Attenuated live varicella virus vaccine(Merck)

RAHWAY, N.J.--( BUSINESS WIRE )--Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced financial results for the fourth quarter of 2024. “ We delivered strong growth in 2024, reflecting demand for our innovative portfolio, including for KEYTRUDA, which continues to benefit more patients with cancer globally, the successful launch of WINREVAIR and strong performance of our Animal Health business,” said Robert M. Davis, chairman and chief executive officer, Merck. " We’re continuing to progress our pipeline, advance key clinical programs and augment our pipeline through promising business development. Our business remains well positioned thanks to the dedication of our talented global team, and I am more confident than ever in our long-term growth potential.” Financial Summary $ in millions, except EPS amounts Fourth Quarter Year Ended 2024 2023 Change Change Ex-Exchange Dec. 31, 2024 Dec. 31, 2023 Change Change Ex-Exchange Sales $15,624 $14,630 7% 9% $64,168 $60,115 7% 10% GAAP net income (loss) 1 3,743 (1,226) N/M N/M 17,117 365 N/M N/M Non-GAAP net income that excludes certain items 1,2* 4,372 66 N/M N/M 19,444 3,837 N/M N/M GAAP EPS 1.48 (0.48) N/M N/M 6.74 0.14 N/M N/M Non-GAAP EPS that excludes certain items 2* 1.72 0.03 N/M N/M 7.65 1.51 N/M N/M *Refer to table on page 9. N/M - not meaningful Generally Accepted Accounting Principles (GAAP) earnings per share (EPS) assuming dilution was $1.48 for the fourth quarter and $6.74 for the full year of 2024. Non-GAAP EPS was $1.72 for the fourth quarter and $7.65 for the full year of 2024. GAAP and non-GAAP EPS in the fourth quarter of 2024 include a charge of $0.23 per share related to the execution of licensing agreements with LaNova Medicines Ltd. (LaNova) and Hansoh Pharma (Hansoh). GAAP loss per share and non-GAAP EPS in the fourth quarter of 2023 include a charge of $1.69 per share related to a collaboration with Daiichi Sankyo. GAAP and non-GAAP EPS for the full years of 2024 and 2023 include charges of $1.28 and $6.21 per share, respectively, related to certain collaborations, licensing agreements and asset acquisitions. Non-GAAP EPS excludes acquisition- and divestiture-related costs, costs related to restructuring programs, and income and losses from investments in equity securities. Non-GAAP EPS in the fourth quarter and full year of 2024 also exclude a benefit due to a reduction in reserves for unrecognized income tax benefits resulting from the expiration of the statute of limitations for assessments related to certain federal tax return years. Non-GAAP EPS for the full year of 2023 also excludes a charge related to settlements with certain plaintiffs in the Zetia antitrust litigation. Fourth-Quarter Sales Performance The following table reflects sales of the company’s top products and significant performance drivers. Fourth Quarter $ in millions 2024 2023 Change Change Ex-Exchange Commentary Total Sales $15,624 $14,630 7% 9% The negative impact of foreign exchange was primarily due to devaluation of Argentine peso, which was largely offset by inflation-related price increases, consistent with practice in that market. Pharmaceutical 14,042 13,141 7% 8% Increase driven by growth in oncology and cardiovascular, partially offset by declines in diabetes, vaccines, immunology and virology. KEYTRUDA 7,836 6,608 19% 21% Growth driven by continued strong global demand from metastatic indications, including increased uptake in bladder and endometrial cancers, as well as increased global uptake in earlier-stage indications, including triple-negative breast cancer and non-small cell lung cancer (NSCLC). The negative impact of foreign exchange was primarily due to devaluation of Argentine peso, which was largely offset by inflation-related price increases. GARDASIL/GARDASIL 9 1,550 1,871 -17% -18% Decline primarily due to lower demand in China, partially offset by higher demand in most international regions, particularly in Japan. PROQUAD, M-M-R II and VARIVAX 594 545 9% 9% Growth primarily due to higher pricing in the U.S. and higher tenders in certain international markets, partially offset by lower demand in the U.S. JANUVIA/JANUMET 487 787 -38% -36% Decline primarily due to lower pricing in the U.S., as well as ongoing generic competition in many international markets and supply constraints in China. BRIDION 449 429 5% 5% Growth primarily due to higher demand in the U.S., partially offset by generic competition in certain international markets, particularly in Japan and Europe. Lynparza* 365 315 16% 18% Growth primarily due to higher global demand. Lenvima* 255 226 13% 14% Growth primarily due to timing of shipments in certain international markets. PREVYMIS 215 175 23% 23% Growth primarily due to higher demand in most markets, particularly in the U.S. WINREVAIR 200 - - - Represents continued uptake since second-quarter launch in the U.S. VAXNEUVANCE 161 176 -9% -9% Decline primarily driven by lower demand in the U.S. due to competition, partially offset by continued uptake from launches in Europe and the Asia Pacific region. WELIREG 160 72 122% 123% Growth primarily driven by higher demand in the U.S., largely attributable to ongoing uptake of a new indication. SIMPONI - 171 N/M N/M Marketing rights in former Merck territories reverted to Johnson & Johnson on Oct. 1, 2024. Animal Health 1,397 1,278 9% 13% Growth primarily driven by higher pricing for both Livestock and Companion Animal product portfolios, as well as sales related to July 2024 acquisition of Elanco aqua business and higher demand for Livestock products. Approximately 3 percentage points of the negative impact of foreign exchange were due to devaluation of Argentine peso, which were largely offset by inflation-related price increases. Livestock 889 808 10% 14% Growth primarily driven by higher demand for poultry products, sales related to acquisition of Elanco aqua business, as well as higher pricing across the portfolio. Companion Animal 508 470 8% 10% Growth primarily driven by higher pricing across the product portfolio. Sales of BRAVECTO were $209 million and $197 million in current and prior year quarters, respectively, which represented growth of 6%, or 10% excluding impact of foreign exchange. Other Revenues** 185 211 -13% 3% Decline primarily due to impact of revenue-hedging activities and lower revenues from third-party manufacturing arrangements, partially offset by payments received for out-licensing arrangements and higher royalty income. *Alliance revenue for this product represents Merck’s share of profits, which are product sales net of cost of sales and commercialization costs. **Other revenues are comprised primarily of revenues from third-party manufacturing arrangements and miscellaneous corporate revenues, including revenue-hedging activities. N/M – not meaningful Full-Year Sales Performance The following table reflects sales of the company’s top products and significant performance drivers. Year Ended $ in millions Dec. 31, 2024 Dec. 31, 2023 Change Change Ex- Exchange Total Sales $64,168 $60,115 7% 10% Pharmaceutical 57,400 53,583 7% 10% KEYTRUDA 29,482 25,011 18% 22% GARDASIL/GARDASIL 9 8,583 8,886 -3% -2% PROQUAD, M-M-R II and VARIVAX 2,485 2,368 5% 5% JANUVIA/JANUMET 2,268 3,366 -33% -29% BRIDION 1,764 1,842 -4% -3% Lynparza* 1,311 1,199 9% 11% Lenvima* 1,010 960 5% 6% LAGEVRIO 964 1,428 -33% -28% VAXNEUVANCE 808 665 22% 23% PREVYMIS 785 605 30% 33% ROTATEQ 711 769 -8% -7% SIMPONI** 543 710 -24% -23% WELIREG 509 218 133% 133% WINREVAIR 419 - - - Animal Health 5,877 5,625 4% 8% Livestock 3,462 3,337 4% 9% Companion Animal 2,415 2,288 6% 7% Other Revenues*** 891 907 -2% 4% *Alliance revenue for this product represents Merck’s share of profits, which are product sales net of cost of sales and commercialization costs. **Marketing rights in former Merck territories reverted to Johnson & Johnson on Oct. 1, 2024. ***Other revenues are comprised primarily of revenues from third-party manufacturing arrangements and miscellaneous corporate revenues, including revenue-hedging activities. Full-year 2024 pharmaceutical sales grew 7% to $57.4 billion. Excluding the unfavorable impact of foreign exchange, pharmaceutical sales grew 10%. Approximately 2 percentage points of the negative impact of foreign exchange were due to devaluation of the Argentine peso, which were largely offset by inflation-related price increases, consistent with practice in that