ACE2

Preliminary Q3 2024 PEMGARDA™ (pemivibart) net product revenue of $9.3 million; Invivyd ended Q3 2024 with approximately $107 million in cash and cash equivalentsExpects to finish 2024 with $65 million or more in cash and cash equivalentsWithdraws formal revenue guidance due to recent growth headwind from U.S. FDA’s late-Q3 2024 warning on potential for substantially reduced activity of pemivibart through the PEMGARDA Fact Sheet and other media based on contested, third-party, non-peer-reviewed, non-reproducible, virologic activity data from a non-pemivibart antibodyTargets near-term (1H 2025) profitability with existing cash and cash equivalents, anticipated growth of net product revenue, and various operational efficiency improvementsFurther update to be provided on Q3 2024 earnings call WALTHAM, Mass., Oct. 29, 2024 (GLOBE NEWSWIRE) -- Invivyd, Inc. (Nasdaq: IVVD), a biopharmaceutical company devoted to delivering protection from serious viral infectious diseases, today reported preliminary third quarter (Q3) 2024 financial results and an update to prior financial guidance. Invivyd previously guided to $150-200 million in PEMGARDA™ (pemivibart) net product revenue in 2024 and expected to finish the year with $75 million or more in cash and cash equivalents. This previous guidance did not contemplate the impact on product growth at the most critical moment in Invivyd’s financial year arising from the U.S. Food and Drug Administration (FDA) updating the PEMGARDA Fact Sheet for Healthcare Providers (HCPs) (Fact Sheet) in late-Q3 2024 to include a link to contested, third-party, non-peer-reviewed, non-reproducible, virologic activity data from a non-pemivibart antibody and convey that, based on such data, PEMGARDA may have substantially reduced susceptibility to the currently circulating SARS-CoV-2 variants (i.e., KP.3.1.1). Invivyd previously communicated this unfortunate and unnecessary action that created doubt regarding the antiviral activity of PEMGARDA against current circulating variants. This resulted in confusion in the HCP and vulnerable population communities and an uncertainty regarding the pursuit of additional options for protection against COVID-19 for certain immunocompromised people. Though not at previously anticipated rates, PEMGARDA net product revenue grew through the third quarter and continues to grow. Now with a corrected PEMGARDA Fact Sheet removing the aforementioned data reference and including authentic pemivibart data reflecting ongoing neutralization activity against currently circulating variants tested in line with prior variants represented in the CANOPY Phase 3 clinical trial, Invivyd expects ongoing commercial optimization to drive growth. Based on currently available information, the company is announcing preliminary Q3 2024 PEMGARDA net product revenue of $9.3 million. The company estimates that Invivyd ended Q3 2024 with approximately $107 million in cash and equivalents, and, with anticipated growth of net product revenue and various operational efficiencies underway, believes existing cash and cash equivalents will be sufficient to fund operations through profitability. These estimates are preliminary and are subject to change upon completion of the company’s financial closing procedures for Q3 2024, and finalization of the unaudited interim condensed consolidated financial statements. “At the Infectious Disease (ID) Week meeting in mid-October, we were thrilled to engage with healthcare professionals in an effort to alleviate any remaining confusion regarding the antiviral activity of PEMGARDA, and to share the positive, CANOPY clinical trial data and the safety data included in the Fact Sheet,” said Tim Lee, Chief Commercial Officer of Invivyd. “Today’s announcement of a new exploratory analysis from the CANOPY Phase 3 clinical trial showing that PEMGARDA provided substantial protection from symptomatic COVID-19 versus placebo in immunocompetent participants through month twelve, absent additional dosing and with minimal drug concentration, only amplifies our confidence about re-accelerating our product growth in COVID-19 pre-exposure prophylaxis (PreP) in the appropriate immunocompromised population. It also reflects our potential to deliver meaningful protection to certain immunocompromised adults and adolescents who may not mount a strong enough immune response to vaccines alone. This population must not be forgotten.” “We continue to feel confident about the demand for PEMGARDA and our ability to return to strong product growth despite the unnecessary headwinds inflicted by the Agency,” said Marc Elia, Chairman of the Invivyd Board of Directors. “While we are engaged in discussions with key leaders at the FDA, it is clear that we must also focus on what matters most: the vulnerable populations who deserve more protection against this devastating virus, especially as we enter the winter season where many individuals hope to gather with friends and family. Our confidence in the ultimate commercial potential of PEMGARDA is unchanged.” About InvivydInvivyd, Inc. (Nasdaq: IVVD) is a biopharmaceutical company devoted to delivering protection from serious viral infectious diseases, beginning with SARS-CoV-2. The company’s proprietary INVYMAB™ platform approach combines state-of-the-art viral surveillance and predictive modeling with advanced antibody engineering. INVYMAB is designed to facilitate the rapid, serial generation of new monoclonal antibodies (mAbs) to address evolving viral threats. In March 2024, Invivyd received emergency use authorization (EUA) from the U.S. FDA for its first mAb in a planned series of innovative antibody candidates. Visit https://invivyd.com/ to learn more. About PEMGARDA PEMGARDA™ (pemivibart) is a half-life extended investigational monoclonal antibody (mAb). PEMGARDA was engineered from adintrevimab, Invivyd’s investigational mAb that has a robust safety data package and provided evidence of clinical efficacy in a global Phase 2/3 clinical trial for the prevention and treatment of COVID-19. PEMGARDA has demonstrated in vitro neutralizing activity against