to an artisan of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the combined teachings of Yao, O'Shea and DeHart, Yao, Avitabile et al, Kousoulas 2008, Abdeljelil et al 2013, Han, Muggeridge and Hillen and Berenes as recited supra to arrive at the claim 23-25 invention on treating of the solid tumor and breast cancer in a mouse model/clinical trial in patient to arrive at the inventions of claims 23-25. An artisan of ordinary skills would have been motivated to demonstrate the efficacy of the tetracycline operator comprising regulatable oncolytic HSV type 1 recombinant virus of instant claim 2 for safety, and further clinical trials for commercial success. There would have been a reasonable expectation of success to arrive at the inventions of claims 23-25 given the disclosures available in the art as recited supra and the disclosures of Yao 2010 and Yao et al 2008. This is analogous to some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the inventions of claims 23-25. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) (see MPEP § 2143, example of rationales, G).
Double Patenting
13. The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
14. Claims 2, 6, 9-10, 13, and 15-17 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 9, 11, 13, 17, 26, 29, 32-33 and 39 of copending Application No. 17/776, 828 in view of Yao 2008 (US20080008686A1, published 10 Jan 2008) and further in view of O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Avitabile et al 2003 (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology, 444(2013)263–273), Han et al 2012 (published in PNAS, 109 (48) p 19798-19803), Muggeridge et al 2004 (published in Virology 328 (2004) 244 – 253) and Hillen and Berenes (published Ann Review Microbiology, 1994, 48), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Dolter et al (Published December 1994, Journal of Virology, 68 (12) pages 8277-8281), Bertrand et al 2010 (Journal of Virology, 2010, vol 84 (1) p. 109–118), Hiroaki and Hideaki (US20210386807A1, published 16 December 2021, with an earlier priority filed application JP2018-203553 of 30 October 2018), Pearson and Bertrand 2014 (US8771698B2, 07/08/2014), and Carmichael et al 2018 (PLoS Pathog 14(5): e1007054).
Both the instant claims 2, 6, 9-10, 13, and 15-17 and co-pending claims 1, 9, 11, 13, 17, 26, 29, 32-33 and 39 of copending application No. 17/776, 828 are drawn to a tetracycline inducible oncolytic HSV comprising recombinant DNA comprising regulatory elements for transcription (promoters) and replication, a gene sequence encoding tetracycline repressor operably linked to an HSV immediate-early promoter, wherein the gene sequence is located at the ICP0 locus, a variant gene that increases syncytium formation as compared to wild type HSV-1 or HSV-2, gK, a UL24, does not encode functional ICP0 or and ICP34.5 gene, tetracycline operator sequences comprises two Op2 repressor binding sites, the HSV immediate-early promoter is selected from the group consisting of: ICP0 promoter and ICP4 promoter, the recombinant DNA is part of the HSV-1 genome or HSV-2 genome, polypeptode encoded e.g. IL2, IL12, IL15, an anti-PD1 antibody, a composition, a pharmaceutical composition, and a method of treating a cancer.
The differences between instant claims in the examined application compared to a claim in the reference patent or copending application is that the instant claims are drawn to an oncolytic HSV comprising at least two variant genes as compared to wild type, wherein the HSV-1, or HSV-2, at least two variant genes are glycoprotein K (gK) variant and UL24 gene variant, wherein said oncolytic HSV does not encode functional ICP0 and functional ICP34.5 protein. The copending application claims are drawn to an oncolytic HSV comprising a variant gene selected from gK, gB, a UL24 variant and UL20 gene variant. In claim 1, the HSV comprises a gene sequence encoding a functional ICP34.5 protein.
The combined teachings as applied for 35 USC 103 obviousness rejection of claims, as recited supra, are incorporated here by reference in entirety including obviousness analysis, motivation and reasonable expectation to arrive at the instant claims 2, 6, 9-10, 13, and 15-17. Therefore, the instant claims 2, 6, 9-10, 13, and 15-17 are obvious variants of co-pending claims 1, 9, 11, 13, 17, 26, 29, 32-33 and 39 of copending application No. 17/776, 828 in view of the applied prior art teachings.
This is a provisional nonstatutory double patenting rejection.