market. Pharmaceutical sales growth was primarily driven by higher sales in oncology, particularly KEYTRUDA and WELIREG, as well as increased alliance revenue from Reblozyl and Lynparza. Higher sales in the cardiovascular franchise, reflecting the successful launch of WINREVAIR, as well as higher sales of certain hospital acute care products, particularly PREVYMIS, also drove revenue growth in 2024. Pharmaceutical sales growth in 2024 was partially offset by lower sales of JANUVIA and JANUMET, primarily reflecting lower pricing in the U.S. and generic competition in many international markets, lower sales of the COVID-19 medication LAGEVRIO, lower sales of GARDASIL/GARDASIL 9 and lower sales of SIMPONI and REMICADE, reflecting the transfer of marketing rights in former Merck territories back to Johnson & Johnson. Full-year 2024 Animal Health sales grew 4% to $5.9 billion. Excluding the unfavorable impact of foreign exchange, Animal Health sales grew 8%. Approximately 2 percentage points of the negative impact of foreign exchange were due to devaluation of the Argentine peso, which were largely offset by inflation-related price increases, consistent with practice in that market. Full-year sales growth was primarily driven by higher pricing across both the Companion Animal and Livestock product portfolios, and higher demand for poultry and swine products, as well as sales related to the acquisition of the Elanco aqua business. Sales of BRAVECTO were $1.1 billion in 2024, which represented growth of 6%, or 8% excluding the impact of foreign exchange. Fourth-Quarter and Full-Year Expense, EPS and Related Information The table below presents selected expense information. $ in millions GAAP Acquisition- and Divestiture- Related Costs 3 Restructuring Costs (Income) Loss From Investments in Equity Securities Non- GAAP 2 Fourth Quarter 2024 Cost of sales $3,828 $701 $121 $- $3,006 Selling, general and administrative 2,864 29 16 - 2,819 Research and development 4,585 12 (1) - 4,574 Restructuring costs 51 - 51 - - Other (income) expense, net 126 (31) - 152 5 Fourth Quarter 2023 Cost of sales $3,911 $454 $117 $- $3,340 Selling, general and administrative 2,804 24 29 - 2,751 Research and development 9,628 790 - - 8,838 Restructuring costs 255 - 255 - - Other (income) expense, net 78 (35) - (61) 174 $ in millions GAAP Acquisition- and Divestiture- Related Costs 3 Restructuring Costs (Income) Loss From Investments in Equity Securities Certain Other Items Non- GAAP 2 Year Ended December 31, 2024 Cost of sales $15,193 $2,409 $495 $- $ - $12,289 Selling, general and administrative 10,816 117 83 - - 10,616 Research and development 17,938 72 1 - - 17,865 Restructuring costs 309 - 309 - - - Other (income) expense, net (24) (79) - 45 - 10 Year Ended December 31, 2023 Cost of sales $16,126 $2,018 $211 $- $- $13,897 Selling, general and administrative 10,504 86 122 - - 10,296 Research and development 30,531 819 1 - - 29,711 Restructuring costs 599 - 599 - - - Other (income) expense, net 466 (47) - (279) 573 219 GAAP Expense, EPS and Related Information Gross margin was 75.5% for the fourth quarter of 2024 compared with 73.3% for the fourth quarter of 2023. The increase was primarily due to the favorable effects of product mix (including lower royalty rates related to KEYTRUDA and GARDASIL/GARDASIL 9) and foreign exchange, partially offset by higher manufacturing-related costs (including inventory write-offs) and higher amortization of intangible assets. Gross margin was 76.3% for the full year of 2024 compared with 73.2% for the full year of 2023. The increase was primarily due to the favorable effects of product mix (including lower royalty rates related to KEYTRUDA and GARDASIL/GARDASIL 9) and foreign exchange, partially offset by higher amortization of intangible assets, as well as higher restructuring costs (primarily reflecting asset impairment charges) and higher manufacturing-related costs (including inventory write-offs). Selling, general and administrative (SG&A) expenses were $2.9 billion in the fourth quarter of 2024, an increase of 2% compared with the fourth quarter of 2023. The increase was primarily due to higher promotional and selling costs, partially offset by the favorable impact of foreign exchange and lower restructuring costs. Full-year 2024 SG&A expenses were $10.8 billion, an increase of 3% compared with full-year 2023. The increase was primarily due to higher administrative, promotional, selling and acquisition-related costs, partially offset by the favorable impact of foreign exchange and lower restructuring costs. Research and development (R&D) expenses were $4.6 billion in the fourth quarter of 2024, a decrease of 52% compared with the fourth quarter of 2023. R&D expenses were $17.9 billion for the full year of 2024, a decrease of 41% compared with the full year of 2023. The declines in both the fourth quarter and full year of 2024 were primarily due to lower charges for business development activity, lower intangible asset impairment charges, and the favorable impact of foreign exchange, partially offset by increased compensation and benefit costs and higher clinical development spending. Other (income) expense, net, was $126 million of expense in the fourth quarter of 2024 compared with $78 million of expense in the fourth quarter of 2023. The unfavorability was primarily due to net losses from investments in equity securities compared with net income from investments in equity securities in the prior year quarter, partially offset by lower foreign exchange losses and lower net interest expense. Other (income) expense, net, was $24 million of income in the full year of 2024 compared with $466 million of expense in the full year of 2023, primarily due to a $572.5 million charge in 2023 related to settlements with certain plaintiffs in the Zetia antitrust litigation. The favorability was also due to $170 million of income related to the expansion of an existing development and commercialization agreement with Daiichi Sankyo, as well as lower foreign exchange losses in 2024. Other (income) expense, net, in the full year of 2024 was unfavorably affected by lower net income from investments in equity securities and higher net interest expense compared with 2023. The effective tax rates of 10.2% and 14.1% for the fourth quarter and full year of 2024, respectively, include a 6.2 percentage point favorable impact and a 2.6 percentage point favorable impact, respectively, due to a reduction in reserves for unrecognized income tax benefits, resulting from the expiration of the statute of limitations for assessments related to certain federal tax return years. GAAP EPS was $1.48 for the fourth quarter of 2024 compared with a loss per share of $0.48 for the fourth quarter of 2023, primarily driven by lower charges for business development transactions, operational strength in the business, lower intangible asset impairment charges, and a benefit from the expiration of the statute of limitations for assessments related to the 2020 federal tax return year. GAAP EPS was $6.74 for the full year of 2024 compared with EPS of $0.14 for the full year of 2023. The increase was primarily driven by lower charges for business development transactions, operational strength in the business, lower intangible asset impairment charges, a benefit from the expiration of the statute of limitations for the 2020 and 2019 federal tax return years, and a charge in the prior year for settlements with certain plaintiffs in the Zetia antitrust litigation, partially offset by the unfavorable effect of foreign exchange. Non-GAAP Expense, EPS and Related Information Non-GAAP gross margin was 80.8% for the fourth quarter of 2024 compared with 77.2% for the fourth quarter of 2023. Non-GAAP gross margin was 80.8% for the full year of 2024 compared with 76.9% for the full year of 2023. The non-GAAP gross margin improvements in both the fourth quarter and full year of 2024 were primarily due to the favorable effects of product mix (including lower royalty rates related to KEYTRUDA and GARDASIL/GARDASIL 9) and foreign exchange, partially offset by higher manufacturing-related costs (including inventory write-offs). Non-GAAP SG&A expenses were $2.8 billion in the fourth quarter of 2024, an increase of 2% compared with the fourth quarter of 2023. Non-GAAP SG&A expenses were $10.6 billion for the full year of 2024, an increase of 3% compared with the full year of 2023. The increases were