major SARS-CoV-2 variants, including JN.1. PEMGARDA targets the SARS-CoV-2 spike protein receptor binding domain (RBD), thereby inhibiting virus attachment to the human ACE2 receptor on host cells. PEMGARDA (pemivibart) injection (4500 mg), for intravenous use is an investigational mAb that has not been approved, but has been authorized for emergency use by the U.S. FDA under an EUA for the pre-exposure prophylaxis (prevention) of COVID-19 in adults and adolescents (12 years of age and older weighing at least 40 kg) who have moderate-to-severe immune compromise due to certain medical conditions or receipt of certain immunosuppressive medications or treatments and are unlikely to mount an adequate immune response to COVID-19 vaccination. Recipients should not be currently infected with or have had a known recent exposure to an individual infected with SARS-CoV-2. PEMGARDA is not authorized for use for treatment of COVID-19 or post-exposure prophylaxis of COVID-19. Pre-exposure prophylaxis with PEMGARDA is not a substitute for vaccination in individuals for whom COVID-19 vaccination is recommended. Individuals for whom COVID-19 vaccination is recommended, including individuals with moderate-to-severe immune compromise who may derive benefit from COVID-19 vaccinations, should receive COVID-19 vaccination. In individuals who have recently received a COVID-19 vaccine, PEMGARDA should be administered at least 2 weeks after vaccination. Anaphylaxis has been observed with PEMGARDA and the PEMGARDA Fact Sheet for Healthcare Providers includes a boxed warning for anaphylaxis. The most common adverse events (all grades, incidence ≥2%) observed in participants who have moderate-to-severe immune compromise treated with PEMGARDA included systemic and local infusion-related or hypersensitivity reactions, upper respiratory tract infection, viral infection, influenza-like illness, fatigue, headache, and nausea. For additional information, please see the PEMGARDA full product Fact Sheet for Healthcare Providers, including important safety information and boxed warning. To support the EUA for PEMGARDA, an immunobridging approach was used to determine if PEMGARDA may be effective for pre-exposure prophylaxis of COVID-19. Immunobridging is based on the serum virus neutralizing titer-efficacy relationships identified with other neutralizing human mAbs against SARS-CoV-2. This includes adintrevimab, the parent mAb of pemivibart, and other mAbs that were previously authorized for EUA. There are limitations of the data supporting the benefits of PEMGARDA. Evidence of clinical efficacy for other neutralizing human mAbs against SARS-CoV-2 was based on different populations and SARS-CoV-2 variants that are no longer circulating. Further, the variability associated with cell-based EC50 value determinations, along with limitations related to pharmacokinetic data and efficacy estimates for the mAbs in prior clinical trials, impact the ability to precisely estimate protective titer ranges. Additionally, certain SARS-CoV-2 viral variants may emerge that have substantially reduced susceptibility to PEMGARDA, and PEMGARDA may not be effective at preventing COVID-19 caused by these SARS-CoV-2 viral variants. The emergency use of PEMGARDA is only authorized for the duration of the declaration that circumstances exist justifying the authorization of the emergency use of drugs and biological products during the COVID-19 pandemic under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or authorization revoked sooner. PEMGARDA is authorized for use only when the combined national frequency of variants with substantially reduced susceptibility to PEMGARDA is less than or equal to 90%, based on available information including variant susceptibility to PEMGARDA and national variant frequencies. Cautionary Note Regarding Forward Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as “anticipates,” “believes,” “could,” “expects,” “estimates,” “intends,” “potential,” “preliminary,” “projects,” and “future” or similar expressions (as well as other words or expressions referencing future events, conditions or circumstances) are intended to identify forward-looking statements. Forward-looking statements include statements concerning, among other things, the company’s preliminary third quarter 2024 results; the company’s expectation regarding its cash and cash equivalents balance at the end of 2024; the company’s aim for near-term profitability, expectations regarding the commercialization of PEMGARDA, and plans for operational efficiency improvements; the company’s belief that its existing cash and cash equivalents, and anticipated growth of net product revenue and various operational efficiencies, will be sufficient to fund operations through profitability; the company’s expectations regarding its interactions with the FDA; the company’s ongoing research and clinical development activities, as well as future potential research and clinical development efforts; the company’s expectations regarding potential PEMGARDA product growth in the appropriate immunocompromised population, and the company’s potential to deliver meaningful protection to certain immunocompromised adults and adolescents who may not mount a strong enough immune response to vaccines alone; the company’s belief in the ultimate commercial potential of PEMGARDA; the potential of PEMGARDA as a mAb for pre-exposure prophylaxis (prevention) of COVID-19 in certain adults and adolescents who have moderate-to-severe immune compromise; the company’s devotion to delivering protection from serious viral infectious diseases, beginning with SARS-CoV-2; the design of the company’s INVYMAB platform approach to facilitate the rapid, serial generation of new mAbs to address evolving viral threats; the company’s business strategies and objectives; and other statements that are not historical fact. The company may not actually achieve the plans, intentions or expectations disclosed in the company’s forward-looking statements and you should not place undue reliance on the company’s forward-looking statements. These forward-looking