15. Claims 2-3, 6, 8-10, 13, 15, 17, and 23-25 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-7, 10-13, 15-16, 19-21-23, and 25-26 of copending Application No. 17/296, 879 in view of Yao 2008 (US20080008686A1, published 10 Jan 2008) and further in view of O'Shea and DeHart (WO2017189754A1, published 02 November 2017), Avitabile et al 2003 (published June 2003, Journal of Virology, 77: 6836-6844), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology, 444(2013)263–273), Han et al 2012 (published in PNAS, 109 (48) p 19798-19803), Muggeridge et al 2004 (published in Virology 328 (2004) 244 – 253) and Hillen and Berenes (published Ann Review Microbiology, 1994, 48), Kousoulas 2008 (WO2008141151A2, published 11/20/2008), Abdeljelil et al 2013 (Virology444(2013)263–273), Dolter et al (Published December 1994, Journal of Virology, 68 (12) pages 8277-8281), Bertrand et al 2010 (Journal of Virology, 2010, vol 84 (1) p. 109–118), Hiroaki and Hideaki (US20210386807A1, published 16 December 2021, with an earlier priority filed application JP2018-203553 of 30 October 2018), and Pearson and Bertrand 2014 (US8771698B2, 07/08/2014), and Carmichael et al 2018 (PLoS Pathog 14(5): e1007054).
Both the instant claims 2-3, 6, 8-10, 13, 15, 17, and 23-25 and co-pending claims claims 1-7, 10-13, 15-16, 19-21-23, and 25-26 of copending application No. 17/296, 879 are drawn to a tetracycline inducible oncolytic HSV comprising recombinant DNA comprising regulatory elements for transcription (promoters) and replication, a gene sequence encoding tetracycline repressor operably linked to an HSV immediate-early promoter, wherein the gene sequence is located at the ICP0 locus, a variant gene that increases syncytium formation as compared to wild type HSV-1 or HSV-2, gK, a UL24, gK variant gene amino acid substation (instant claim 3 verses copending claim 2) does not encode functional ICP0 or and ICP34.5 gene, tetracycline operator sequences comprises two Op2 repressor binding sites, the HSV immediate-early promoter is selected from the group consisting of: ICP0 promoter and ICP4 promoter, the recombinant DNA is part of the HSV-1 genome or HSV-2 genome, polypeptode encoded e.g. IL2, IL12, IL15, an anti-PD1 antibody, a composition, a pharmaceutical composition, and a method of treating a cancer.
The differences between instant claims in the examined application compared to a claim in the reference patent or copending application is that the instant claims are drawn to an oncolytic HSV comprising at least two variant genes as compared to wild type, wherein the HSV-1, or HSV-2, at least two variant genes are glycoprotein K (gK) variant and UL24 gene variant, wherein said oncolytic HSV does not encode functional ICP0 and functional ICP34.5 protein. The copending application claims are drawn to an oncolytic HSV comprising a variant gene selected from gK, gB, a UL24 variant and UL20 gene variant. In claim 1, the HSV comprises a gene sequence encoding a functional ICP34.5 protein.
The combined teachings as applied for 35 USC 103 obviousness rejection of claims, as recited supra, are incorporated here by reference in entirety including obviousness analysis, motivation and reasonable expectation to arrive at the instant claims 2-3, 6, 8-10, 13, 15, 17, and 23-25. Therefore, the instant claims 2-3, 6, 8-10, 13, 15, 17, and 23-25 are obvious variants of co-pending application No. 17296879 claims 1-7, 10-13, 15-16, 19-21-23, and 25-26 in view of the applied prior art teachings.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
16. Applicant’s arguments/remarks filed on 08/13/2025 with respect to Final rejection office action have been fully considered and the response is given below.
17. Rejection of claims 17, and 23-25 is withdrawn in view of applicant’s arguments and applicant’s amendment of the claims and in vitro experimental data of the claimed oncolytic HSV infection effects on human breast cancer cell line MDA-MB 231 and MCF7.
18. Applicant’s arguments with respect to claims 2, 3, 4, 6-10, 13, 15-17, 23-25 have been considered but are moot because the new ground of rejection based on amendment of independent claim 2 reciting a claim limitation at least two variant genes gK and variant UL24 does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
19. No claim is allowed.
20. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Nakashima and Chiocca (US20180133269A1, published 17 May 2018). Oncolytic HSV 1 vector and methods of use. Discloses oncolytic HSV-1, inter alia, with a deletion or inactivating mutation in both copies of the gene encoding ICP34.5 gene.
Bertrand L, Pearson A. The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin. J Gen Virol. 2008;89(Pt 5):1142-1151.
Okubo Y et al 2016. Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus. J Virol. 2016 Nov 28;90(24):11096-11105. Okubo et al teaches that membrane fusion is the key process required for both initial entry of t