primarily due to higher promotional and selling costs and, for the full year, higher administrative costs, partially offset by the favorable impact of foreign exchange. Non-GAAP R&D expenses were $4.6 billion in the fourth quarter of 2024, a decrease of 48% compared with the fourth quarter of 2023. Non-GAAP R&D expenses were $17.9 billion for the full year of 2024, a decrease of 40% compared with the full year of 2023. The declines in both the fourth quarter and full year of 2024 were primarily due to lower charges for business development activity and the favorable impact of foreign exchange, partially offset by increased compensation and benefit costs and higher clinical development spending. Non-GAAP other (income) expense, net, was $5 million of expense in the fourth quarter of 2024 compared with $174 million of expense in the fourth quarter of 2023. The favorability was primarily due to lower foreign exchange losses and lower net interest expense. Non-GAAP other (income) expense, net, was $10 million of expense in the full year of 2024 compared with $219 million of expense in the full year of 2023. The favorability was primarily due to $170 million of income related to the expansion of an existing development and commercialization agreement with Daiichi Sankyo, as well as lower foreign exchange losses in 2024, partially offset by higher net interest expense. The non-GAAP effective tax rate was 16.2% for the fourth quarter and 16.8% for the full year of 2024. Non-GAAP EPS was $1.72 for the fourth quarter of 2024 compared with $0.03 for the fourth quarter of 2023. Non-GAAP EPS was $7.65 for the full year of 2024 compared with EPS of $1.51 for the full year of 2023. The increase in both periods was primarily driven by lower charges for business development transactions and operational strength in the business. The unfavorable effect of foreign exchange partially offset the increase in the full year. A reconciliation of GAAP to non-GAAP net income (loss) and earnings (loss) per share is provided in the table that follows. Fourth Quarter Year Ended $ in millions, except EPS amounts 2024 2023 Dec. 31, 2024 Dec. 31, 2023 EPS GAAP EPS $1.48 $(0.48) $6.74 $0.14 Difference 0.24 0.51 0.91 1.37 Non-GAAP EPS that excludes items listed below 2 $1.72 $0.03 $7.65 $1.51 Net Income (Loss) GAAP net income (loss) 1 $3,743 $(1,226) $17,117 $365 Difference 629 1,292 2,327 3,472 Non-GAAP net income that excludes items listed below 1,2 $4,372 $66 $19,444 $3,837 Excluded Items: Acquisition- and divestiture-related costs 3 $711 $1,233 $2,519 $2,876 Restructuring costs 187 401 888 933 Loss (income) from investments in equity securities 152 (61) 45 (279) Charge for Zetia antitrust litigation settlements - - - 573 Decrease to net income/increase to net loss before taxes 1,050 1,573 3,452 4,103 Estimated income tax (benefit) expense 4 (421) (281) (1,125) (631) Decrease to net income/increase to net loss $629 $1,292 $2,327 $3,472 Pipeline and Portfolio Highlights Merck made important advancements in its broad, diverse pipeline, meeting significant regulatory and clinical milestones throughout the fourth quarter. In oncology, Merck announced positive topline results from the pivotal Phase 3 MK-3475A-D77 trial evaluating the noninferiority of subcutaneous pembrolizumab and berahyaluronidase alfa, in combination with chemotherapy, versus intravenous (IV) KEYTRUDA administered with chemotherapy, for the first-line treatment of adult patients with metastatic NSCLC. Subcutaneous pembrolizumab and berahyaluronidase alfa has the potential to improve the patient experience and increase access for patients and health care providers compared to IV administration. Merck presented new data across multiple hematologic malignancies at the American Society of Hematology Annual Meeting and Exposition in December 2024, including promising Phase 2 data for its investigational antibody-drug conjugate zilovertamab vedotin for the treatment of patients with previously untreated diffuse large B-cell lymphoma. With more than 20 abstracts presented, the data showcased Merck’s continued progress in advancing clinical research for its expanding and diverse hematology pipeline. Merck also achieved several key regulatory milestones in the U.S., Europe, Japan and China. Highlights include the U.S. Food and Drug Administration (FDA) granting Breakthrough Therapy designation to sacituzumab tirumotecan (sac-TMT) for the treatment of certain patients with previously treated advanced or metastatic nonsquamous NSCLC with epidermal growth factor receptor (EGFR) mutations. Additionally, Merck received new approvals for KEYTRUDA-based regimens in Japan and China, as well as for WELIREG and Lynparza in China. In vaccines and infectious diseases, the FDA accepted the Biologics License Application (BLA) for clesrovimab, an investigational prophylactic long-acting monoclonal antibody designed to protect infants from respiratory syncytial virus (RSV) disease during their first RSV season and set a Prescription Drug User Fee Act (PDUFA) date of June 10, 2025. This regulatory milestone marks important progress toward having clesrovimab available in time for the 2025-26 RSV season. The filing was based on results from the pivotal Phase 2b/3 study of clesrovimab in infants for the prevention of RSV that was presented at ID Week 2024. In addition, Merck announced topline results from two pivotal Phase 3 trials of the investigational, once-daily, oral, two-drug, single-tablet regimen of doravirine/islatravir (DOR/ISL) in adults with virologically suppressed HIV-1 infection, in line with Merck’s commitment to help address the needs of people living with HIV. In January 2025, Merck also received expanded approval in China for GARDASIL. It is now the first HPV vaccine approved in China for the prevention of certain HPV-related cancers and diseases in males 9-26 years of age. In addition, the European Union’s (EU) Committee for Medicinal Products for Human Use (CHMP) recommended the approval of CAPVAXIVE for pneumococcal vaccination in adults, with a final decision for EU approval expected in the second quarter of 2025. In cardiovascular disease, Merck announced positive topline results from the Phase 3 ZENITH study, evaluating WINREVAIR in adults with pulmonary arterial hypertension (PAH) with World Health Organization (WHO) Group 1 functional class (FC) III or IV at high risk of mortality. Based on the positive results of an interim analysis, an independent data monitoring committee recommended that the study be stopped early due to overwhelming efficacy. In addition, in January 2025, Merck announced the Phase 3 HYPERION study evaluating WINREVAIR in newly diagnosed adults with PAH with FC II or III at intermediate or high risk of disease progression was also stopped early based on the positive results from the interim analysis of the ZENITH trial and a review of the totality of data from the WINREVAIR clinical program to date. All participants in both the ZENITH and HYPERION studies will be offered the opportunity to receive WINREVAIR as part of the open-label, long-term extension study, SOTERIA. Merck continued to execute on its business development strategy. The company announced the closing of an exclusive global license for MK-2010, a novel investigational PD-1/VEGF bispecific antibody from LaNova. Merck also entered into an exclusive global license agreement with Hansoh to evaluate MK-4082, an investigational preclinical oral small molecule glucagon-like peptide (GLP-1) receptor agonist. Notable recent news releases on Merck’s pipeline and portfolio are provided in the table that follows. Oncology FDA Granted Breakthrough Therapy Designation to sac-TMT for the Treatment of Certain Patients With Previously Treated Advanced or Metastatic Nonsquamous NSCLC With EGFR Mutations (Read Announcement) FDA Granted Priority Review to Merck’s Application for WELIREG for the Treatment of Patients With Advanced Pheochromocytoma and Paraganglioma (Read Announcement) Merck Received Positive EU CHMP Opinion for WELIREG as Treatment for Adult Patients With Certain Types of Von Hippel-Lindau (VHL) Disease-Associated Tumors and for Certain Previously Treated Adult Patients With Advanced RCC, Based on Results From Phase 2 LITESPARK-004 and Phase 3 LITESPARK-005 Trials (Read Announcement) Merck Received Positive EU CHMP Opinion for KEYTRUDA Plus Chemotherapy as First-Line Treatment for Adult Patients With Unresectable Non-Epithelioid