statements involve risks and uncertainties that could cause the company’s actual results to differ materially from the results described in or implied by the forward-looking statements, including, without limitation: uncertainties regarding the company’s expectations, projections and estimates regarding future costs and expenses, future revenue, capital requirements, and the availability of and the need for additional financing; whether the company’s cash and cash equivalents are sufficient to support its operating plan for as long as anticipated; uncertainties regarding market acceptance, payor coverage or future sales and revenue generated by PEMGARDA; uncertainties regarding the potential advantages from the company’s planned operational efficiency improvements; the timing, progress and results of the company’s discovery, preclinical and clinical development activities; the risk that results of nonclinical studies or clinical trials may not be predictive of future results, and interim data are subject to further analysis; unexpected safety or efficacy data observed during preclinical studies or clinical trials; the predictability of clinical success of the company’s product candidates based on neutralizing activity in nonclinical studies; potential variability in neutralizing activity of product candidates tested in different assays, such as pseudovirus assays and authentic assays; the company’s reliance on third parties with respect to virus assay creation and product candidate testing and with respect to its clinical trials; variability of results in models used to predict activity against SARS-CoV-2 variants; whether pemivibart or any other product candidate is able to demonstrate and sustain neutralizing activity against major SARS-CoV-2 variants, particularly in the face of viral evolution; how long the EUA granted by the FDA for PEMGARDA will remain in effect and whether the EUA is revised or revoked by the FDA; the company’s ability to build and maintain sales, marketing and distribution capabilities to successfully commercialize PEMGARDA; uncertainties related to the regulatory authorization or approval process, and available development and regulatory pathways for authorization or approval of the company’s product candidates; the ability to maintain a continued acceptable safety, tolerability and efficacy profile of any product candidate following regulatory authorization or approval; changes in the regulatory environment; changes in expected or existing competition; the complexities of manufacturing mAb therapies; the company’s ability to leverage its INVYMAB platform approach to facilitate the rapid, serial generation of new mAbs to address evolving viral threats; any legal proceedings or investigations relating to the company; any change in the preliminary estimates of the company’s third quarter 2024 results upon completion of the company’s financial closing procedures, and finalization of the unaudited interim condensed consolidated financial statements; the company’s ability to continue as a going concern; and whether the company has adequate funding to meet future operating expenses and capital expenditure requirements. Other factors that may cause the company’s actual results to differ materially from those expressed or implied in the forward-looking statements in this press release are described under the heading “Risk Factors” in the company’s Annual Report on Form 10-K for the year ended December 31, 2023 and the company’s Quarterly Report on Form 10-Q for the quarter ended June 30, 2024, each filed with the Securities and Exchange Commission (SEC), and in the company’s other filings with the SEC, and in its future reports to be filed with the SEC and available at www.sec.gov. Forward-looking statements contained in this press release are made as of this date, and Invivyd undertakes no duty to update such information whether as a result of new information, future events or otherwise, except as required under applicable law. This press release contains hyperlinks to information that is not deemed to be incorporated by reference in this press release. Contacts:Media Relations(781) 208-1747media@invivyd.com Investor Relations(781) 208-1747investors@invivyd.com

Following strong protection (84% relative risk reduction versus placebo) demonstrated through month 6 with pemivibart, CANOPY clinical trial participants were followed for an additional six-month period (months 7-12), with no additional doses, to assess safety and efficacy as drug concentrations declined over timeIn the six-month off-drug follow-up period (months 7-12), substantially reduced concentrations of PEMGARDA reduced the risk of symptomatic COVID-19 by 64% compared to placebo in an immunocompetent adult population, for total twelve-month risk reduction of 76% following two initial pemivibart doses (nominal p <0.0001)Months 7-12 follow-up period included the U.S. summer KP.3 and KP.3.1.1 dominant wave, per CDC surveillanceNo new safety observations occurred months 7-12Across all 12 months of the CANOPY clinical trial, Cohort B placebo arm participants experienced an 18% rate of PCR-confirmed symptomatic COVID-19, excluding reinfections, indicative of continued, widespread, uncontrolled virus transmission in the U.S. WALTHAM, Mass., Oct. 29, 2024 (GLOBE NEWSWIRE) -- Invivyd, Inc. (Nasdaq: IVVD), a biopharmaceutical company devoted to delivering protection from serious viral infectious diseases, today announced positive 12-month exploratory clinical efficacy data from the company’s ongoing CANOPY Phase 3 clinical trial of pemivibart, a half-life extended investigational monoclonal antibody (mAb), for the pre-exposure prophylaxis (PrEP) of COVID-19. The exploratory clinical efficacy data from follow-up months 7-12 in CANOPY Cohort B, a placebo-controlled cohort of immunocompetent individuals at risk for symptomatic COVID-19 disease due to regular unmasked face-to-face indoor interactions, showed a relative risk reduction (RRR) of 64% in the pemivibart arm compared to placebo in the likelihood of trial participants contracting confirmed symptomatic