Emerged HA and NA Mutants of the Pandemic Influenza H1N1 Viruses with Increasing Epidemiological Significance in Taipei and Kaohsiung, Taiwan, 2009–10

The 2009 influenza pandemic provided an opportunity to observe dynamic changes of the hemagglutinin (HA) and neuraminidase (NA) of pH1N1 strains that spread in two metropolitan areas -Taipei and Kaohsiung. We observed cumulative increases of amino acid substitutions of both HA and NA that were higher in the post–peak than in the pre-peak period of the epidemic. About 14.94% and 3.44% of 174 isolates had one and two amino acids changes, respective, in the four antigenic sites. One unique adaptive mutation of HA2 (E374K) was first detected three weeks before the epidemic peak. This mutation evolved through the epidemic, and finally emerged as the major circulated strain, with significantly higher frequency in the post-peak period than in the pre-peak (64.65% vs 9.28%, p<0.0001). E374K persisted until ten months post-nationwide vaccination without further antigenic changes (e.g. prior to the highest selective pressure). In public health measures, the epidemic peaked at seven weeks after oseltamivir treatment was initiated. The emerging E374K mutants spread before the first peak of school class suspension, extended their survival in high-density population areas before vaccination, dominated in the second wave of class suspension, and were fixed as herd immunity developed. The tempo-spatial spreading of E374K mutants was more concentrated during the post–peak (p = 0.000004) in seven districts with higher spatial clusters (p<0.001). This is the first study examining viral changes during the naïve phase of a pandemic of influenza through integrated virological/serological/clinical surveillance, tempo-spatial analysis, and intervention policies. The vaccination increased the percentage of E374K mutants (22.86% vs 72.34%, p<0.001) and significantly elevated the frequency of mutations in Sa antigenic site (2.36% vs 23.40%, p<0.001). Future pre-vaccination public health efforts should monitor amino acids of HA and NA of pandemic influenza viruses isolated at exponential and peak phases in areas with high cluster cases

A Study of the Doctrine of Equivalence on Biosimilars Based on the Patent Infringement in the context of Generics –From U.S. Perspectives