Malignant Pleural Mesothelioma, Based on Results From Phase 2/3 IND.227/KEYNOTE-483 Trial (Read Announcement) KEYTRUDA Approved in China in Combination With Chemotherapy as Neoadjuvant Treatment, Then Continued as Monotherapy After Surgery as Adjuvant Treatment for Patients With Resectable Stage II, IIIA or IIIB NSCLC, Based on Results From Phase 3 KEYNOTE-671 Trial (Read Announcement) WELIREG Approved in China for Treatment of Adult Patients With Certain Types of VHL Disease-Associated Tumors, Based on Results From Phase 2 LITESPARK-004 Trial (Read Announcement) Merck Announced Phase 3 MK-3475A-D77 Trial of Subcutaneous Pembrolizumab With Berahyaluronidase Alfa Met Primary Endpoints (Read Announcement) Merck Announced Phase 3 KEYLYNK-001 Trial Met Primary Endpoint of Progression-Free Survival in Patients With Advanced Epithelial Ovarian Cancer (Read Announcement) Lynparza Demonstrated Clinically Meaningful Prolonged Survival Benefit in Early Breast Cancer, Based on Results From Phase 3 OlympiA Trial (Read Announcement) Investigational Zilovertamab Vedotin in Combination With R-CHP Demonstrated Complete Response Rate of 100% at 1.75 MG/KG Dose in Previously Untreated Patients With Diffuse Large B-Cell Lymphoma, Based on Results From Phase 2 WaveLINE-007 Trial (Read Announcement) Vaccines Merck Announced FDA Acceptance of BLA for Clesrovimab (MK-1654), an Investigational Long-Acting Monoclonal Antibody Designed to Protect Infants From RSV Disease During Their First RSV Season; FDA Set PDUFA Date of June 10, 2025 (Read Announcement) Merck Received Expanded Approval of GARDASIL for Males in China (Read Announcement) Merck Received Positive EU CHMP Opinion for CAPVAXIVE for Pneumococcal Vaccination in Adults (Read Announcement) Merck Presented New Data From GARDASIL 9 Studies Reinforcing Importance of Gender-Neutral HPV Vaccination in Adults Up to Age 45 at International Papillomavirus Conference 2024 (Read Announcement) Cardiovascular Merck Announced Pivotal Phase 3 ZENITH Trial Evaluating WINREVAIR Met Primary Endpoint at Interim Analysis (Read Announcement) Merck Announced Decision to Stop Phase 3 HYPERION Trial Evaluating WINREVAIR Early and Move to Final Analysis (Read Announcement) Infectious Diseases Merck Announced Topline Results From Pivotal Phase 3 Trials Evaluating Investigational, Once-Daily, Oral, Two-Drug, Single-Tablet Regimen of Doravirine/Islatravir (DOR/ISL) for the Treatment of Adults With Virologically Suppressed HIV-1 Infection (Read Announcement) Full-Year 2025 Financial Outlook The following table summarizes the company’s full-year financial outlook. Full Year 2025 Sales * $64.1 billion to $65.6 billion Non-GAAP Gross margin 2 Approximately 82.5% Non-GAAP Operating expenses 2** $25.4 billion to $26.4 billion Non-GAAP Other (income) expense, net 2 $300 million to $400 million expense Non-GAAP Effective tax rate 2 16.0% to 17.0% Non-GAAP EPS 2*** $8.88 to $9.03 Share count (assuming dilution) Approximately 2.53 billion *The company does not have any non-GAAP adjustments to sales. **Includes $300 million for an anticipated milestone payment to LaNova associated with the technology transfer for MK-2010 expected to be completed in 2025. Outlook does not assume any additional significant potential business development transactions. ***Includes expected one-time charge of approximately $0.09 per share related to the $300 million milestone payment to LaNova upon completion of the technology transfer for MK-2010. Merck has not provided a reconciliation of forward-looking non-GAAP gross margin, non-GAAP operating expenses, non-GAAP other (income) expense, net, non-GAAP effective tax rate and non-GAAP EPS to the most directly comparable GAAP measures, given it cannot predict with reasonable certainty the amounts necessary for such a reconciliation, including intangible asset impairment charges, legal settlements, and income and losses from investments in equity securities either owned directly or through ownership interests in investment funds, without unreasonable effort. These items are inherently difficult to forecast and could have a significant impact on the company’s future GAAP results. Merck anticipates full-year 2025 sales to be between $64.1 billion and $65.6 billion, including a negative impact of foreign exchange of approximately 2% at mid-January 2025 exchange rates. This sales range reflects a decision to temporarily pause shipments of GARDASIL/GARDASIL 9 into China beginning February 2025 through at least mid-year. Merck’s full-year non-GAAP effective income tax rate is expected to be between 16.0% and 17.0%. Merck expects full-year 2025 non-GAAP EPS to be between $8.88 and $9.03, including a negative impact of foreign exchange of approximately $0.35 per share. This range includes an expected one-time charge of $300 million, or approximately $0.09 per share, related to a milestone payment to LaNova, which will be recognized upon completion of the technology transfer for MK-2010. In 2024, non-GAAP EPS of $7.65 was negatively impacted by a net charge of $1.28 per share related to certain asset acquisitions, licensing agreements and collaborations. Consistent with past practice, the financial outlook does not assume additional significant potential business development transactions. Earnings Conference Call Investors, journalists and the general public may access a live audio webcast of the earnings conference call on Tuesday, Feb. 4, at 9 a.m. ET via this weblink . A replay of the webcast, along with the sales and earnings news release, supplemental financial disclosures, and slides highlighting the results, will be available at www.merck.com . All participants may join the call by dialing (800) 369-3351 (U.S. and Canada Toll-Free) or (517) 308-9448 and using the access code 9818590. About Merck At Merck, known as MSD outside of the United States and Canada, we are unified around our purpose: We use the power of leading-edge science to save and improve lives around the world. For more than 130 years, we have brought hope to humanity through the development of important medicines and vaccines. We aspire to be the premier research-intensive biopharmaceutical company in the world – and today, we are at the forefront of research to deliver innovative health solutions that advance the prevention and treatment of diseases in people and animals. We foster a diverse and inclusive global workforce and operate responsibly every day to enable a safe, sustainable and healthy future for all people and communities. For more information, visit www.merck.com and connect with us on X (formerly Twitter) , Facebook , Instagram , YouTube and LinkedIn . Forward-Looking Statement of Merck & Co., Inc., Rahway, N.J., USA This news release of Merck & Co., Inc., Rahway, N.J., USA (the “company”) includes “forward-looking statements” within the meaning of the safe harbor provisions of the U.S. Private Securities Litigation Reform Act of 1995. These statements are based upon the current beliefs and expectations of the company’s management and are subject to significant risks and uncertainties. There can be no guarantees with respect to pipeline candidates that the candidates will receive the necessary regulatory approvals or that they will prove to be commercially successful. If underlying assumptions prove inaccurate or risks or uncertainties materialize, actual results may differ materially from those set forth in the forward-looking statements. Risks and uncertainties include but are not limited to, general industry conditions and competition; general economic factors, including interest rate and currency exchange rate fluctuations; the impact of pharmaceutical industry regulation and health care legislation in the United States and internationally; global trends toward health care cost containment; technological advances, new products and patents attained by competitors; challenges inherent in new product development, including obtaining regulatory approval; the company’s ability to accurately predict future market conditions; manufacturing difficulties or delays; financial instability of international economies and sovereign risk; dependence on the effectiveness of the company’s patents and other protections for innovative products; and the exposure to litigation, including patent litigation, and/or regulatory actions. The company undertakes no obligation to publicly update any forward-looking statement, whether as a result of new