COVID-19. Continued protection over this follow-up period, absent continued dosing, was a prespecified exploratory endpoint and, added to initial 180-day clinical efficacy observations, generated an overall 12-month protection rate of 76% following two doses of pemivibart (administered on Days 1 and 90), nominally significant at <0.0001. Clinical efficacy data are displayed in the table below. Placebo Arm Pemivibart Arm Nominal SubjectsCOVID-19 CasesAttack Rate SubjectsCOVID-19 CasesAttack Rate RRR*** p valueOn-drug Period 0-6 months*1601911.9% 31761.9% 84% <0.0001 Long-term Follow-up Period Following Cessation of Dosing 0-9 months1602012.5% 31782.5% 80% <0.00010-12 months1602918.1% 317144.4% 76% <0.00017-12 months**133107.5% 28982.8% 64% 0.0285 * Period includes US JN.1 wave ** Period includes US KP.3 and KP.3.1.1 wave *** Relative risk reduction excludes deaths unrelated to COVID-19 Pemivibart has an estimated in vivo half-life of approximately 45 days, and pemivibart IC50 values against circulating viruses are tested routinely, allowing for the calculation of the average serum virus neutralizing antibody (sVNA) titer over time, the measure of how much antiviral activity pemivibart confers to treated participants. Previously published literature1 indicates likely robust protection from symptomatic COVID-19 at sVNA titer levels well below the levels delivered by the authorized PEMGARDA (pemivibart) dosing regimen. In this updated CANOPY exploratory analysis, the 64% RRR (excluding deaths unrelated to COVID-19) that residual circulating pemivibart delivered versus placebo in months 7 through 12 occurred at calculated average titers of only approximately 7% of the average titers during the active dosing period. This was attributable to the expected decline in pemivibart serum concentrations following cessation of dosing, along with modest change to assessed pemivibart IC50 in the transition from the JN.1 dominant virus to the KP lineage dominant circulating viruses. “Today’s data underline our high confidence in the clinical benefit that mAbs like pemivibart can confer even at very low serum concentrations and associated antiviral titers,” commented Robert Allen, Ph.D., Chief Scientific Officer of Invivyd. “Engineered mAbs are designed to confer a step change in protection compared to the protection possible from native immune response to infection and vaccination boosts2. We believe that the clinical risk reduction generated at such low titers in this CANOPY clinical trial follow-up, which builds on now longstanding published data, implies that the modest changes (less than ~10 fold change) in pemivibart IC50 observed against emerging variants are of questionable significance.” “Our company’s goal is to provide high quality options for protection from SARS-CoV-2. Today’s exploratory clinical efficacy data update from our Phase 3 trial is critical, as it adds contemporary context to longstanding data showing strong protection from COVID-19 disease at relatively low sVNA titers with mAbs,” commented Marc Elia, Chairman of the Invivyd Board of Directors. “These consistent observations now span years and countless virus lineages and mutations, and, in toto, suggest that Invivyd, pending alignment with regulators, can develop and commercialize low-dose, high potency, long-acting mAbs that can have the same route of administration and system-friendliness as COVID-19 vaccine boosts, but with the goal of higher, more consistent, and longer-acting protection. Our current pipeline molecule, VYD2311, is currently in Phase 1 testing including an intramuscular (IM) administration in anticipation of executing against this exact goal.” The safety profile of pemivibart over the 12-month study period was reassuring with no new trends or safety signals observed for treatment-emergent adverse events (TEAEs) or infusion-related reactions or hypersensitivity reactions since the disclosure of the 6-month CANOPY data. Over the 12 months, in Cohort A, an open-label single-arm cohort, the most common TEAEs (>5%) included upper respiratory tract infection (URTI) (10.1%), viral infection (8.8%), urinary tract infection (UTI) (5.6%), and influenza-like illness (5.2%). As previously disclosed, anaphylaxis was observed in 4 participants (0.6%) in Cohort A – 2 participants during the first infusion and 2 participants during the second infusion; two reactions were life-threatening, and all led to permanent discontinuation of pemivibart. Infusion-related reactions and hypersensitivity reactions within 24 hours of dosing pemivibart were observed in 8.2% and 3.9% of participants in Cohort A after the initial dose and redose, respectively, and were generally mild to moderate in severity. Over the 12 months, in Cohort B, the most common TEAEs in the pemivibart arm included viral infection (8.5%), URTI (8.5%), and influenza-like illness (5.7%), with higher percentages in the placebo arm. No participants developed anaphylaxis. Infusion-related reactions and hypersensitivity reactions within 24 hours of dosing pemivibart were observed in 1.3% and 2.5% of participants in Cohort B after initial dose and redose, respectively, and were all mild or moderate in severity. Serious Adverse Events (SAEs) occurring in >1 Cohort A participant included pneumonia (n=3), anaphylactic reaction (n=2), hypotension (n=2), pyelonephritis (n=2), syncope (n=2), and UTI (n=2). No SAEs occurred in >1 participant in either treatment arm in Cohort B. These data support that the safety profile for pemivibart remains consistent with the PEMGARDA Fact Sheet for Healthcare Providers and the known safety profile of other SARS-CoV-2-directed mAbs. CANOPY Cohort B exploratory clinical efficacy results from months 7-12 provide data from reduced concentration of pemivibart and reduced sVNA titers in a contemporary immunocompetent population that had acquired prior immune exposure from either vaccination or natural infection and overlapped with the height of the Summer 2024 KP.3 and KP3.1.1 wave that saw a surge in COVID-19 cases nationwide in the U.S. By contrast, ancestral clinical trials of prior COVID-19 PrEP