BPCIA在2010年三月生效後，生物相似藥廠商開始可以利用簡化的文件向美國食品和藥物管理局（FDA）申請藥品許可證，這個新醫療法賦予FDA決定如何落實法案的權力。基於不同生物製劑之間缺乏比較性這個已知的事實，加上公眾安全的考量，在還沒有累積大量經驗可以歸納出哪些是比較分析必要的資訊之前，FDA會保守的要求生物相似藥廠商以BPCIA提出申請時，必須提供臨床試驗資料來證明與參照藥品之間沒有臨床上有意義的差異。 雖然BPCIA給出了解決專利糾紛的框架，俗稱專利舞蹈(patent dance)，依照目前聯邦巡迴上訴法院對BPCIA的解釋，認為BPCIA法案不強制生物相似藥申請者遵循其規定之專利糾紛解決程序，雖然就目前的最新發展來看，迴避專利舞蹈可以避免一些程序上的麻煩，但真正參照藥品廠商和生物相似藥公司的輸贏仍是在訴訟戰場上見真章。 美國FDA在2015年3月6日核准了的一個生物相似藥-Zarxio( filgrastim-sndz)，目前尚不清楚均等論這種不確定性在生物相似藥上影響的程度，但藉由簡化新藥申請上市的小分子藥物所涉入的侵權訴訟做有限度的推論可以發現，小分子藥物的均等謬論案件是牽涉到外圍專利，當專利不再提供足夠的誘因去激勵專利權人時，學名藥廠商就會贏得均等論謬論案件。因為生物製劑是一種製程決定的產物，因此其專利通常是集中在製程。以BPCIA和專利法為框架來分析過去的相關侵權訴訟，可以預測生物相似藥廠商在轉化前步驟、轉化步驟、調劑、或包裝做改變，其成功的機會較大，而在細胞培養會純化步驟做改變，成功的機會最小。然而，最終還是要看法院將來如何解決生物相似藥的侵權問題，各方都要意識到科學與法律議題的複雜性，及妥適解決侵權訴訟的重要性。 台灣廠商要進入生物相似藥的領域，是困難重重的。生物相似藥的開發及法規成本，不如想像中低，鑒於蛋白質藥『產能』一直被看作是市場發展受阻的主要原因，藥廠委外合作(CRO、CMO或NRDO) 的模式能快速與國際藥廠接軌，逐步奠定台灣在藥物開發的供應鏈合作利基並提昇國際知名度。The Biologics Price Competition and Innovation of 2009 was activated on March in 2010. Now the US Food and Drug Administration (FDA) can approve biosimilars and was empowered to how to practice. Given the known issues with lack of comparability between different biologics preparations, and the Agency’s strong interest in protecting public safety, it is probable that, until it has developed a body of experience with regards to the amount and kind of data needed to make comparability evaluations, the FDA will adopt a conservative approach and require at least some clinical studies before approving biologics under BPCIA. Though BPCIA provide the frame for resolving patent issues, that is so-called patent dance, Federal Circuit said that parties were not compelled to dance. Thus the law uncertainty was shifted to patent infringement. FDA approved the first biosimilar, Zarxio (filgrastim-sndz), on 6, March, 2015. It is unclear how biosimilar will be treated in court based on doctrine of equivalence. Based on the experience from generics, courts tends to adjust the scope of equivalents to improve the correspondence between patent scope and desired patent incentives. In contrast, biologics is path depended. That is to say process decided what biologics would be. Both the BPCIA and patent law guide the shape of infringement suits. Follow-on biologics companies will be most successful when they make a change in the pretransformation process, the transformation process, the formulation, or the packaging. They will be least successful when they make a change in the cell culture conditions or the purification process. It remains to be seen how courts will address issues of infringement for follow-on biologics, but all parties should be aware of the complexity of the scientific and legal issues and the importance of addressing them properly. The cost for development and the complexity of regulation in biosimilars were tremendously high. Thus it is difficult for biopharmaceutical industries in Taiwan to enter this field. In the light of unmet production capacity in protein drug, pharmaceutical industries in Taiwan could apply the mode of CRO, CMO or NRDO to integrate into global biopharmaceutical community

Toward a pan-SARS-CoV-2 vaccine targeting conserved epitopes on spike and non-spike proteins for potent, broad and durable immune responses.

BackgroundThe SARS-CoV-2 non-Spike (S) structural protein targets on nucleocapsid (N), membrane (M) and envelope (E), critical in the host cell interferon response and memory T-cell immunity, are grossly overlooked in COVID vaccine development. The current Spike-only vaccines bear an intrinsic shortfall for promotion of a fuller T cell immunity. Vaccines designed to target conserved epitopes could elicit strong cellular immune responses that would synergize with B cell responses and lead to long-term vaccine success. We pursue a universal (pan-SARS-CoV-2) vaccine against Delta, Omicrons and ever-emergent new mutants.Methods and findingsWe explored booster immunogenicity of UB-612, a multitope-vaccine that contains S1-RBD-sFc protein and sequence-conserved promiscuous Th and CTL epitope peptides on the Sarbecovirus N, M and S2 proteins. To a subpopulation (N = 1,478) of infection-free participants (aged 18-85 years) involved in a two-dose Phase-2 trial, a UB-612 booster (third dose) was administered 6-8 months after the second dose. The immunogenicity was evaluated at 14 days post-booster with overall safety monitored until the end of study. The booster induced high viral-neutralizing antibodies against live Wuhan WT (VNT50, 1,711) and Delta (VNT50, 1,282); and against pseudovirus WT (pVNT50, 11,167) vs. Omicron BA.1/BA.2/BA.5 variants (pVNT50, 2,314/1,890/854), respectively. The lower primary neutralizing antibodies in the elderly were uplifted upon boosting to approximately the same high level in young adults. UB-612 also induced potent, durable Th1-oriented (IFN-γ+-) responses (peak/pre-boost/post-boost SFU/106 PBMCs, 374/261/444) along with robust presence of cytotoxic CD8+ T cells (peak/pre-boost/post-boost CD107a+-Granzyme B+, 3.6%/1.8%/1.8%). This UB-612 booster vaccination is safe and well tolerated without SAEs.ConclusionsBy targeting conserved epitopes on viral S2, M and N proteins, UB-612 could provide potent, broad and long-lasting B-cell and T-cell memory immunity and offers the potential as a universal vaccine to fend off Omicrons and new VoCs without resorting to Omicron-specific immunogens.Trial registrationClinicalTrials.gov ID: NCT04773067; ClinicalTrials.gov ID: NCT05293665; ClinicalTrials.gov ID: NCT05541861