information, future events or otherwise. Additional factors that could cause results to differ materially from those described in the forward-looking statements can be found in the company’s Annual Report on Form 10-K for the year ended December 31, 2023 and the company’s other filings with the Securities and Exchange Commission (SEC) available at the SEC’s Internet site ( www.sec.gov ). Appendix Generic product names are provided below. Pharmaceutical BRIDION (sugammadex) CAPVAXIVE (Pneumococcal 21-valent Conjugate Vaccine) GARDASIL ( Human Papillomavirus Quadrivalent [Types 6, 11, 16 and 18] Vaccine, Recombinant ) GARDASIL 9 (Human Papillomavirus 9-valent Vaccine, Recombinant) JANUMET (sitagliptin and metformin HCl) JANUVIA (sitagliptin) KEYTRUDA (pembrolizumab) LAGEVRIO (molnupiravir) Lenvima (lenvatinib) Lynparza (olaparib) M-M-R II (Measles, Mumps and Rubella Virus Vaccine Live) PREVYMIS ( letermovir ) PROQUAD (Measles, Mumps, Rubella and Varicella Virus Vaccine Live) Reblozyl (luspatercept) REMICADE (infliximab) ROTATEQ (Rotavirus Vaccine, Live, Oral, Pentavalent) SIMPONI (golimumab) VARIVAX (Varicella Virus Vaccine Live) VAXNEUVANCE ( Pneumococcal 15-valent Conjugate Vaccine ) WELIREG ( belzutifan ) WINREVAIR (sotatercept-csrk) Animal Health BRAVECTO (fluralaner) 1 Net income (loss) attributable to Merck & Co., Inc. 2 Merck is providing certain 2024 and 2023 non-GAAP information that excludes certain items because of the nature of these items and the impact they have on the analysis of underlying business performance and trends. Management believes that providing this information enhances investors’ understanding of the company’s results because management uses non-GAAP results to assess performance. Management uses non-GAAP measures internally for planning and forecasting purposes and to measure the performance of the company along with other metrics. In addition, annual employee compensation, including senior management’s compensation, is derived in part using a non-GAAP pretax income metric. This information should be considered in addition to, but not as a substitute for or superior to, information prepared in accordance with GAAP. For a description of the non-GAAP adjustments, see Table 2a attached to this release. 3 Reflects expenses related to business combinations, including the amortization of intangible assets, intangible asset impairment charges, and expense or income related to changes in the estimated fair value measurement of liabilities for contingent consideration. Also includes integration, transaction and certain other costs associated with acquisitions and divestitures, as well as amortization of intangible assets related to collaborations and licensing arrangements. 4 Includes the estimated tax impacts on the reconciling items based on applying the statutory rate of the originating territory of the non-GAAP adjustments, as well as a $260 million benefit and a $519 million benefit in the fourth quarter and full year of 2024, respectively, due to reductions in reserves for unrecognized income tax benefits resulting from the expiration of the statute of limitations for assessments related to certain federal tax return years. The benefit recognized in the fourth quarter of 2024 relates to the 2020 federal tax return year and the benefit for the full year of 2024 relates to both the 2020 and 2019 federal tax return years. MERCK & CO., INC. CONSOLIDATED STATEMENT OF OPERATIONS - GAAP (AMOUNTS IN MILLIONS, EXCEPT PER SHARE FIGURES) (UNAUDITED) Table 1 GAAP % Change GAAP % Change 4Q24 4Q23 Full Year 2024 Full Year 2023 Sales $ 15,624 $ 14,630 7% $ 64,168 $ 60,115 7% Costs, Expenses and Other Cost of sales 3,828 3,911 -2% 15,193 16,126 -6% Selling, general and administrative 2,864 2,804 2% 10,816 10,504 3% Research and development 4,585 9,628 -52% 17,938 30,531 -41% Restructuring costs 51 255 -80% 309 599 -48% Other (income) expense, net 126 78 62% (24 ) 466 * Income (Loss) Before Taxes 4,170 (2,046 ) * 19,936 1,889 * Income Tax Provision (Benefit) 425 (821 ) 2,803 1,512 Net Income (Loss) 3,745 (1,225 ) * 17,133 377 * Less: Net Income Attributable to Noncontrolling Interests 2 1 16 12 Net Income (Loss) Attributable to Merck & Co., Inc. $ 3,743 $ (1,226 ) * $ 17,117 $ 365 * Earnings (Loss) per Common Share Assuming Dilution (1) $ 1.48 $ (0.48 ) * $ 6.74 $ 0.14 * Average Shares Outstanding Assuming Dilution (1) 2,537 2,533 2,541 2,547 Tax Rate 10.2 % 40.1 % 14.1 % 80.0 % * 100% or greater (1) Because the company recorded a net loss in the fourth quarter of 2023, no potential dilutive common shares were used in the computation of loss per common share assuming dilution as the effect would have been anti-dilutive. MERCK & CO., INC. FOURTH QUARTER AND FULL YEAR 2024 GAAP TO NON-GAAP RECONCILIATION (AMOUNTS IN MILLIONS, EXCEPT PER SHARE FIGURES) (UNAUDITED) Table 2a GAAP Acquisition and Divestiture Related Costs (1) Restructuring Costs (2) (Income) Loss from Investments in Equity Securities Certain Other Items Adjustment Subtotal Non-GAAP Fourth Quarter Cost of sales $ 3,828 701 121 822 $ 3,006 Selling, general and administrative 2,864 29 16 45 2,819 Research and development 4,585 12 (1) 11 4,574 Restructuring costs 51 51 51 – Other (income) expense, net 126 (31) 152 121 5 Income Before Taxes 4,170 (711) (187) (152) (1,050) 5,220 Income Tax Provision (Benefit) 425 (111) (3) (17) (3) (33) (3) (260) (4) (421) 846 Net Income 3,745 (600) (170) (119) 260 (629) 4,374 Net Income Attributable to Merck & Co., Inc. 3,743 (600) (170) (119) 260 (629) 4,372 Earnings per Common Share Assuming Dilution $ 1.48 (0.23) (0.07) (0.04) 0.10 (0.24) $ 1.72 Tax Rate 10.2% 16.2% Full Year Cost of sales $ 15,193 2,409 495 2,904 $ 12,289 Selling, general and administrative 10,816 117 83 200 10,616 Research and development 17,938 72 1 73 17,865 Restructuring costs 309 309 309 – Other (income) expense, net (24) (79) 45 (34) 10 Income Before Taxes 19,936 (2,519) (888) (45) (3,452) 23,388 Income Tax Provision (Benefit) 2,803 (461) (3) (135) (3) (10) (3) (519) (4) (1,125) 3,928 Net Income 17,133 (2,058) (753) (35) 519 (2,327) 19,460 Net Income Attributable to Merck & Co., Inc. 17,117 (2,058) (753) (35) 519 (2,327) 19,444 Earnings per Common Share Assuming Dilution $ 6.74 (0.81) (0.30) (0.01) 0.21 (0.91) $ 7.65 Tax Rate 14.1% 16.8% Only the line items that are affected by non-GAAP adjustments are shown. Merck is providing certain non-GAAP information that excludes certain items because of the nature of these items and the impact they have on the analysis of underlying business performance and trends. Management believes that providing non-GAAP information enhances investors’ understanding of the company’s results because management uses non-GAAP measures to assess performance. Management uses non-GAAP measures internally for planning and forecasting purposes and to measure the performance of the company along with other metrics. In addition, annual employee compensation, including senior management’s compensation, is derived in part using a non-GAAP pretax income metric. The non-GAAP information presented should be considered in addition to, but not as a substitute for or superior to, information prepared in accordance with GAAP. (1) Amounts included in cost of sales primarily reflect expenses for the amortization of intangible assets. Amounts included in selling, general and administrative expenses reflect integration, transaction and certain other costs related to acquisitions and divestitures. Amounts included in research and development expenses primarily reflect the amortization of intangible assets. Additionally, research and development expenses for the full year includes Animal Health intangible asset impairment charges. Amounts included in other (income) expense, net, primarily reflect royalty income and a decrease in the estimated fair value measurement of liabilities for contingent consideration related to the prior termination of the Sanofi-Pasteur MSD joint venture. (2) Amounts primarily include employee separation costs, accelerated depreciation and asset impairments associated with facilities to be closed or divested related to activities under the company's formal restructuring programs. (3) Represents the estimated tax impacts on the reconciling items based on applying the statutory rate of the originating territory of the non-GAAP adjustments. (4) Represents benefits recorded in the fourth quarter and full year due to reductions in reserves