candidate mAbs were performed in populations naïve to vaccination or infection. The months’ 7-12 CANOPY Cohort B clinical efficacy exploratory data announced today complement the 180-day clinical efficacy exploratory data demonstrating potential signals of clinical protection from symptomatic COVID-19 shared previously. The company expects the full data set to be provided in an upcoming scientific publication. About InvivydInvivyd, Inc. (Nasdaq: IVVD) is a biopharmaceutical company devoted to delivering protection from serious viral infectious diseases, beginning with SARS-CoV-2. The company’s proprietary INVYMAB™ platform approach combines state-of-the-art viral surveillance and predictive modeling with advanced antibody engineering. INVYMAB is designed to facilitate the rapid, serial generation of new monoclonal antibodies (mAbs) to address evolving viral threats. In March 2024, Invivyd received emergency use authorization (EUA) from the U.S. FDA for its first mAb in a planned series of innovative antibody candidates. Visit https://invivyd.com/ to learn more. About PEMGARDA PEMGARDA™ (pemivibart) is a half-life extended investigational monoclonal antibody (mAb). PEMGARDA was engineered from adintrevimab, Invivyd’s investigational mAb that has a robust safety data package and provided evidence of clinical efficacy in a global Phase 2/3 clinical trial for the prevention and treatment of COVID-19. PEMGARDA has demonstrated in vitro neutralizing activity against major SARS-CoV-2 variants, including JN.1. PEMGARDA targets the SARS-CoV-2 spike protein receptor binding domain (RBD), thereby inhibiting virus attachment to the human ACE2 receptor on host cells. PEMGARDA (pemivibart) injection (4500 mg), for intravenous use is an investigational mAb that has not been approved, but has been authorized for emergency use by the U.S. FDA under an EUA for the pre-exposure prophylaxis (prevention) of COVID-19 in adults and adolescents (12 years of age and older weighing at least 40 kg) who have moderate-to-severe immune compromise due to certain medical conditions or receipt of certain immunosuppressive medications or treatments and are unlikely to mount an adequate immune response to COVID-19 vaccination. Recipients should not be currently infected with or have had a known recent exposure to an individual infected with SARS-CoV-2. PEMGARDA is not authorized for use for treatment of COVID-19 or post-exposure prophylaxis of COVID-19. Pre-exposure prophylaxis with PEMGARDA is not a substitute for vaccination in individuals for whom COVID-19 vaccination is recommended.  Individuals for whom COVID-19 vaccination is recommended, including individuals with moderate-to-severe immune compromise who may derive benefit from COVID-19 vaccinations, should receive COVID-19 vaccination.  In individuals who have recently received a COVID-19 vaccine, PEMGARDA should be administered at least 2 weeks after vaccination. Anaphylaxis has been observed with PEMGARDA and the PEMGARDA Fact Sheet for Healthcare Providers includes a boxed warning for anaphylaxis. The most common adverse events (all grades, incidence ≥2%) observed in participants who have moderate-to-severe immune compromise treated with PEMGARDA included systemic and local infusion-related or hypersensitivity reactions, upper respiratory tract infection, viral infection, influenza-like illness, fatigue, headache, and nausea. For additional information, please see the PEMGARDA full product Fact Sheet for Healthcare Providers, including important safety information and boxed warning. To support the EUA for PEMGARDA, an immunobridging approach was used to determine if PEMGARDA may be effective for pre-exposure prophylaxis of COVID-19. Immunobridging is based on the serum virus neutralizing titer-efficacy relationships identified with other neutralizing human mAbs against SARS-CoV-2. This includes adintrevimab, the parent mAb of pemivibart, and other mAbs that were previously authorized for EUA. There are limitations of the data supporting the benefits of PEMGARDA. Evidence of clinical efficacy for other neutralizing human mAbs against SARS-CoV-2 was based on different populations and SARS-CoV-2 variants that are no longer circulating. Further, the variability associated with cell-based EC50 value determinations, along with limitations related to pharmacokinetic data and efficacy estimates for the mAbs in prior clinical trials, impact the ability to precisely estimate protective titer ranges. Additionally, certain SARS-CoV-2 viral variants may emerge that have substantially reduced susceptibility to PEMGARDA, and PEMGARDA may not be effective at preventing COVID-19 caused by these SARS-CoV-2 viral variants. The emergency use of PEMGARDA is only authorized for the duration of the declaration that circumstances exist justifying the authorization of the emergency use of drugs and biological products during the COVID-19 pandemic under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or authorization revoked sooner. PEMGARDA is authorized for use only when the combined national frequency of variants with substantially reduced susceptibility to PEMGARDA is less than or equal to 90%, based on available information including variant susceptibility to PEMGARDA and national variant frequencies. About CANOPY The ongoing CANOPY Phase 3 clinical trial is designed to evaluate the safety and tolerability of pemivibart and to assess immunobridging from pemivibart to certain historical data from the company’s previous Phase 2/3 clinical trial of adintrevimab (ADG20) for the prevention of symptomatic COVID-19 (EVADE). Additionally, there are pre-specified exploratory endpoints through three, six and twelve months to evaluate clinical efficacy of pemivibart compared to placebo in the prevention of RT-PCR-confirmed symptomatic COVID-19. The latest analysis from the Phase 3 CANOPY clinical trial includes 365-day data. The CANOPY clinical trial enrolled participants in two cohorts: Cohort A is a single-arm, open-label trial in adults who have moderate-to-severe immune compromise including complex underlying medical conditions. Cohort