Locations of changed amino acid residues of HA1 and HA2 of pH1N1 virus in Taiwan.

<p><b>A</b>. Globular head of HA1. Site Ca (Red); Site Cb Orange); Site Sa (Green); Site Sb (Blue). <b>B</b>. Stalk region of HA2. <b>C</b>. Early isolate with E374E residue. <b>D.</b> Late isolate with E374K. All the figures were generated and rendered with the use of MacPyMOL (<a href="http://wwwpymolorg" target="_blank">http://wwwpymolorg</a>). Numbers of amino acids represents the order of the amino acid taking out signal peptide (17 amino acids) and then counting the numbers from initial codon.</p

Weekly distributions of positive rates of pH1N1 isolated from NTUH and YGH in Taiwan, 2009–2010.

<p>NTUH: National Taiwan University Hospital located in Taipei; YGH: Yuan's General Hospital (YGH) located in Kaohsiung. The time frames of different public health prevention/control measures was indicated with arrows.</p

Global spatial autocorrelation analysis of E374K in Taipei metropolitan area, 2009.

<p>Global spatial autocorrelation analysis of E374K in Taipei metropolitan area, 2009.</p

Univariate analysis of the factors associated with the frequency of the pH1N1 HA E374K mutants isolated in Taiwan, 2009–2010.

<p>*: Based on the positive rate detected by RT-PCR or viral culture.</p

Odds ratios for the pH1N1-HA E374K mutants isolated in Taipei City by multivariate logistic regression analysis.

<p>Abbreviation : CI, confidence interval; OR: Odds ratio.</p><p>*p<0.05.</p

Comparison of the percentages of the pH1N1-E374K mutants in HA2 and other variants at the four antigenic sites and their relationships with different intervention strategies in various epidemic periods, 2009–2010.

1<p>. The effect of intervention of antiviral agent on the percentages of E374K mutants: Before (0%, 0/17) vs after (40.78%, 73/179), p<0.001 (Fisher's exact test).</p>2<p>. The effect of intervention of vaccination on the percentages of E374K mutants: Before (22.86%, 32/140) vs after (72.34%, 41/56), p<0.001 (Fisher's exact test).</p>3<p>. The effect of intervention of antiviral agent on the percentages of the pH1N1 variants with substitutions at four antigenic sites (Ca, Cb, Sa, Sb): Before (29.41%, 5/17) vs after (17.83%, 28/157), p = 0.324 (Fisher's exact test).</p>4<p>. The effect of intervention of vaccination on the percentages of the pH1N1 variants with substitutions at four antigenic sites (Ca, Cb, Sa, Sb): Before (11.86%, 18/118) vs after (25%, 15/56), p = 0.097 (Fisher's exact test).</p><p>*: Two strains with double mutations in Ca and Sa sites.</p><p>**: Three strains with double mutations in Ca and Sa sites.</p

Serological comparison in the antibody responses reacted with the pH1N1 isolates with various amino acid substitutions at different antigenic sites by hemagglutination inhibition (HI) tests.

1<p>ID numbers of the tested human serum samples.</p>2<p>ATCC IRR FR188 and.</p>3<p>ATCC IRR FR-187 in the 2010–2011 WHO Influenza Regent kit for diagnosis of influenza virus from the WHO Collaborating Centers for Surveillance, Epidemiology and control of Influenza at the U.S. Centers for Disease Control and Prevention.</p>4<p>HI serotiters: The highest dilution of human serum samples showed complete inhibition of 4 HA units of pH1N1 viruses.</p