for unrecognized income tax benefits resulting from the expiration of the statute of limitations for assessments related to federal income tax return years. The benefit recognized in the fourth quarter relates to the 2020 federal tax return year and the benefit recognized for the full year relates to both the 2020 and 2019 federal tax return years. MERCK & CO., INC. FRANCHISE / KEY PRODUCT SALES (AMOUNTS IN MILLIONS) (UNAUDITED) Table 3 2024 2023 4Q Full Year 1Q 2Q 3Q 4Q Full Year 1Q 2Q 3Q 4Q Full Year Nom % Ex-Exch % Nom % Ex-Exch % TOTAL SALES (1) $15,775 $16,112 $16,657 $15,624 $64,168 $14,487 $15,035 $15,962 $14,630 $60,115 7 9 7 10 PHARMACEUTICAL 14,006 14,408 14,943 14,042 57,400 12,721 13,457 14,263 13,141 53,583 7 8 7 10 Oncology Keytruda 6,947 7,270 7,429 7,836 29,482 5,795 6,271 6,338 6,608 25,011 19 21 18 22 Alliance Revenue – Lynparza (2) 292 317 337 365 1,311 275 310 299 315 1,199 16 18 9 11 Alliance Revenue – Lenvima (2) 255 249 251 255 1,010 232 242 260 226 960 13 14 5 6 Welireg 85 126 139 160 509 42 50 54 72 218 122 123 133 133 Alliance Revenue – Reblozyl (3) 71 90 100 110 371 43 47 52 70 212 58 58 75 75 Vaccines (4) Gardasil/Gardasil 9 2,249 2,478 2,306 1,550 8,583 1,972 2,458 2,585 1,871 8,886 -17 -18 -3 -2 ProQuad/M-M-R II/Varivax 570 617 703 594 2,485 528 582 713 545 2,368 9 9 5 5 Vaxneuvance 219 189 239 161 808 106 168 214 176 665 -9 -9 22 23 RotaTeq 216 163 193 139 711 297 131 156 185 769 -25 -25 -8 -7 Pneumovax 23 61 59 68 74 263 96 92 140 85 412 -12 -12 -36 -34 Hospital Acute Care Bridion 440 455 420 449 1,764 487 502 424 429 1,842 5 5 -4 -3 Prevymis 174 188 208 215 785 129 143 157 175 605 23 23 30 33 Dificid 73 92 96 79 340 65 76 74 87 302 -9 -9 13 13 Zerbaxa 56 62 64 70 252 50 54 53 61 218 14 16 16 18 Noxafil 56 45 41 36 177 60 55 51 46 213 -23 -17 -17 -8 Cardiovascular Winrevair 70 149 200 419 - - - - Alliance Revenue - Adempas/Verquvo (5) 98 106 102 109 415 99 68 92 108 367 1 1 13 13 Adempas (6) 70 72 72 73 287 59 65 65 66 255 11 9 12 14 Virology Lagevrio 350 110 383 121 964 392 203 640 193 1,428 -37 -37 -33 -28 Isentress/Isentress HD 111 89 102 92 394 123 136 119 105 483 -13 -7 -18 -14 Delstrigo 56 60 65 69 249 44 50 54 54 201 28 29 24 26 Pifeltro 42 39 42 40 163 34 38 37 33 142 20 20 15 15 Neuroscience Belsomra 46 53 78 45 222 56 63 58 54 231 -17 -17 -4 1 Immunology Simponi 184 172 189 543 180 180 179 171 710 N/M N/M -24 -23 Remicade 39 35 41 114 51 48 45 43 187 N/M N/M -39 -36 Diabetes (7) Januvia 419 405 278 232 1,334 551 511 581 547 2,189 -58 -56 -39 -36 Janumet 251 224 204 255 935 329 354 255 240 1,177 7 11 -21 -16 Other Pharmaceutical (8) 576 573 644 713 2,510 626 560 568 576 2,333 25 25 8 10 ANIMAL HEALTH 1,511 1,482 1,487 1,397 5,877 1,491 1,456 1,400 1,278 5,625 9 13 4 8 Livestock 850 837 886 889 3,462 849 807 874 808 3,337 10 14 4 9 Companion Animal 661 645 601 508 2,415 642 649 526 470 2,288 8 10 6 7 Other Revenues (9) 258 222 227 185 891 275 122 299 211 907 -13 3 -2 4 N/M - Not Meaningful Sum of quarterly amounts may not equal year-to-date amounts due to rounding. (1) Only select products are shown. (2) Alliance Revenue represents Merck’s share of profits, which are product sales net of cost of sales and commercialization costs. (3) Alliance Revenue represents royalties. (4) Total Vaccines sales were $3,424 million, $3,656 million, $3,675 million and $2,693 million in the first, second, third and fourth quarter of 2024, respectively, and $3,133 million, $3,557 million, $4,002 million and $2,962 million in the first, second, third and fourth quarter of 2023, respectively. (5) Alliance Revenue represents Merck's share of profits from sales in Bayer's marketing territories, which are product sales net of cost of sales and commercialization costs. (6) Net product sales in Merck's marketing territories. (7) Total Diabetes sales were $745 million, $715 million, $592 million and $546 million in the first, second, third and fourth quarter of 2024, respectively, and $950 million, $951 million, $924 million and $876 million in the first, second, third and fourth quarter of 2023, respectively. (8) Includes Pharmaceutical products not individually shown above. (9) Other Revenues are comprised primarily of revenues from third-party manufacturing arrangements and miscellaneous corporate revenues, including revenue-hedging activities. Other Revenues related to the receipt of upfront and milestone payments for out-licensed products were $61 million, $15 million, $15 million and $15 million in the first, second, third and fourth quarter of 2024, respectively, and $51 million, $3 million and $65 million in the first, second and third quarter of 2023, respectively.

摘要:核酸技术彻底改变了疫苗开发，使得RNA和DNA疫苗的设计和生产能够快速进行，以预防和治疗疾病。针对COVID-19的mRNA和质粒DNA疫苗的成功部署进一步验证了这项技术。目前，由于其更高的有效性，mRNA平台占据主导地位，而DNA平台正在快速进化，因为它拥有独特的优势，有可能克服与mRNA平台相关的问题。为了帮助理解这两种疫苗平台的最新表现，并认识到它们在未来的临床潜力，本综述比较了文献中目前已知的mRNA和DNA疫苗的优势和缺点，包括开发时间线、财务成本、分发便利性、有效性、安全性和产品的监管批准。此外，综述还讨论了正在进行的临床试验、改进策略，以及RNA和DNA平台的替代疫苗设计。 1.引言 核酸疫苗因其在临床试验中对多种疾病（包括癌症和SARS-CoV2等病毒感染）的治疗或预防显示出有希望的结果，最近受到了极大关注。与传统的基于蛋白质或病毒的疫苗相比，核酸疫苗拥有众多优势，这些优势已经并将继续使它们成为疾病控制和治疗最有希望的平台。与基于蛋白质和病毒的疫苗生产所需的在孵化鸡蛋或培养细胞中的漫长过程不同，核酸疫苗的开发和制造简单快捷，并且可以迅速扩大生产规模，特别是在应对新兴大流行病时。核酸疫苗的生产过程中不包含传染性病原体，这也消除了不完全灭活病毒或纯化的问题，并确保了它们在人类中的安全性。此外，核酸疫苗的基因序列可以轻松修改以针对特定病原体和疾病，使它们能够适应新兴传染病和快速演变的病原体，或者适应癌症等异质性疾病中的个性化疫苗。除了安全性和生产便利性，核酸疫苗能够通过MHC I类和II类分子呈现抗原，并诱导宿主免疫系统的细胞和体液反应，而蛋白质疫苗在诱导CD8+细胞毒性T淋巴细胞反应方面效率较低，激活这些细胞需要MHC I分子呈现抗原。同样重要的是，核酸疫苗避免了当前基于鸡蛋的疫苗中病毒适应在鸡蛋中复制的问题，这可能导致关键抗原位点的变化，干扰保护性免疫反应。核酸疫苗的另一个优势是，与基于蛋白质或病毒的传统疫苗相比，它们在最终溶液产品中的添加剂更少。传统疫苗的溶液通常含有MF59、铝盐和CpG等佐剂。Saponin作为佐剂被添加到Novavax（一种针对COVID-19的蛋白质亚单位疫苗）的溶液中。Thimerosal有时被用作传统疫苗的防腐剂，以防止细菌或真菌污染。尽管没有证据表明疫苗中使用Thimerosal是有害的，但公众担心Thimerosal可能导致儿童自闭症等神经系统疾病。相比之下，Moderna或Pfizer的COVID-19 mRNA疫苗的溶液中不包含额外的佐剂或防腐剂。用于mRNA传递的脂质纳米颗粒（LNPs）也作为疫苗佐剂。此外，疫苗溶液通常含有稳定剂。明胶是病毒疫苗溶液中常用的稳定剂（例如，Flumist®用于流感，M-M-R® II用于麻疹、腮腺炎和风疹，Varivax®用于水痘）以保护病毒在制造和运输过程中免受冻干或解冻的损害。接种疫苗的患者血液中可能产生抗明胶抗体，这可能导致严重的过敏反应。Polysorbate 80是另一种用于基于蛋白质和病毒的疫苗（例如，HPV疫苗、Novavax和J&J/Janssen COVID-19疫苗）溶液中的稳定剂，也可能在一些患者中引起过敏反应。相比之下，Moderna和Pfizer的COVID-19 mRNA疫苗使用蔗糖作为mRNA疫苗溶液中的稳定剂，没有数据显示蔗糖的存在与任何疫苗相关的不良反应有关。然而，需要注意的是，mRNA疫苗含有聚乙二醇（PEG）在脂质纳米颗粒中用于稳定脂质结构，这被认为是引起mRNA疫苗在临床上观察到的严重过敏反应的潜在因素之一。为了解决这个问题，已经开发了新的聚合物，如聚（寡（乙二醇）甲基醚甲基丙烯酸酯）（POEGMA）作为PEG的替代品，可能稳定LNPs并减少免疫原性（见第2.4.2节）。 目前，两种主要类型的核酸疫苗在研究实验室和临床上越来越受到关注，分别是DNA疫苗和RNA疫苗，它们分别以DNA和mRNA作为主要疫苗成分。RNA和DNA疫苗都通过使用宿主自身的蛋白质合成机制来产生“抗原”，这些抗原是病毒或致病细胞表面可以被宿主免疫系统识别并触发免疫原特异性免疫反应的免疫原性蛋白。这个过程模仿了自然感染而不引起疾病，允许身体以受控的方式发展免疫力。DNA疫苗通常通过将编码抗原的基因片段克隆到一个圆形载体中（如质粒和微环DNA）来创建，这些载体可以在哺乳动物细胞中高效表达，并通过肌肉内和皮内注射等途径将修饰后的DNA构建物传递给宿主。宿主细胞，例如肌肉内传递中的肌细胞，将摄取DNA，读取其序列中的遗传信息，并合成编码的抗原（图1）。RNA疫苗可以通过mRNA和其他RNA载体（如自复制RNA和环状RNA（见第3.3节））来创建。在这种情况下，RNA而不是DNA负责携带生产抗原所需的遗传信息。在抗原呈递细胞（APCs）如树突状细胞（DC）和巨噬细胞的帮助下，产生的抗原被宿主免疫系统识别为外来物，并将对其产生免疫反应。对于抗病毒疫苗，将产生能够识别和中和病毒的抗体。对于所有疫苗，T细胞也被激活以帮助破坏感染的细胞或带有目标抗原的细胞。 RNA和DNA疫苗都需要被传递到细胞中以表达抗原。单独的裸DNA已在许多临床试验中作为疫苗使用，并被证明是有用的，尽管需要进一步提高裸DNA疫苗的传递效率和因此的免疫原性，以扩大其应用。与通常直接注射到宿主中的DNA疫苗不同，RNA疫苗（例如mRNA）通常被包裹在脂质纳米颗粒（LNPs）中，这有助于保护RNA并促进其进入细胞和内质网逃逸。这些LNPs通常是免疫原性的，并表现出对RNA疫苗的重要佐剂作用。在被注射部位附近的细胞摄取后，RNA从LNPs中释放出来，进入细胞的细胞质中进行后续的翻译和呈递过程，这些过程与DNA疫苗相似（图1）。 图1. mRNA和DNA疫苗的作用途径。（1）mRNA和DNA疫苗可以通过肌肉注射或其他方法给药，之后疫苗将被宿主的体细胞内化（2）。mRNA和DNA疫苗编码抗原的遗传信息，并依赖宿主自身的蛋白质合成机制来产生抗原。mRNA需要在进入宿主细胞后从其递送载体中释放出来，例如LNP，然后直接翻译成蛋白质抗原。质粒DNA疫苗需要被运送到细胞核中，DNA中的遗传信息才能被读取并转录成mRNA。之后，mRNA从细胞核中出来并翻译成编码的蛋白质抗原。（3）合成的抗原被转移到抗原呈递细胞（APCs）中，这些细胞迁移到次级淋巴器官，通过MHC分子与T细胞受体（TCR）之间的相互作用以及共刺激分子之间的相互作用（例如APC表面的CD86和T细胞上的CD28等受体）来（4）激活T细胞。激活的CD4+ T细胞分泌促炎细胞因子以激活负责（5）建立针对抗原的体液免疫反应的B细胞。 RNA和DNA疫苗平台在包括寨卡病毒、流感、狂犬病等疾病的临床前和临床研究中都显示出了有希望的结果。正在进行的DNA和RNA疫苗的大量临床试验强调了它们在未来医学中的重要角色。然而，这两种疫苗平台也有限制，这些限制目前阻碍了它们充分发挥潜力，并在临床试验中造成了许多失败。由于这两种疫苗在疫苗接种中相对较新，缺乏来自已完成的后期临床试验的足够支持数据，而且目前可用的长期安全性和有效性数据非常有限，因此很难判断哪一种在临床使用中具有更大的潜力。此外，这两种方法都在不断地被开发和优化，这增加了未来DNA和RNA疫苗之间胜者的不确定性。 迄今为止，大多数数据都是通过质粒DNA和基于mRNA的疫苗产生的。因此，本综述聚焦于讨论和比较当前质粒DNA和mRNA疫苗的成就和正在进行的发展，尽管也提到了其他平台。目的是提供洞见，了解它们在预防和治疗疾病方面可能产生重大影响的潜力。 2.mRNA和DNA疫苗的优势和考虑因素 mRNA疫苗高效、安全且多功能。最近，由于在COVID-19大流行期间成功应用于减少可能导致患者住院或死亡的严重病例，它们成为了热门话题。这一成功也减弱了对DNA疫苗的热情。 尽管目前产生的热情较少，DNA疫苗有一些独特的优势，可能克服mRNA疫苗的局限性。它们在热稳定性上更强，保质期更长。因此，它们不需要冷链基础设施进行运输和储存，这降低了疫苗分发的成本，并允许在基础设施有限的地区获得疫苗。此外，DNA疫苗可以在细胞中提供比mRNA更持久的基因表达，这可能会延长对抗原的免疫反应。DNA疫苗更便宜且更容易扩大生产规模。此外，裸DNA疫苗可以直接交付，而不需要与生物材料（如LNPs）包装，这与mRNA疫苗相比进一步简化和降低了设计和制造过程的成本。没有包装材料中的炎症因子，DNA疫苗更安全，因为它们会引起较少的不良副作用。 此外，最近一项关于COVID-19疫苗的临床前研究表明，DNA和mRNA平台之间存在不同的免疫反应。DNA疫苗触发了更强的T细胞反应，而mRNA引发了更强的体液反应，这表明每个平台可能特别适合针对特定疾病。为了深入理解两种疫苗接种方法，我们从几个不同的方面系统比较了mRNA和DNA疫苗的优势和劣势：开发时间线和财务成本、储存和运输要求、剂量和保护效力、监管批准以及安全性和副作用。 2.1.开发时间线和财务成本 与mRNA和DNA疫苗的设计和生产相关的时间和财务成本取决于各种因素，如病原体类型、监管要求和全球需求。这两种疫苗平台有很多共同点。