B is a randomized, placebo-controlled cohort that enrolled adults without moderate-to-severe immune compromise who are at risk of acquiring COVID-19 due to regular unmasked face-to-face interactions in indoor settings. Cautionary Note Regarding Forward Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as “anticipates,” “believes,” “could,” “expects,” “estimates,” “intends,” “potential,” “projects,” and “future” or similar expressions (as well as other words or expressions referencing future events, conditions or circumstances) are intended to identify forward-looking statements. Forward-looking statements include statements concerning, among other things, the company’s ongoing research and clinical development activities, as well as future potential research and clinical development efforts; the potential clinical benefit that mAbs like PEMGARDA (pemivibart) can confer, even at very low serum concentrations and associated antiviral titers; the possibility for Invivyd to develop and commercialize low-dose, high potency, long-acting mAbs with the same route of administration and system-friendliness as COVID-19 vaccine boosts, but higher, more consistent, and longer-acting protection; the company’s expectation to provide a full data set from the CANOPY clinical trial in an upcoming scientific publication; the potential of PEMGARDA as a mAb for pre-exposure prophylaxis (prevention) of COVID-19 in certain adults and adolescents who have moderate-to-severe immune compromise; the company’s devotion to delivering protection from serious viral infectious diseases, beginning with SARS-CoV-2; the design of the company’s INVYMAB platform approach to facilitate the rapid, serial generation of new mAbs to address evolving viral threats; the company’s plans for a series of innovative antibody candidates and its goals for pipeline molecule VYD2311; and other statements that are not historical fact. The company may not actually achieve the plans, intentions or expectations disclosed in the company’s forward-looking statements and you should not place undue reliance on the company’s forward-looking statements. These forward-looking statements involve risks and uncertainties that could cause the company’s actual results to differ materially from the results described in or implied by the forward-looking statements, including, without limitation: the timing, progress and results of the company’s discovery, preclinical and clinical development activities; the risk that results of nonclinical studies or clinical trials may not be predictive of future results, and interim data are subject to further analysis; unexpected safety or efficacy data observed during preclinical studies or clinical trials; the predictability of clinical success of the company’s product candidates based on neutralizing activity in nonclinical studies; potential variability in neutralizing activity of product candidates tested in different assays, such as pseudovirus assays and authentic assays; the company’s reliance on third parties with respect to virus assay creation and product candidate testing and with respect to its clinical trials; variability of results in models used to predict activity against SARS-CoV-2 variants; whether pemivibart, VYD2311, or any other product candidate is able to demonstrate and sustain neutralizing activity against major SARS-CoV-2 variants, particularly in the face of viral evolution; how long the EUA granted by the FDA for PEMGARDA will remain in effect and whether the EUA is revised or revoked by the FDA; the company’s ability to build and maintain sales, marketing and distribution capabilities to successfully commercialize PEMGARDA; uncertainties related to the regulatory authorization or approval process, and available development and regulatory pathways for authorization or approval of the company’s product candidates; the ability to maintain a continued acceptable safety, tolerability and efficacy profile of any product candidate following regulatory authorization or approval; changes in the regulatory environment; changes in expected or existing competition; the complexities of manufacturing mAb therapies; the company’s ability to leverage its INVYMAB platform approach to facilitate the rapid, serial generation of new mAbs to address evolving viral threats; any legal proceedings or investigations relating to the company; the company’s ability to continue as a going concern; and whether the company has adequate funding to meet future operating expenses and capital expenditure requirements. Other factors that may cause the company’s actual results to differ materially from those expressed or implied in the forward-looking statements in this press release are described under the heading “Risk Factors” in the company’s Annual Report on Form 10-K for the year ended December 31, 2023 and the company’s Quarterly Report on Form 10-Q for the quarter ended June 30, 2024, each filed with the Securities and Exchange Commission (SEC), and in the company’s other filings with the SEC, and in its future reports to be filed with the SEC and available at www.sec.gov. Forward-looking statements contained in this press release are made as of this date, and Invivyd undertakes no duty to update such information whether as a result of new information, future events or otherwise, except as required under applicable law. This press release contains hyperlinks to information that is not deemed to be incorporated by reference in this press release. Contacts:Media Relations(781) 208-1747media@invivyd.com Investor Relations(781) 208-1747investors@invivyd.com 1Schmidt, et al. Antibody-Mediated Protection Against Symptomatic COVID-19 Can Be Achieved at Low Serum Neutralizing Titers. Sci. Transl. Med. 15, eadg2783 (2023); Stadler, et al. Monoclonal Antibody Levels and Protection From COVID-19, Nat. Commun., 14:4545 (2023) 2 https://www.cdc.gov/acip/downloads/slides-2024-06-26-28/03-COVID-Link-Gelles-508.pdf