与传统的基于蛋白质和病毒的疫苗相比，它们的生产相对较快，因为它们的生产不需要使用受精鸡蛋或培养细胞。mRNA和DNA疫苗都需要一个类似的初始步骤，即确定正在开发的疫苗的抗原的基因序列（图2）。对于DNA疫苗，基因序列将被克隆到DNA载体中，如质粒DNA，并伴有选定的启动子和增强子以实现优化的转录。DNA载体首先在细菌中扩增，然后从细菌培养中收获。分离和纯化后，它们被重新溶解在水溶液中，准备用于给药。 图2. mRNA和DNA疫苗的生产。（左）生产mRNA-LNP疫苗的一种常见方法。（1）将目标抗原的遗传序列插入到一个环形DNA载体中。（2）然后在细菌中扩增环形DNA并分离出来。（3）线性化环形DNA模板后，遗传序列被（4）体外的噬菌体聚合酶转录成mRNA，并且（5）通过高性能液相色谱（HPLC）纯化mRNA转录本。（6）mRNA与脂质在微流体混合器中迅速混合，导致mRNA被脂质瞬间包裹并沉淀为自组装的纳米颗粒（mRNA-LNPs）。（7）通过透析或过滤纯化mRNA-LNP溶液，以去除非水溶剂和未包裹的mRNA。（8）纯化的mRNA疫苗产品在无菌瓶中包装，供使用前准备。（右）生产DNA疫苗的一种方法。（1）将目标抗原的遗传序列插入到质粒DNA载体中。（2）然后在细菌中扩增质粒DNA并分离出来。（3）通过HPLC纯化质粒DNA后，（4）得到的DNA疫苗产品在无菌瓶中包装，供使用前准备。 mRNA疫苗的设计需要相同的初始步骤，即基因序列鉴定。但是，mRNA疫苗的生产涉及更多的步骤，因为它依赖于双链线性DNA作为模板，如线性化的质粒DNA，其本身可以作为DNA疫苗。模板包含感兴趣的序列和通常来源于T3、T7或SP6噬菌体的DNA依赖性RNA聚合酶启动子。mRNA是通过体外转录（IVT）从模板产生的。通过在DNA模板的3′端包含或在IVT后通过酶反应，将聚(A)尾添加到mRNA转录本。由于mRNA分子不稳定，并且在宿主内给药后高度易受核酸酶降解，裸mRNA很少用作疫苗。相反，mRNA被包裹在纳米颗粒（NPs）中，如LNPs，以稳定mRNA，保护其免受降解，并促进其进入细胞。用于mRNA疫苗的LNPs，如针对COVID-19大流行的Comirnaty（辉瑞-BioNTech）和Spikevax（Moderna），通常包含4种不同类型的脂质：可电离阳离子脂质、中性脂质、胆固醇和聚乙二醇化脂质。脂质与纯化的mRNA在微流体混合器中迅速混合，形成mRNA-LNP复合物。经过透析或过滤以去除未复合的成分后，mRNA-LNP重新分散在通常含有磷酸二氢钾、磷酸二氢钠或Tris（三甲胺）（pH 7-8）的盐溶液中。NPs的设计和筛选是耗时的，因为已经开发了成千上万的NPs用于不同的目的，选择最佳的一种用于特定的mRNA疫苗取决于目标组织/器官和细胞类型、mRNA大小和序列、期望的佐剂作用以及可接受的炎症水平。通过传统实验筛选NP库是识别特定设置中最佳配方的常见方式，但可能成本高昂且耗时。随着机器学习和计算方法的最新进展，这一问题可以得到缓解，这些进展有潜力显著加快这一过程并降低成本。 除了NPs的设计和筛选，通常用于LNPs的材料，如可电离脂质，可能很昂贵。Moderna mRNA疫苗的脂质成本为每剂2.85美元。从2020年7月到2022年7月，美国政府花费了253亿美元购买辉瑞和Moderna COVID-19疫苗，平均每剂20.69美元。没有政府支持，美国那些无保险和保险不足的个人可能面临财务障碍。生活在低收入和中等收入国家的人的成本障碍将更高。mRNA疫苗的成本效益因此仍然是一个问题，需要解决。 由于不需要LNPs，DNA疫苗的开发和生产成本明显低于mRNA疫苗，但由于与临床前和临床试验以及政府法规相关的成本，仍然不特别便宜。在印度获批紧急使用的COVID-19 DNA疫苗ZyCov-D的报告价格为每剂3.57美元。 随着越来越多的mRNA和DNA疫苗进入临床试验和公共使用，预计它们的价格将下降，但也应该考虑大幅降低设计、制造和原材料成本的策略，特别是对于mRNA-LNP疫苗。 2.2.运输和储存要求 为了防止mRNA和LNPs的结构变化，mRNA疫苗必须在（超）低温下长期储存和运输。目前Moderna和辉瑞的COVID-19 mRNA疫苗分别需要在-15至-25°C和-60至-90°C之间冷冻储存。对于短期储存，Moderna mRNA COVID-19疫苗在室温下稳定长达12小时，在2-8°C下稳定长达30天；辉瑞-BioNTech mRNA COVID-19疫苗在室温下稳定长达2小时，在2-8°C下稳定长达5天。两种疫苗的温度要求差异的确切原因尚待确定。此外，已经表明，在25°C下储存7天的mRNA LNPs导致转基因表达比在-30°C下储存的减少90%以上。 关于mRNA疫苗在延长储存期间的稳定性以及LNPs中mRNA封装对疫苗稳定性的影响，公开可用的数据很少。保持mRNA疫苗在（超）低温下增加了储存和运输的成本，并限制了mRNA疫苗在全球所有地区或国家的可获得性。这可能导致疫苗获取的不平等，导致疫苗接种率和医疗结果的不均衡。解决这个问题的一个潜在解决方案是对mRNA-LNPs进行冻干，因为Muramatsu等人已经证明冻干的mRNA-LNPs可以在室温下保持物理化学稳定性长达12周，在4°C下超过24周，而不损害它们在小鼠中的免疫原性。Popowski等人也表明，冻干的mRNA疫苗干粉在室温下稳定一个月。在含有蔗糖的溶液中储存mRNA-LNP产品也可以提高其稳定性。为确保有效的全球分发，疫苗应具有足够长的保质期，理想情况下在冰箱可达到的温度（2-8°C）或以上。在这方面，DNA疫苗比mRNA疫苗更好，因为DNA的卓越热稳定性允许它们在4°C甚至环境温度下运输和储存，使它们成为控制资源有限的发展中国家和农村地区传染病传播最有希望的候选疫苗。例如，在印度获得紧急使用授权的COVID-19 DNA疫苗ZyCoV-D，可以在2-8°C下储存，并且在至少三个月内稳定在25°C，保留美国食品药品监督管理局（FDA）要求的所有规格。乙型肝炎的重组DNA疫苗（Engerix-B）可以在45°C下储存一周或在37°C下储存一个月。涉及138名健康成年人的临床研究结果表明，与在4°C的正常储存条件相比，这两种储存方式都没有改变疫苗诱导的保护性抗体滴度。DNA疫苗的显著热稳定性使得运输和储存变得容易，减轻了与冷链要求相关的挑战，并最小化了因冷链中断而导致的疫苗浪费风险。 2.3.剂量和保护效力 当前的DNA疫苗在临床试验中比mRNA疫苗具有更低的效力，并且需要更高的剂量来产生有效的免疫反应。对于初次接种COVID-19疫苗（附录表1），辉瑞的mRNA疫苗以两剂，每剂30微克的形式给予，Moderna mRNA疫苗包含两剂100微克的剂量；相比之下，ZyCoV-D被批准为三剂方案，每剂含有2毫克质粒DNA，分别是辉瑞和Moderna疫苗mRNA剂量的100倍和30倍。据报道，ZyCoV-D的两剂方案，每剂3毫克，在临床试验中产生了相当的保护，但低于6毫克的总剂量仍需验证有效的疫苗接种。 最初的单价Moderna和辉瑞mRNA疫苗在2020年的3期临床试验中对COVID-19的效力相似（附录表1），Moderna疫苗在预防COVID-19疾病方面的有效性为94.1%，在预防严重病例方面的有效性为100%，辉瑞疫苗在预防COVID-19方面的有效性为95%。然而，尽管使用了更高的剂量，ZyCoV-D的效力较低——在接种两剂后预防COVID-19的有效性为66.6%，在预防严重和中度病例方面的有效性为100%（附录表1）。值得注意的是，ZyCoV-D的3期临床试验发生在印度Delta株流行期间，这可能是单价DNA疫苗相对较低效力的直接原因。 两种疫苗平台之间在剂量和效力方面的巨大差异主要是由于DNA传递在启动抗原生产之前的细胞内障碍更大。无论注射途径如何，DNA疫苗都需要克服细胞膜和核膜的生理屏障，才能到达细胞转录机制。之后，从DNA转录的mRNA离开细胞核并在细胞质中进行翻译。相比之下，mRNA疫苗在被翻译成目标抗原之前不需要到达细胞核，LNP配方中的可电离阳离子脂质可以大大促进细胞表面的跨膜运输和细胞内mRNA的内质网逃逸。因此，开发策略以克服DNA疫苗传递的生理屏障对于提高其效力和减少所需剂量至关重要。 基于mRNA和DNA疫苗治疗癌症的临床数据也已经发表，尽管与COVID-19疫苗研究相比，涉及的患者数量较少。在针对多种自体癌症的1/2期试验（NCT03480152）中，包括肝细胞癌（HCC），编码新抗原的mRNA疫苗每两周肌肉注射一次，最多4剂，剂量为0.13毫克或0.39毫克。所有接种疫苗的患者（100%，2名患者中的2名在任何剂量水平）显示出循环抗原特异性T细胞的增加。然而，他们中没有人（在任何剂量水平）显示出对治疗的临床反应（客观肿瘤退缩）。 在完成的1期研究（NCT00199849）中，使用NY-ESO-1质粒DNA疫苗（pPJV7611）治疗表达NY-ESO-1或LAGE-1抗原的各种肿瘤患者，如非小细胞肺癌（NSCLC）。参与者在近距离的不同位置通过粒子介导的表皮传递接受pPJV7611。第一组参与者接受了4×1微克pPJV7611在4个不同位置的传递；第二组参与者接受了8×1微克pPJV7611在8个位置的传递；第三组参与者接受了2×4微克pPJV7611在2个不同位置和4个不同时间（第1、3、5和8天）的传递。治疗在4周和8周后重复。研究报告表明，在第一次疫苗治疗后13周，所有三个队列中对肿瘤反应的受试者数量为0（0.0%）。在多达13周内具有NY-ESO-1特异性CD4+T细胞免疫的受试者数量分别为第一队列3（100.0%），第二队列5（83.3%），第三队列7（100.0%）。在多达13周内具有NY-ESO-1特异性CD8+T细胞免疫的受试者数量分别为第一队列1（33.3%），第二队列0（0.0%），第三队列1（25.0%）。 这些研究中取得的有限效力可能归因于给予患者的质粒DNA或mRNA疫苗的低剂量以及缺乏与其他免疫治疗剂的联合使用；否则，在临床研究中观察到了有利的结果。在针对晚期HCC的1/2期试验（NCT04251117）中，一种编码多达40个新抗原的个性化治疗性癌症DNA疫苗（GNOS-PV02）与编码白介素-12（pIL12）的质粒共同配制，并以1毫克GNOS-PV02和0.34毫克pIL12的剂量每3周一次皮内注射给参与者，随后进行电穿孔。在前四次治疗中，参与者接受了1毫克GNOS-PV02和0.34毫克pIL12的剂量，随后每9周一次直至第二年结束，之后每12周一次直至第五年结束。此外，参与者每3周接受一次200毫克的PD-1抑制剂pembrolizumab治疗，为期2年。治疗结果很有希望，因为新抗原特异性T细胞反应率为86.4%（22名可评估患者中的19名），客观反应率为30.6%（36名患者中的11名），其中8.3%（36名患者中的3名）实现了完全反应。 在另一项1/2期研究（NCT03164772）中，NSCLC受试者要么接受Durvalumab（一种抗PD-L1抗体）和包含6个编码不同肿瘤相关抗原（MUC1、Survivin、NY-ESO-1、5 T4、MAGE-C2和MAGE-C1）成分的BI 1361849 mRNA疫苗的联合治疗（A组），要么接受Durvalumab、Tremelimumab（一种抗CTLA-4抗体）和BI 1361849 mRNA疫苗的联合治疗（B组）。BI 1361849 mRNA疫苗的每个成分分别以80微克的剂量给予两次。在研究期间，每位参与者共接受了12次mRNA皮内注射（总共960微克mRNA）。A组估计的中位无进展生存期为2个月，B组为1.8个月。在第8周无进展的受试者数量分别为A组11（47.8%）和B组11（32.4%）。这些数字在8周到第24周之间随时间单调下降。 2.4.不良事件和安全数据 2.4.1.常见观察到的不良事件 mRNA和DNA疫苗相关的不良事件（AEs）通常与注射外来物质有关，如核酸、水溶液和脂质纳米颗粒（LNPs），尽管在一些研究中，ZyCoV-D疫苗注射相关的AEs低发生率归因于使用无针皮内注射系统而非针头。大多数mRNA或DNA疫苗接种相关的AEs轻度至中度。大多数可用数据基于COVID-19疫苗的临床研究。比较两种平台COVID-19疫苗的表现，DNA疫苗在诱发和非诱发AEs的严重程度和发生率方面比mRNA疫苗展现出更好的安全档案。在mRNA-1273的3期临床试验（附录表2）中，接种组比安慰剂组更频繁地报告诱发AEs，第一剂后（87.8%对48%）和第二剂后（92.2%对42.8%）。并且局部和全身AEs的严重程度和发生率在第二剂后比第一剂后增加。在ZyCoV-D的3期临床试验（附录表2）中，接种组（4.49%）和安慰剂组（4.47%）之间诱发AEs的发生相似。中期分析还表明，疫苗作为加强剂对接受者来说耐受性良好，因为与第一剂相比，第三剂后AEs的频率没有增加，大多数报告的事件是在第一剂而不是第二剂或第三剂后。这与mRNA-1273试验的结果形成对比，表明重复接种会引发更强的诱发AEs。mRNA-1273和ZyCoV-D的非诱发AEs与相应的安慰剂组相似，但mRNA-1273的发生率高（23.9%）比ZyCoV-D试验（3.27%）。有趣的是，尽管ZyCoV-D使用的核酸剂量是mRNA-1273的30倍，但ZyCoV-D的AEs严重程度和发生率更低。这可能归因于mRNA-1273中使用的LNP在ZyCoV-D中不存在。然而，重要的是要注意，这两项临床试验由不同的科学家在不同的国家和人类群体中进行，这可能会使不同疫苗报告的AEs之间的比较复杂化。与ZyCoV-D类似，另一种DNA疫苗COVID-eVax在1期试验中显示出在第二次免疫后全身和局部诱发AEs的发生率比第一次后低。第一次后局部为35-87%，全身为40-50%，第二次后局部为20-55%，全身为30-45%，取决于疫苗剂量。这些发生率高于ZyCov-D，可能由于COVID-eVax使用了不同的给药程序：肌肉内针头注射后通过移动电脉冲发生器进行电穿孔，这些程序更具侵入性和炎症性。DNA疫苗作为加强剂比mRNA疫苗更易被耐受的原因尚不清楚，了解它们的分子机制对于设计更好的平台很重要。对于mRNA和DNA疫苗在预防除COVID-19以外的疾病方面的安全性数据也有一些，但不多。例如，一项mRNA疫苗已在临床试验中用于预防狂犬病。在10%的接受者中观察到3级全身AEs，这些被归因于mRNA疫苗的炎症特性。 2.4.2.其他安全问题 除了常见的AEs外，关于DNA疫苗的一个主要担忧是其可能被整合到受体细胞的基因组中，因为它们需要进入细胞核以表达抗原。已经进行了大量研究以确定DNA整合到人类染色体的风险，从而激活致癌基因或增加染色体不稳定性。这些研究的数据表明，这种风险低于由自发基因组突变引起的风险。人类临床试验研究进一步证实了DNA疫苗的安全性和耐受性，尽管仍需要更多的长期临床数据来完全解决这一安全问题。另一方面，mRNA疫苗在患者中相对安全，因为它们在宿主中的保留时间相对较短，mRNA不太可能进入细胞核。对于mRNA疫苗，超敏反应是一个主要问题。它们是由某些材料如非天然脂质的传递载体引起的。在一项临床前研究中，Ndeupen等人表明，一些已经过仔细表征并在临床前mRNA疫苗研究中广泛使用的商业LNPs具有高度炎症性，并导致动物中高死亡率（约80%）。还有临床证据表明，COVID-19的一些mRNA疫苗中的聚乙二醇（PEG）可以诱导血液中的抗PEG抗体，导致过敏反应。然而，报告的结果相互矛盾。Carreno等人的研究表明，BNT162b2接种患者的血清中未检测到PEG特异性抗体，并且过敏反应与mRNA-1273诱导的抗PEG抗体之间没有明显的相关性。此外，mRNA疫苗接种诱导的抗PEG抗体对疫苗效力的影响仍然未知。Kozma等人的一项研究表明，这些抗PEG抗体可能会降低疫苗效力，而Guerrini等人和Ju等人的其他研究表明，抗PEG抗体对mRNA疫苗产生的抗刺突中和抗体没有影响。因此，PEG对mRNA疫苗的潜在不良影响仍然是一个活跃的研究和辩论领域。需要更多的研究和监测数据来对这个问题得出明确结论。与此同时，已经开发了下一代PEG衍生物，如POEGMA，作为改善药物和基因传递的替代聚合物。这些聚合物具有与PEG相似的功能属性，但引起的抗PEG抗体较少。它们可能被用来稳定mRNA疫苗。mRNA和DNA疫苗的另一个潜在安全问题与反复接种可能产生的抗mRNA或DNA反应和诱导全身自身免疫疾病有关。但发表的研究有限，以解决这个问题。一项关于癌症DNA疫苗的1期临床试验报告称，在66名注册患者中，没有检测到抗双链DNA抗体或PBMCs中的质粒，并且报告称没有接受者出现自身免疫疾病的症状，但需要更大规模的临床研究的证据来完全解决这一问题。总体而言，迄今为止mRNA和DNA疫苗显示出良好的安全档案，并且由于安全问题而退出临床试验的疫苗候选者很少。有必要进行更多的研究和分析，以全面评估安全方面，为在行业和临床中建立更好的标准和法规。 2.5.临床使用的批准 由Zydus Cadila开发的ZyCoV-D获得了印度药品监管总局（DCGI）在2021年8月批准的紧急使用限制，迄今为止，它是唯一获得批准的DNA疫苗。它包含一个编码SARS-CoV-2的刺突（S）蛋白和免疫球蛋白E（IgE）信号肽的单一质粒DNA，以促进转染细胞的分泌。 康希诺（研究名称：BNT162b2）由辉瑞-BioNTech开发，于2020年12月获得美国FDA的紧急使用授权（EUA），适用于成人，并于2021年8月获得同一机构的批准，用于预防16岁及以上个体的COVID-19。斯皮克瓦克斯（研究名称：mRNA-1273）由Moderna开发，于2020年12月获得美国FDA的EUA，适用于18岁及以上个体。其在成人中的完全批准于2022年1月获得。斯皮克瓦克斯和康希诺及其后续加强疫苗的更详细的临床试验和批准历史在图3中说明。到目前为止，它们是唯一获批用于常规临床使用的mRNA疫苗。更多的DNA和mRNA疫苗用于预防和治疗许多其他疾病，目前处于不同的临床试验阶段，并已显示出令人兴奋的疗效和安全数据。我们将在第3.1节中更详细地讨论正在进行和已完成的临床试验。作为首批展示强大保护效果并获得批准的疫苗，mRNA疫苗在COVID-19大流行期间受到了公众和研究领域的前所未有的关注。它们在大流行情况下的历史性批准以及后来在预防疾病传播方面的出色表现，使它们立即比DNA疫苗处于更有利的位置。当前的资金和研究重点显然倾向于mRNA疫苗，原因之一是更高的效力和较少的公众对基因插入突变的担忧。然而，考虑到上述讨论的所有DNA疫苗的独特优势，mRNA疫苗的成功不应成为忽视DNA疫苗的理由。同样，正如我们社会在应对COVID-19大流行中的经验所证明的那样，平行开发多种替代疫苗平台也很重要，在大流行之前，mRNA疫苗也基本上被忽视，不被认为是与基于蛋白质和病毒的疫苗相竞争的。 图3. COVID-19 mRNA和DNA疫苗的临床试验及里程碑。辉瑞-BioNTech于2020年4月开始对其COVID-19 mRNA疫苗候选物BNT162b2进行I/II期临床试验，随后在2020年7月进行了II/III期试验。BNT162b2（商品名：Comirnaty）是首个获得美国紧急使用授权（EUA）的COVID-19疫苗——成人使用的EUA于2020年12月授予。2021年8月，FDA完全批准Comirnaty用于16岁及以上个体。Moderna于2020年3月开始对其COVID-19 mRNA疫苗候选物mRNA-1273进行I期临床试验，5月进行II期试验，7月进行III期试验。美国FDA于2020年12月授予mRNA-1273（商品名：Spikevax）紧急使用授权，紧随Comirnaty之后获得紧急使用授权。2022年1月，FDA完全批准Spikevax用于成人。印度制药公司Zydus Cadila于2020年7月开始对其COVID-19 DNA疫苗候选物ZyCoV-D进行I/II期临床试验，并于2021年1月进行III期试验。2021年8月，印度药品监管总局（DCGI）批准ZyCoV-D在印度限制性紧急使用。 3.mRNA和DNA疫苗的正在进行开发 本节重点讨论已完成和正在进行的mRNA和DNA疫苗的临床试验结果，这些结果可能影响它们未来的临床使用，改进空间，以及新平台的开发。 3.1.已完成和正在进行的临床试验 尽管目前获批的mRNA和DNA疫苗仅用于COVID-19，但正在进行的研究正在扩大它们的应用范围，以应对其他疾病。在COVID-19 mRNA和DNA疫苗取得胜利后，核酸疫苗（图4）的临床试验数量和规模都有了显著增加。 图4. mRNA/DNA疫苗技术的临床应用。mRNA和DNA疫苗已在临床试验中广泛测试，用于治疗癌症和预防传染病，如SARS-CoV-2、流感、呼吸道合胞病毒（RSV）、人类免疫缺陷病毒（HIV）、寨卡病毒（ZIKV）。目前涉及传染病临床试验的mRNA和DNA疫苗编码一种或少数几种蛋白（抗原），例如SARS-CoV-2的刺突蛋白、流感的血凝素（HA）、RSV的融合（F）糖蛋白、HIV的包膜（Env）蛋白、寨卡病毒的前膜和包膜（prME）蛋白以及寨卡病毒类病毒颗粒（VLP）。对于当前临床试验中的癌症疫苗，mRNA和DNA编码的蛋白包括个性化的新抗原、致癌病毒抗原（例如HPV E6和E7致癌蛋白）以及肿瘤相关抗原（TAA）。此外，mRNA和DNA已被用作遗传佐剂，编码细胞因子，如白介素-12（IL-12）和粒细胞-巨噬细胞集落刺激因子（GM-CSF），以增强对抗原的免疫反应。 3.1.1.mRNA疫苗的临床试验 mRNA疫苗涉及的临床试验目标包括流感、疟疾、呼吸道合胞病毒（RSV）、人类免疫缺陷病毒（HIV）、巨细胞病毒（CMV）、结核病、狂犬病和寨卡病毒（ZIKV）（附录表3）。此外，mRNA疫苗已在临床上用于癌症治疗，如非小细胞肺癌（NSCLC）、结直肠癌、胰腺腺癌、黑色素瘤、胃肠癌、三阴性乳腺癌、卵巢癌等（附录表3）。 在撰写本综述时，目前有301项mRNA疫苗的临床试验正在进行，15项试验已被终止或暂停，129项已完成。它们大多数处于1期、2期或1/2期。所有24项已完成的3期或4期试验都与SARS-CoV-2有关。目前在3期试验中活跃并有积极中期结果的候选疫苗之一是Moderna的mRNA-1345（ConquerRSV），用于对抗RSV感染。该疫苗基于LNP平台，编码一种名为预融合F糖蛋白的RSV蛋白。尽管大多数以前的针对RSV的mRNA疫苗临床试验由于中和抗体产量不足而失败，Moderna最近（2023年1月）宣布了其ConquerRSV 3期关键效力试验的积极数据，涉及超过37,000名60岁及以上的成年人，声称“已经实现了主要效力终点，包括针对RSV相关的下呼吸道感染（RSV-LRTD）的疫苗效力为83.7%（95.88% CI：66.1%，92.2%；p < 0.0001），如定义的两个或更多症状。”2023年1月，美国FDA授予mRNA-1345突破性疗法认定，适用于60岁及以上的成年人。辉瑞和Moderna都已启动了基于mRNA的流感疫苗的临床研究。mRNA-1010是Moderna评估的五种流感疫苗候选之一，是一种四价疫苗，编码来自四种季节性流感病毒的血凝素（HA）蛋白，包括A/H1N1、A/H3N2、B/Yamagata和B/Victoria谱系。2023年2月，Moderna宣布了mRNA-1010 3期临床试验的中期安全性和免疫原性结果。根据新闻稿，与活性疫苗对照相比，mRNA-1010在A/H3N2和A/H1N1的血清转换率上表现出优越性，在A/H3N2的几何平均滴度（GMT）比率上表现出优越性，并且在A/H1N1的GMT比率上表现出非劣性。然而，对于流感B/Victoria和B/Yamagata谱系菌株，血清转换率或GMT比率的非劣性未达到。另一种四价流感疫苗候选是辉瑞开发的modRNA。正在进行的一项3期主动对照研究（NCT05540522）涉及53,200名参与者，旨在评估modRNA的安全性和效力，并预计将在2024年完成。 3.1.2.DNA疫苗的临床试验 DNA疫苗的临床试验针对的传染病包括SARS-CoV-2、寨卡病毒、乙型肝炎、流感、HIV等（附录表4）。值得注意的是，DNA疫苗在临床试验中用于治疗癌症已有相对较长的历史，如三阴性乳腺癌、前列腺癌、黑色素瘤、人乳头瘤病毒（HPV）、宫颈癌和卵巢癌（附录表4）。但很少有完成和正在进行的癌症DNA疫苗试验通过了2期。 一个例子是针对晚期、erb-b2受体酪氨酸激酶2（ERBB2）阳性乳腺癌的治疗性DNA疫苗（pNGVL3-hICD）的1期试验，共有66名患者。该试验在2001年至2010年间在美国华盛顿州的Fred Hutchinson癌症中心进行，监测疫苗毒性10年。试验中使用的疫苗是一种质粒DNA，编码ERBB2的细胞内域（ICD）；它以递增剂量（10 μg、100 μg、500 μg）皮内注射，固定剂量的粒细胞-巨噬细胞集落刺激因子（GM-CSF）蛋白（100 μg）作为佐剂。结果分析表明，大多数患者对ERBB2 ICD的1型免疫反应呈剂量依赖性；反应显著且持久，并与外周血液中中央记忆T细胞的增加相关。疫苗在患者中耐受性良好——大多数与疫苗相关的AEs为1级和2级。在患者的PBMCs中未检测到抗双链DNA抗体或疫苗质粒，并且没有接受者出现自身免疫疾病的症状。在根据不同剂量组的73.5-118.6周的中位随访期间，只有一名患者（在100 μg剂量组）在注册后2.2年发展为可能与疫苗接种相关的淋巴细胞性结肠炎。接种疫苗后一年以上观察到的其他AEs不被认为是与疫苗接种相关的。这些观察结果表明，用治疗性ERBB2 ICD DNA疫苗免疫具有有利的短期和长期安全档案，以及显著、持久的Th1反应水平。因此，DNA疫苗目前正在低表达ERBB2的患者中进行随机2期试验（NCT05163223）。 直接比较DNA和mRNA疫苗的试验清楚地指出，mRNA目前比DNA更受青睐——几乎所有处于第三阶段的核酸疫苗试验都是基于mRNA而非DNA，这表明了早期mRNA试验取得了更好的结果。mRNA试验通常规模更大，表明资金更容易获得，公众对mRNA疫苗的接受率更高。然而，由于RNA和DNA平台都在快速发展，未来情况可能会改变。考虑到它们的独特优势，如果DNA疫苗的效力得到提高，特别是在针对特定疾病的应用场景中，DNA疫苗可能是一个更好的选择。 3.2.疫苗改进 核酸疫苗的历史相对较短，但仍在积极开发中，以满足广泛的应用需求，并在效力、安全性和稳定性方面进行优化。还进行了大量研究，以确定mRNA和DNA疫苗的物理、化学和生物特性，以及免疫反应的分子机制。正在进行的研究将解决当前的限制，并创造一个临床应用的格局，展示针对特定疾病mRNA和DNA最合适的平台。 3.2.1.mRNA疫苗的改进 mRNA疫苗的改进主要集中在RNA及其序列修饰、LNP递送系统、抗原的选择和组合以及佐剂。多个mRNA结构元素已成为修饰的目标，包括非翻译区（UTRs）、5′端帽、开放阅读框架（ORF）和聚（A）尾。为了减少mRNA的免疫原性并增强翻译效率，通常使用诸如5-甲基胞嘧啶（m5C）和假尿苷（ψ）核苷等碱基对修饰。这些修饰的核苷酸能够避开由干扰素（IFNs）诱导的抗病毒途径，这些途径特别针对并降解入侵的mRNA。结果，核苷酸修饰的mRNA疫苗大大增加了抗原的翻译效率，并诱导较少的非期望炎症反应。 其他修饰，如密码子优化和N1-甲基假尿苷（1 mΨ）核苷替换，也已进行以改善mRNA翻译。密码子优化是必要的，因为通过不同的密码子分配编码相同蛋白的mRNA序列可能具有截然不同的蛋白表达水平。因此，用哺乳动物细胞中常用的密码子替换稀有密码子可以增加抗原产量。 还进行了大量研究以优化mRNA疫苗的另一个重要组成部分，即LNPs。除了在生理环境中保护mRNA免受核酸酶降解和克服细胞内递送障碍外，LNPs还被发现对某些mRNA疫苗表现出强烈的佐剂活性。已经设计和使用了不同的LNPs进行mRNA疫苗制备，并且正在开发更多。选择具有上述最佳功能的同时，在人体中具有低毒性的LNP是一个复杂但值得的任务。为了避免上述LNPs的不良影响，也在探索其他mRNA递送策略，如基于细胞外囊泡、类脂材料、合成聚合物和生物材料。细胞外囊泡是自然分泌的，并被哺乳动物细胞用作细胞间通信的方式，特别是癌细胞。因此它们可以很容易地被重新用作mRNA和其他治疗剂的递送工具，具有出色的生物相容性和安全性，在某些情况下还具有组织靶向效果。 将针对不同抗原的mRNA组合在一个疫苗产品中，可能对单一病原体或一系列病原体诱导更广泛的免疫反应。最近，Arevalo等人已经证明，由20种编码不同血凝素的mRNA组成的多价mRNA疫苗可以保护动物免受所有已知的甲型和乙型流感病毒亚型。除了诱导广泛的交叉反应性免疫反应外，这种通用疫苗策略还可以大大减少针对一组病原体的疫苗接种复杂性，从而节省疫苗开发和部署所需的时间、金钱和努力。 3.2.2.DNA疫苗的改进 DNA疫苗的改进可以分为四个主要类别：抗原的选择和组合、DNA构建设计、DNA递送和佐剂的结合。 第一类别中的策略对DNA疫苗来说并不独特。它们与基于RNA的疫苗相似。目标是确定最佳抗原或抗原组合以实现有效的疫苗接种。 DNA构建设计涉及优化抗原的密码子组成、不同序列的最佳组合以递送几个抗原或融合蛋白与单一载体，以及结合特定启动子、增强子和DNA核靶向序列。例如，使用结合病毒和真核元素的混合启动子，以防止纯病毒启动子可能发生的转录沉默。一个特别针对APCs的启动子允许将抗原和佐剂表达限制在特定细胞类型，如DCs，从而防止调节细胞类型如骨髓源抑制细胞（MDSCs）的耐受。 Ross等人和Bros等人已经证明了使用Fascin-1启动子在小鼠和人类中实现DCs的转录靶向的可行性，Sudowe等人在随后的研究中将Fascin-1启动子应用于DCs靶向DNA疫苗的设计。 将不同抗原通过连接子组合在一个疫苗中，可以用来诱导广泛的CD4+/CD8+ T细胞反应。 与编码不变链的序列融合可以增强抗原加载到APCs表面表达的MHC II分子上。 DNA比mRNA更难递送，因为DNA需要进入哺乳动物细胞的核以表达抗原，这对mRNA疫苗来说不是必需的。在已经开发的不同方法中，电穿孔（也称为“电转染”）迄今为止是最有效和方便的方法。它基于脉冲电场，暂时在细胞质膜中创建孔，允许小的、不透过膜的分子进入细胞质。但较大的分子，如质粒和mRNA主要通过电脉冲触发的内吞作用进入细胞。电穿孔越来越多地用于人类临床试验，以增强DNA疫苗针对不同疾病的效果，包括SARS-Cov-2、HIV和HPV。Dross等人还使用基因枪递送DNA疫苗给猕猴，并证明了与mRNA疫苗相当的免疫反应。 用于改善DNA跨细胞膜递送的其他常见方法基于由各种材料组成的纳米载体，如脂质、聚合物、蛋白质和无机材料。还开发了策略以提高DNA跨核膜递送的效率。例如，Dean等人发现将DNA核靶向序列纳入DNA载体中，使其能够与具有核定位信号（NLS）的内源性蛋白结合，通过核孔复合体（NPCs）促进DNA进入细胞核。 抑制进入细胞的DNA疫苗的溶酶体降解也是提高疫苗效力的目标。然而，截至本综述之时，上述讨论的方法尚未获得FDA批准用于改善人类DNA疫苗递送。 第四类别的策略是将佐剂纳入DNA疫苗中，以增强APC激活和T细胞吸引及极化。佐剂可以是化学药品，如氢氧化铝，或遗传物质，如免疫刺激性的2′,3′-环鸟苷酸-腺苷酸（cGAMP）。常见的佐剂还包括促炎细胞因子，如GM-CSF。这些细胞因子可以直接作为蛋白质添加，或通过与DNA疫苗共给药的表达载体编码。探索当前佐剂的使用和设计新的强效佐剂可以显著增强DNA疫苗的效果。同时，选择临床用途的佐剂需要额外考虑，特别是在儿童、老年人和有特殊医疗状况的个体，如自身免疫疾病、糖尿病和充血性心力衰竭患者的疫苗接种中。 3.3.核酸疫苗的替代设计 除了广泛使用的mRNA-LNP和质粒DNA平台外，还有激动人心的核酸疫苗新设计正在出现。对于RNA平台，它们包括自我复制RNA（saRNA），由于其在细胞内自我复制的能力，可以在低剂量下导致更高和更长时间的抗原产生，以及与线性RNA相比具有增强稳定性的环状RNA（图5）。DNA平台的新设计包括小圈DNA（mcDNA）和纳米质粒DNA载体，由于它们的较小骨架尺寸，它们显著提高了递送效率和抗原产生水平，以及可以在不整合到基因组中的情况下实现高抗原表达水平的环状DNA载体，因为它在染色体外停留和复制。在此，我们将讨论一些有潜力开启RNA和DNA疫苗新时代的有前景的设计。 图5. 传统mRNA疫苗与自扩增RNA（saRNA）和环状RNA（circRNA）疫苗的比较。(A) 传统mRNA呈线性格式。mRNA进入宿主细胞后（1），被核糖体用于翻译蛋白抗原（2）。同时，由于mRNA的线性结构，它们容易受到核酸酶（包括内切核酸酶和外切核酸酶）的降解（3）。因此，传统mRNA在宿主细胞中的半衰期短，蛋白抗原表达有限。(B) saRNAs包含来自自复制单链RNA病毒的遗传工程复制子。saRNA进入宿主细胞后（1），通过RNA依赖的RNA聚合酶（RdRP）复合体在宿主细胞中进行自我扩增（2）。这导致宿主细胞中抗原编码mRNA的数量更多、可用时间更长，从而增加了蛋白抗原的产生并延长了其持续时间（3）。与mRNA类似，saRNA也可以被核酸酶降解（4）。(C) circRNA是共价连接的，并包含IRES序列以实现内部核糖体进入。circRNA进入宿主细胞后（1），被用于翻译蛋白抗原（2）。circRNA可以被内切核酸酶降解（3），但由于其共价闭合的环状结构，降解速度比mRNA和saRNA慢（4）。因此，circRNA更稳定，在宿主细胞中的半衰期更长。从这些RNA翻译的蛋白抗原将被转移到抗原呈递细胞（图中未显示）进行处理，并在MHC复合体上呈现以激活宿主的免疫反应。UTR，非翻译区。CDS，编码序列。IRES，内部核糖体进入位点。 3.3.1.自我复制RNA疫苗 自我复制RNA（saRNA）是一种替代非复制mRNA的RNA疫苗平台。它是为了解决传统mRNA疫苗由于mRNA在细胞内半衰期短导致的抗原产生不足的问题，需要重复接种以诱导足够的免疫原性。saRNA包含从自我复制的单链RNA病毒衍生的遗传工程复制子，通常是正链α病毒，如委内瑞拉马脑炎病毒（VEEV）、辛德比斯病毒（SINV）和Semliki森林病毒（SFV）。它们可以作为病毒复制子颗粒（VRPs）递送，saRNA包装在病毒颗粒中。由于缺陷的包膜结构，结果VRPs在最初感染细胞后无法形成传染性病毒颗粒；只有saRNA可以在宿主细胞中进一步扩增。saRNA的细胞内扩增是通过由正链α病毒复制酶基因编码的RNA依赖性RNA聚合酶（RdRP）复合物实现的，它在原位扩增合成的转录物。为了避免病毒载体的隐患，采用了与mRNA递送中使用的非病毒制剂相似的各种非病毒制剂，如阳离子脂质、LNPs、聚合物和硫酸鱼精蛋白，以及电穿孔等物理技术，作为施用saRNA疫苗的替代方法。例如，Hekele等人已经证明了针对H7N9流感的LNP配方saRNA疫苗在小鼠中足够的免疫原性。由于自我复制能力，saRNA可以在比传统mRNA疫苗更低的剂量下施用，以达到类似的抗原表达水平。与每剂需要30和100 µg mRNA的辉瑞和Moderna COVID-19 mRNA-LNP疫苗相比，最近针对COVID-19 saRNA-LNP疫苗（COVAC1）的临床试验仅在2剂方案中每剂使用了0.1-10 µg，并在接种者中诱导了良好的（但不是100%）血清转化。在免疫后6周，接受5.0和10.0 µg疫苗的组别达到了最高的血清转化率（87%）。数据表明，saRNA疫苗在低剂量需求方面有潜力超越传统mRNA疫苗，从而允许剂量节省策略、更好的耐受性和更低的制造成本。尽管需要进一步优化以达到其全部能力，辉瑞公司开发的saRNA疫苗（PF-07852352, PF-07836391, PF-07836394, PF-07836395, PF-07836396, 和 PF-07867246）已进入I期临床试验（NCT05227001）。我们预计未来几年将有更多的saRNA疫苗被设计和临床测试。 3.3.2.小圈和纳米质粒DNA疫苗 小圈DNA（mcDNA）因其与传统质粒DNA疫苗相比的显著特点，目前正在广泛研究其在疫苗接种中的使用。mcDNA消除了质粒DNA中对细菌扩增必需但对哺乳动物细胞表达不必要的原核序列，从而产生了更小的DNA疫苗，提高了基因递送效率和编码抗原的表达。mcDNA疫苗也比质粒DNA疫苗更安全，因为它们避免了对宿主免疫系统识别的原核序列的炎症反应，并防止接受者获得由质粒DNA中包含的抗生素选择标记基因诱导的抗生素抗性。然而，复杂的生产和纯化过程以及与制造相关的高成本目前限制了mcDNA在疫苗接种中的使用。mcDNA通常通过在E. coli中通过一个重组酶如ϕC31整合酶和一个限制酶（RE）如I-SceI进行分子内重组产生，这些酶通过一个可诱导的基因表达系统激活。重组酶促进识别序列（attB和attP）的位点特异性重组，导致形成两个环状DNA分子：具有真核表达盒的mcDNA和包含细菌骨架的小质粒（MP）。然后MP被诱导的RE降解；mcDNA被提取和纯化，其程序与常规质粒DNA类似。为了改进mcDNA生产，正在探索新的方法。例如，Jiang等人开发了一个mcDNA平台，允许整个质粒DNA在体内通过Cre重组酶介导自发转化为mcDNA，并证明了mcDNA平台在保护鸡免受新城疫病毒中的效力。在另一项研究中，Kay等人构建了一个经过基因改造的E. coli菌株，可以通过稳定表达可诱导的ϕC31整合酶和I-SceI同源内切酶高效生产纯化的mcDNAs。到目前为止，mcDNA疫苗尚未在临床上进行评估。 纳米质粒载体被开发出来，旨在解决mcDNA的制造限制，同时保留其优势。纳米质粒使用RNA/RNA相互作用的非编码选择标记系统（“RNA-OUT”）来消除传统质粒骨架中对抗生素抗性标记的需求。由于其较小的骨架大小（<500 bp），纳米质粒载体已显示出在体外和体内都具有强大的转染效率，这一效率比使用传统质粒DNA高出多达十倍。由于这两个优势，它们已被用于流感[222]、委内瑞拉马脑炎病毒（VEEV）和埃博拉病毒的疫苗的临床前研究，并与传统质粒DNA疫苗相比显示出增强的免疫反应。EG-70，一种编码视黄酸诱导基因I（RIG-I）激动剂和IL-12的纳米质粒产品，目前正在进行针对非肌层浸润性膀胱癌（NMIBC）患者的I/II期临床试验（NCT04752722）。 4.结论性评论 核酸疫苗是研究和开发中的一个令人兴奋的新领域，其应用范围从预防各种传染病到治疗癌症。在COVID-19大流行之前，核酸在疫苗领域的潜力在很大程度上被忽视了，但基于mRNA和DNA的COVID-19疫苗的批准代表了它们成为与传统病毒或蛋白基础疫苗相比的替代和更好的平台的革命性突破。使用核酸技术在有效性、安全性以及简单性和快速设计和制造方面提供了几个优势。 随着核酸疫苗受到广泛关注，了解哪些平台在特定应用中更合适至关重要。通过本综述中对mRNA和DNA平台的全面比较，平均来看，目前的mRNA疫苗比DNA疫苗具有更高的临床影响，特别是在COVID-19大流行期间。从技术上讲，mRNA疫苗在抗原表达和免疫系统激活方面更有效，需要的剂量更低，并且公众对插入性突变风险的担忧较少。另一方面，DNA疫苗拥有许多其他优势，包括低成本的生产、运输和储存，以及低报告的不良事件（AEs）。 目前，RNA和DNA疫苗都在经历快速的演变，两个平台上都出现了令人兴奋的新技术，这些技术将提高有效性并影响未来在预防、治疗和控制各种疾病中平台的偏好。与此同时，由于RNA和DNA疫苗在临床开发和使用中的历史相对较短，需要更多的研究来优化平台，收集长期的有效性和安全性数据，并了解抗原呈递和免疫反应的分子机制。 识别微信二维码，添加生物制品圈小编，符合条件者即可加入 生物制品微信群！ 请注明：姓名+研究方向！ 版 权 声 明 本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。