摘要：改进的疫苗接种需要更好地传递抗原并激活自然免疫反应。在这里，我们报告了一种脂质纳米颗粒系统，该系统具有携带包括mRNA和蛋白质在内的抗原的能力，通过表面装饰有刺突蛋白形成病毒样结构，展示了对SARS-CoV-2变体的应用。该策略使用Omicron BA.1上的S1蛋白在表面传递XBB.1的S1蛋白mRNA。病毒样颗粒通过表面S1蛋白与ACE2或DC-SIGN受体的相互作用，能够在人类呼吸道上皮细胞和巨噬细胞中特异性增强mRNA的表达。通过相同的受体结合，展示了巨噬细胞和树突状细胞的激活。与单独的mRNA和蛋白质疫苗相比，蛋白质和mRNA的结合在BALB/c小鼠中增加了抗体反应。我们对这种强大免疫机制的探索表明，它可能涉及交叉呈递给从激活的先天免疫信号到适应性免疫信号的不同亚群的树突状细胞。 1.引言 自2019年底由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引起的2019冠状病毒病（COVID-19）大流行开始以来，已经开发并上市了多种针对该疾病的疫苗。然而，这些疫苗的应用并未完全限制大流行的传播，流行病学调查中发现的抗体水平显示出有限的持续时间和对病毒变种的低效性。这些发现表明，SARS-CoV-2与宿主免疫系统之间的相互作用在很大程度上尚不清楚，需要开发新型疫苗。幸运的是，基于脂质纳米递送系统的成功开发，mRNA疫苗取得了突破性进展，提供了新的技术可能性。通过人工脂质膜与细胞质膜的融合，将编码抗原的mRNA通过脂质纳米颗粒（LNP）传递到细胞内，可在细胞内有效表达病毒刺突蛋白（S蛋白）抗原，并诱导随后的S抗原呈递，通过树突状细胞（DCs）和巨噬细胞激活先天/适应性免疫。 根据病毒免疫学研究，在细胞内病毒复制过程中产生的结构和/或非结构抗原可以被先天免疫系统的细胞模式识别受体在宿主免疫反应的抗原信号刺激的首次事件中识别。这意味着mRNA具有通过这种自然免疫途径引发免疫反应的优势；其策略在某种程度上类似于膜状病毒SARS-CoV-2的感染机制，尽管活病毒通常利用病毒膜中的S蛋白与细胞膜中的ACE2受体特异性结合以实现病毒基因组的内化。 因此，当脂质递送颗粒在其表面装载S蛋白时，这些脂质颗粒可能具有类似病毒的结构（VLS），通过受体靶向细胞。特别是，SARS-CoV-2的S蛋白被发现与DC和巨噬细胞表面表达的ACE2或DC-SIGN受体相互作用。这些发现导致合乎逻辑的推断，如果能够构建所提出的VLS系统，它将不仅与注射局部组织的肌肉细胞或上皮细胞相互作用，还与携带ACE2受体或DC-SIGN的先天免疫细胞（例如DCs和巨噬细胞）。先前报道了一种可吸入的纳米疫苗，其具有仿生冠状病毒结构，模拟病毒遗传物质由脂质体包裹，表面装载有S蛋白的受体结合域（RBDs）作为“刺突”以模拟病毒结构，表明VLS的可能性。因此，本研究的目的是设计一种VLS颗粒来包裹mRNA分子并装载蛋白质，并研究其免疫学特性，特别是其通过表面S1蛋白特异性结合到DCs和巨噬细胞上的DC-SIGN来在小鼠模型中引发免疫反应的能力。所获得的数据表明，通过VLS结构将mRNA特异性地传递到DCs的这种策略与常规mRNA疫苗相比具有非常重要的意义。 2.结果 2.1.VLS的特性描述 SARS-CoV-2 mRNA疫苗的早期开发中一个重要的技术基础是通过静电吸引和氢键将mRNA分子与阳离子脂质结合，这导致了mRNA分子被LNPs包裹。在我们的VLS设计中，需要一个脂质载体来包裹mRNA，并装载表面蛋白，这可能需要更大的阳离子脂质电荷容量以在离子环境中进行电荷复合。为了使脂质颗粒同时携带mRNA和蛋白的能力更强，我们的设计包括了两种阳离子脂质分子，((2-(2-羟乙氧基)乙基)氮杂二基)双(己烷-6,1-二基)双(2-己基癸酸酯)和1,2-二油酰-3-三甲基铵丙烷；一种中心磷脂，1,2-二油酰-磷脂酰胆碱；以及一种聚聚乙二醇(PEG)分子，甲氧基聚(乙二醇)-N-十四烷基十四酰胺-1-2k。通过微流控工艺生产了这种脂质组成和编码SARS-CoV-2 XBB.1株S1蛋白的mRNA的LNP（补充图1a）。这些特征粒子的结构参数（扩展数据表1）和与脂质定量相关的识别mRNA分子（补充图1a）表明了mRNA疫苗的典型特征。此外，这种LNP能够以剂量依赖性的方式将其包裹的mRNA传递到293细胞中（图1a,b）。此外，表达并纯化的S1重组蛋白（补充图1a）被装载在包裹mRNA的脂质颗粒上，以构建具有与mRNA疫苗相似结构特征的VLSs（扩展数据表1）。我们的电子显微镜观察表明装载过程是不可逆的（补充图1b），并且这种VLS的形态特征显示，与单独包裹mRNA的LNP相比，粒子表面有一些凸起，存在差异（图1c）。添加S抗体可以通过抗体桥使颗粒聚集（图1c）。通过免疫沉淀法对VLSs进行结构分析，使用特定的S抗体表明，沉淀的VLS颗粒含有mRNA分子和S1蛋白（图1d）。这些数据支持在我们的设计模型中使用VLS颗粒。 图1 | VLS和LNP的特征。a，荧光显微镜显示LNP、包裹GFP mRNA的LNP和VLS中GFP表达显著。比例尺，250微米。b，通过西方印迹分析检测293T细胞转染LNPs或VLSs 48小时后的S1蛋白。左图：与LNPs相比，293T细胞转染EGFP或S1 mRNA后的S1蛋白表达；右图：转染不同剂量S1 mRNA的VLSs后，293T细胞中S1蛋白的表达。GAPDH作为蛋白装载对照。西方印迹上信号的强度（条带上方）通过ImageJ（版本1.8.0.112）进行密度分析定量。c，LNPs、包裹S1 mRNA的LNPs、VLS和用特定抗S1抗体处理的VLSs的电子显微镜图像。比例尺，200纳米。a-c中的实验均重复了两次。d，总样品、渗透和洗脱中的蛋白（S1或N）和mRNA含量。“IP-S1”表示样品在与特定抗S1抗体孵育过夜后进行了测试，“IP-N”表示样品与抗N抗体孵育。实验装载量相等，并通过西方印迹和SDS-PAGE银染（Input）鉴定。数据以三个独立实验的平均值±标准差表示。 2.2.VLSs使细胞转染并靶向ACE2/DC-SIGN分子 与mRNA疫苗的LNPs相比，当蛋白装载在表面时，VLSs提高了mRNA的传递能力。S1蛋白与ACE2或DC-SIGN分子的结合可能导致VLSs靶向局部组织中具有这两种受体的细胞。我们的研究表明，VLSs不仅能高效转染293细胞（图2a），而且还能增强16HBE细胞培养中的mRNA表达，这不仅显示了目标mRNA的拷贝数（图2b），而且还表达了蛋白质（图2a），并且在添加针对S或ACE2的抗体后呈现恢复（图2c,d）。THP-1细胞也是如此（图2a,b）。基于我们对大约16HBE细胞表达ACE2和THP-1细胞表达ACE2/DC-SIGN的识别数据（补充图2），两种细胞系中mRNA表达的增强可能是由于ACE2或DC-SIGN与S1蛋白的相互作用介导的。此外，用针对DC-SIGN的特定抗体处理THP-1细胞也干扰了VLS mRNA的表达（图2c,d）。这表明VLSs能够通过S1与ACE2和DC-SIGN受体的相互作用，将mRNA传递到人类巨噬细胞中，这与报告的数据一致。此外，表达DC-SIGN的小鼠DCs（JASWIIs）被发现与S1蛋白相互作用（补充图3a）。这些细胞与VLS包裹的EGF或S1 mRNA的转染导致了明显比单独转染LNP包裹的mRNA更大的mRNA表达，因为针对S或DC-SIGN的特定抗体的阻断消除了这种效应（图2c,d和补充图3c）。使用表达DC-SIGN的小鼠RAW264.7巨噬细胞的相同实验也提供了类似的结果（图2c和补充图3b）。基于先前确认的数据，显示人类DCs和巨噬细胞由于表达ACE2/DC-SIGN而被SARS-CoV-2感染，我们的结果表明，VLS与ACE2或DC-SIGN受体的相互作用可能参与了先天免疫的激活。 图2 | VLS通过S1蛋白靶向ACE2/DC-SIGN分子增强细胞转染，实现mRNA传递。a，不同细胞中S1蛋白的表达通过西方印迹进行检测。c，使用不同细胞的受体或S1（N）蛋白的抗体阻断后，通过西方印迹检测VLSs中S1蛋白的表达。LNPs和包裹S1 mRNA的VLSs与293T细胞、16HBE细胞、THP-1细胞、DCs和RAW264.7细胞共孵育。a和c中的实验均重复了两次。b，通过qRT-PCR评估转染到不同细胞的mRNA拷贝数。d，使用细胞受体的抗体阻断或S1（N）蛋白的抗体阻断后，通过qRT-PCR评估转染到细胞的mRNA拷贝数。b、d中，n=3个独立样本，均值±标准差。黄色框架中的蓝色文本显示了不同抗体处理的样本或细胞。显著性差异是通过单因素方差分析（ANOVA）和Tukey的事后检验确定的。 2.3.VLSs与DCs和巨噬细胞的相互作用 先前的数据表明，LNPs对mRNA疫苗在细胞中诱导的强烈转录反应是由于LNPs引起的外源性损伤，被认为是mRNA疫苗的不良反应。我们研究了是否可以通过S1蛋白与受体的结合来降低我们VLS的活性。在我们的研究中，用VLSs处理的16HBE细胞中具有免疫激活作用的一些分子的表达倾向于增加（图3a），在THP-1细胞中检测到免疫调节因子的表达增加和炎症介质如肿瘤坏死因子（TNF）的表达减少（图3b）。此外，在小鼠DCs的JAWSII菌株中，包括TNF和GATA-3在内的分子表达倾向于减少，而干扰素（IFN）家族成员的表达倾向于增加（图3c）。在RAW264.7小鼠巨噬细胞中观察到免疫调节因子和炎症因子的转录概况轻微且平衡的上调（图3d）。有趣的是，上皮细胞中干扰素家族成员的上调表达与DCs和巨噬细胞中的相似（图3a,c）。这些结果表明VLSs在先天免疫中具有某种免疫激活作用，这与mRNA/LNPs不同，并支持我们的假设，即S1蛋白对VLS表面的影响不仅可以导致mRNA传递，还可以激活DC中的DC-SIGN信号通路。 图3 | LNPs和VLSs转染细胞的免疫基因转录。a-d，16HBE（a）、THP-1（b）、JASWII DC（c）和RAW264.7（d）细胞在转染只包裹S1 mRNA的LNPs或同时包裹S1 mRNA和S1蛋白的VLSs后24小时的免疫反应相关主要基因的表达谱。蓝色代表只包裹S1 mRNA的LNPs，黑色代表VLSs。数据以三个独立实验（n=3）的平均值±标准差表示。显著性差异通过单因素方差分析（ANOVA）和Tukey的事后检验确定。 2.4.用VLSs对小鼠进行免疫接种引发免疫信号传导的差异 我们对VLSs与免疫系统之间的相互作用的研究表明，无论VLS中包裹的mRNA编码GFP还是S1蛋白，它都在局部组织中动态表达（补充图4），类似于mRNA疫苗的报告。有趣的是，VLS组小鼠局部组织中S1抗原与DCs或巨噬细胞的共定位率大于mRNA或单独蛋白组小鼠（图4a和补充图5a,b）。在初次免疫后3-7天，VLS组小鼠脾脏中CD86+ DCs与CD83+ DCs的比率，这表明由Toll样受体（TLR）信号刺激的DCs的成熟度，大于mRNA或蛋白组小鼠（图4b）。 脾脏中特定CD4+与CD8+记忆T细胞的比率在增强型VLS组以及接受用VLS预处理的DCs组中也大于其他两组（图4c,d），这表明VLSs通过S1蛋白特异性结合到DC-SIGN受体与小鼠DCs的相互作用，随后mRNA的有效表达增强了DCs的活化效率，随后激活T细胞是显著的。进一步的抗体测量显示，在注射后第21天，接受用VLS预处理的DCs的小鼠的抗体滴度大于接受用mRNA或蛋白疫苗预处理的DCs的小鼠（图4e）。在免疫后24至48小时，对接种实验VLS疫苗的小鼠局部组织中各种重要免疫分子的转录水平进行后续分析，发现与单独用mRNA或蛋白免疫的小鼠相比，与DCs和巨噬细胞活化直接相关的一些功能分子的表达增加，如CXCL10和MHCI（图4f）。CXCL10的千倍增加表明，由VLSs激活的DCs可以被分类为cDCs21。进一步对接种免疫的小鼠脾脏中收集的淋巴细胞在免疫后不同时间点进行的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes）同源转录组分析显示，与mRNA和蛋白组相比，VLS组显示出特征性的动态轮廓（图4g）。所有转录轮廓的变化表明，接种VLSs和mRNA或蛋白的个体宿主免疫反应存在差异。 图4 | VLSs在小鼠中诱导了先天免疫反应。a，代表性共定位率显示，与肌肉注射VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs的小鼠相比，在24小时和48小时后S1表达与DCs/巨噬细胞在肌肉中的情况。b，小鼠在免疫后3天和7天，脾脏中CD86+与CD83+ DCs的比率，这些小鼠接受了VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs的免疫。c，d，在接受加强和DC处理的输血VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs后，特异性记忆T细胞（c）和活化T细胞（d）中带有CD8标记分子的比率。e，在不同处理组DCs输血后第21天的小鼠抗体滴度。f，小鼠肌肉注射VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs后24小时和48小时，与炎症和先天免疫反应相关的主要基因表达谱。g，在初次免疫后7天、14天和28天以及加强免疫后21天，与免疫调节的激活或分化、细胞过程、信号转导和炎症相关的主要基因表达谱的淋巴结。颜色条代表与LNP组数值相比的log2（倍数变化）。颜色越红，基因转录水平上调越明显。组织中炎症细胞因子的相对表达水平通过比较Ct（ΔΔCt）法归一化至空白对照组（LNP）的水平。数据以三个独立实验（n=3）的平均值±标准差表示。显著性差异通过单因素方差分析（ANOVA）和Tukey的事后检验确定。 2.5.VLSs在小鼠中引发更强的免疫反应 基于对我们的VLS疫苗和当前在人群中使用的mRNA疫苗（III期临床试验）（18岁以上）（补充图6）的分析差异，我们的免疫学数据表明，与mRNA或蛋白疫苗中mRNA和蛋白含量相等的VLSs在接种的BALB/c小鼠中引发强大的免疫反应，接种后对Omicron株S抗原的结合抗体水平远高于单独接种mRNA或蛋白的小鼠；高水平维持了八个月以上（图5a），并且小鼠对武汉、Beta和Delta株的S抗原表现出明显的反应性，水平略低（图5b）。使用假病毒和真病毒同步检测变异株的中和抗体也显示了类似的结果（图5c），其中对变异株的中和抗体达到了高于1:103的水平，这在先前对动物和人类的免疫保护研究中被确认为有效。对武汉、Omicron和XBB.1株的RBD的抗体水平在VLS组中大于其他两组（图5d）。与其他两组相比，接种VLS后，尤其是加强免疫后，VLS组小鼠在上呼吸道和血清中分泌的IgA水平增加（图5e）。此外，比较VLS组和mRNA组小鼠淋巴结中活化B细胞的比率表明，VLS组中活化B细胞的比率大于mRNA组（图5f）。重要的是，针对IFNγ和白介素-4（IL-4）的ELISpot检测显示，在免疫后第90天，VLS组的T细胞反应比mRNA和蛋白组更强烈（图5g）。由于观察到IL-4反应的明显加强，同时对这些小鼠的血清中的多种细胞因子进行了检测，结果表明VLS组的大多数细胞因子水平低于mRNA或蛋白组（图5h）。所有数据表明，VLSs引发的免疫反应应与基于VLSs对先天免疫的已知激活效应的mRNAs或蛋白质引发的免疫反应不同。 图5 | VLSs在小鼠中诱导了适应性免疫反应。a,b, 由VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs在小鼠中诱导产生的针对Omicron（a）和野生型（WT）、Beta和Delta（b）株的SARS-CoV-2特异性抗体滴度。样本在初次注射后的第35、49、120、180和240天获取。c, 由VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs在小鼠中诱导产生的针对Omicron真实病毒株BA.5和假病毒的中和抗体（NAb）滴度。d, 由VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs在小鼠中诱导产生的针对Omicron株、XBB.1株和WT株RBD的IgG抗体滴度。e, 由VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs在小鼠中诱导产生的针对Omicron株刺突蛋白的IgA抗体滴度，在免疫后的第21和49天在小鼠BALF（支气管肺泡灌洗液）和血清中。f, 观察到的经VLS和mRNA疫苗处理的小鼠淋巴结中的活化B细胞。g, 在初次免疫后第49天，经肌肉注射VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs的小鼠的IFNγ和IL-4的ELISpot反应。橙色框架代表包裹5、10、20和40μg S1 mRNA的LNPs，从浅到深；蓝色边框代表5、10、20和30μg S1肽；蓝色边框中的橙色框架从浅到深代表不同剂量的RNA和肽的不同组合的LNPs。SFC，形成斑点的细胞。h, 加强免疫后第28天，用VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs免疫的小鼠血清中的细胞因子。所有测定值都通过减去空白小鼠血清中检测到的值来计算。数据以三个独立实验（n=3）的平均值±标准差表示。显著性差异通过单因素方差分析（ANOVA）和Tukey的事后检验确定。 2.6.VLSs在ACE+/+小鼠中引发的有效免疫保护 在我们的病毒挑战测试中，涉及ACE+/+小鼠和仓鼠（补充图7），结果表明VLSs完全保护了挑战的小鼠免受Omicron BA.5株、XBB.1和武汉株的侵害，因为没有观察到症状（扩展数据表2）。在感染期间，在小鼠的各个器官中检测到很少或没有病毒复制（图6a,b和补充图8-10），并且没有检测到组织病理学变化（扩展数据表3）。值得注意的是，在与VLS阴性小鼠相比，用XBB.1挑战的VLS免疫小鼠的主要器官中没有检测到活病毒（图6c）。此外，对BA.5挑战的小鼠的淋巴细胞在第3天进行了转录组分析。结果表明，与功能反应相关的差异表达基因与免疫系统在VLS组明显不同于其他两组（图6d）。B细胞、CD4+细胞和CD8+细胞的分类表明，更多的DEGs百分比集中在CD4+细胞和B细胞群体中（图6d）。这些基因主要是与淋巴细胞增殖相关的转录调节分子。结合ACE+/+小鼠VLS组中出色的免疫保护效果，这种独特的转录组轮廓表明，通过VLSs与DCs的稳健结合，通过系统激活DCs在VLS刺激下基于不同信号的整合效应引发的免疫学结果。 图6 | VLSs对C57-hACE+/+小鼠中Omicron BA.5和XBB.1株挑战的影响。a, 在Omicron BA.5株感染后，通过RT-qPCR测定了肌肉注射VLS、包裹S1肽的LNPs或包裹S1 mRNA的LNPs的hACE+/+小鼠在肺、大脑和脊髓中的病毒载量。b,c, 在Omicron XBB.1株感染后，通过RT-qPCR测定了肌肉注射VLS和LNPs的hACE+/+小鼠在大脑、肺、淋巴、气管和脊髓中的病毒载量（b），并通过病毒斑点形成测定了病毒滴度（c）。基线是根据未感染的阴性小鼠中检测到的值定义的。FFU，聚焦形成单位；dpi，感染后天数。d, 在Omicron BA.5株感染后3天，从小鼠肌肉注射VLSs、包裹S1肽的LNPs或包裹S1 mRNA的LNPs的淋巴结组织中分离的CD8+ T、CD4+ T和B细胞的激活或分化相关的显著差异表达基因。颜色条代表与LNP组相比的log2（倍数变化）。颜色越红，表示基因转录水平上调越明显。数据以三个独立实验（n=3）的平均值±标准差表示。显著性差异通过单因素方差分析（ANOVA）和Tukey的事后检验确定。 3.讨论 在过去几年的COVID-19大流行期间，mRNA疫苗为我们提供了新的疫苗研究技术和疫苗设计和开发的新思路。这种技术方法诱导了对传染因子的生物学模仿，并为下一代COVID-19疫苗的开发提供了新的可能性。基于这一进展，我们的工作建立了一个改进的脂质系统，扩大了抗原携带能力，包括mRNA和蛋白。这个系统依赖于由两种阳离子脂质、一种中心磷脂和一种聚-PEG分子组装成的专门脂质颗粒的结构，该结构由mRNA和病毒蛋白组成。这种脂质系统能够有效地包裹编码S1抗原的mRNA，并在其表面携带S1蛋白。此外，我们的结果揭示了表面S1蛋白能够与ACE2/DC-SIGN受体结合，促进mRNA进入细胞并增加抗原表达，除了通过LNP融合到细胞膜的表达之外。因此，我们进一步表明，VLS表面S1蛋白在引导mRNA/LNPs靶向DCs和巨噬细胞方面发挥重要作用，这导致免疫系统对抗原的摄取显著增加。对这些细胞的转录概况的进一步分析表明，VLS能够以平衡的方式激发多种免疫调节因子和炎症介质的表达的一般趋势与mRNA/LNPs不同，这表明了VLS作为疫苗免疫原的重要性。这种设计通过体内免疫学分析进一步得到验证；我们观察到在接种VLS的个体的局部组织中，S1抗原与DCs或巨噬细胞的共定位程度更高，以及在接种VLS的个体的淋巴器官中活化DCs群体的百分比更高，与接种mRNA/LNPs的个体相比。进一步使用体外用VLS预处理的DCs的BALB/c小鼠进行的调查揭示了VLS组中活化T细胞的比例比mRNA疫苗组更高。重要的是，接种VLS的小鼠表现出比接种mRNA/LNPs的小鼠更强烈的抗体反应和更强的细胞毒性T淋巴细胞反应。有趣的是，检测到了从上呼吸道和血清中分泌的特异性IgA。基于进一步的保护性动物测试，我们发现这种增强的免疫力为对抗Omicron BA.5、XBB.1或武汉株提供了更有效的保护。进一步的调查揭示了在VLS组的ACE+/+小鼠在挑战时CD4+细胞群体中大多数基因的系统上调和B细胞和CD8+细胞群体中50%以上基因的上调的差异。这些发现表明，接种VLS的小鼠具有特征性的系统性免疫反应，可能因为来自先天免疫系统的多种信号的综合效应。这些数据支持我们的推测，即VLSs能够与各种细胞相互作用，这不仅导致mRNA的内源性抗原蛋白表达和细胞内TLRs26识别的mRNA分子，而且还有DCs的不同亚群的质膜中的其他TLRs27识别外源性S1蛋白，类似于与DC-SIGN或ACE2的结合。此外，通过脂质融合转染VLSs的上皮细胞的抗原趋化效应使DCs激活以摄取抗原并产生一定的抗原信号。在VLS免疫期间引发的这些事件可能导致不同亚群的DCs和巨噬细胞对T细胞和B细胞的抗原交叉呈递，并可能导致对VLS免疫的强烈免疫反应（扩展数据图1）。因此，我们假设基于在DCs和巨噬细胞中特异性诱导mRNA表达的策略可能有助于开发病毒样疫苗候选物，使其能够引发综合免疫反应，以提供比当前COVID-19 mRNA疫苗更有效的针对病毒变种的保护。 然而，这项研究缺乏足够的数据来解释VLSs的物理化学结构以及它们与细胞（特别是DCs或巨噬细胞）的相互作用机制、在这些细胞中引发的信号以及VLS免疫个体的系统免疫学过程，这值得进一步深入研究。 4.方法 4.1.伦理声明 四周至五周龄的K18-hACE2 C57BL/6小鼠（人类ACE2基因敲入C57BL/6小鼠）由Cyagen Biosciences提供（动物许可证号码SCXK (Su) 2022-0016）。BALB/c小鼠和七至八周龄的金黄仓鼠从Vital River购买（动物许可证号码SCXK (Jing) 2021-0006和SCXK (Jing) 2021-0011）。所有动物都在特定病原体自由的隔离设施中饲养（实验室许可证号码SYXK (Lu) 2020-0019）。实验动物的照顾和使用遵循“3R”原则和动物福利指南。动物实验过程及与动物相关的护理和福利已经山东威高利彤生物制品有限公司的动物实验伦理委员会审查和批准（批准号码LACUC-RD3-2022-006）。 4.2.细胞系 HEK-293T和16HBE细胞在含有10%胎牛血清（FBS；HyClone，GE Healthcare）、100 U/ml青霉素和100 mg/ml链霉素的Dulbecco改良Eagle培养基（DMEM，Thermo Fisher Scientific）中培养。JASWII树突状细胞在含有20% FBS和5 ng/ml小鼠粒细胞-巨噬细胞集落刺激因子（HY-P7361，MCE）的MEM Alpha（Thermo Fisher Scientific）中培养。RAW264.7细胞在含有10% FBS的最小Eagle培养基（MEM；Thermo Fisher Scientific）中培养。THP-1细胞在含有10% FBS和0.05 mM β-巯基乙醇（M917637，Macklin）的RPMI 1640（Thermo Fisher Scientific）中培养。所有细胞均从ATCC购买，并在37℃、5% CO2条件下维持。 4.3.病毒 新型冠状病Omicron BA.5株（H株）于2022年12月从COVID-19患者中分离出来。完整的基因组序列已上传至NCBI GenBank（OQ179919.1）。新型冠状病KMS-2株（武汉）于2020年2月从云南省传染病医院的COVID-19患者中分离出来。 4.4.动物实验设计 C57BL/6-ACE2（BALB/c或金黄仓鼠）小鼠被随机分为五组（补充图7）。 扩展数据图7| 推测的VLS疫苗与宿主免疫系统相互作用并引发小鼠免疫反应的机制。➀ 注射的VLS疫苗被上皮细胞内吞。从内体逃逸并进入细胞质后，mRNA在核糖体中被翻译成蛋白质。翻译后的抗原蛋白可分泌到细胞外空间。DCs和巨噬细胞可被VLS引起的局部炎症所吸引。分泌的抗原和VLS疫苗表面的抗原均可被招募的DCs和巨噬细胞摄取，随后激活并呈递给Tfh/Th1和B细胞。➁ 注射的VLS疫苗通过S1蛋白特异性结合DC-SIGN或ACE2受体介导，被DCs和巨噬细胞内吞。传递的mRNA被翻译成内源性抗原，可激活DCs和巨噬细胞。这些激活的免疫细胞能够将抗原呈递给Tfh/Th1和B细胞，以激活特异性免疫反应和B细胞的特异性增殖，产生抗体反应，同时引发特异性CTL反应。➂ VLS疫苗表面的S1蛋白抗原作为外源性抗原，被DCs和巨噬细胞识别和内吞，随后激活并呈递给Tfh/Th1和B细胞。这些途径的所有抗原信号都能激活并增强特异性免疫反应。 在A组（免疫实验组，n=35），C57BL/6-hACE2小鼠被分为四组：对照组、VLS组、mRNA组和肽组。VLS包含20μg mRNA和10μg蛋白；mRNA组小鼠接种20μg mRNA，肽组小鼠接种10μg蛋白。所有小鼠在初次免疫后的第21天进行加强免疫。在初次免疫后的24、48和72小时，从免疫部位获取组织，进行qRT-PCR测量细胞因子表达的免疫荧光检测。此外，在初次免疫后的3、7和14天以及加强免疫后的28天，从淋巴结中分离出B细胞、CD4+ T细胞和CD8+ T细胞，进行转录组测序。 在B组（免疫实验组，n=36），C57BL/6-hACE2小鼠被分为三组：对照组、VLS组和mRNA组。用于探索mRNA分子与传递系统的组织部位的mRNA。在肌肉注射后的第1、3、5和7天收集心脏、肝脏、脾脏、肾脏、肺、大脑和淋巴结，进行组织免疫荧光检测。 在C组（免疫实验组，n=72），BALB/c小鼠被分为12组：N1-N12。N1-N4为mRNA疫苗组，N5-N8为VLS疫苗组，N9-N12为肽疫苗组。N1-N4分别包含5、10、20和40μg mRNA。N5-N8包含5μg mRNA和5μg肽、10μg mRNA和5μg肽、10μg mRNA和10μg肽、20μg mRNA和10μg肽。N9-N12分别包含5μg、10μg、20μg和30μg肽。所有小鼠在初次免疫后的第21天进行加强免疫。在免疫后的第35、49、120、180和240天，从尾静脉采集血液样本进行抗体检测。加强免疫后的第28天，收集血液进行中和抗体和结合抗体测试，脾脏进行淋巴细胞分离和ELISpot检测。同时，比较SARS-CoV-2疫苗的免疫滴度。 在D组（免疫保护实验组；C57BL/6-hACE2，n=75；金黄仓鼠，n=40），加强免疫后的第28天，对照组、VLS组、mRNA组和肽组小鼠分别通过鼻内途径用SARS-CoV-2 Omicron BA.5（10^4.5 50%细胞培养感染剂量（CCID50））、SARS-CoV-2野生型（10^4 CCID50）或SARS-CoV-2 Omicron XBB.1（10^4.5 CCID50）进行挑战。在病毒感染后的第3、5和7天收集心脏、肝脏、脾脏、肾脏、肺、气管、大脑、脊髓、淋巴结和性器官，进行病毒载量定量。同时，在挑战后第3天，从C57BL/6-hACE2小鼠的淋巴结中分离出B细胞、CD4+ T细胞和CD8+ T细胞，进行转录组测序。 在E组（输血实验，n=27），DCs用LNPs-肽、LNPs-mRNA和VLSs处理12小时。体外处理的DCs（每只小鼠3×10^5个细胞）通过尾静脉输注到小鼠体内。在移植后的第3和5天获取脾脏和腹股沟淋巴结，进行流式细胞术和qRT-PCR。输血后21天测量抗体滴度。上述动物挑战实验由武汉生物制品研究所委托进行。 4.5.LNP传递系统的合成和配方 根据((2-(2-羟乙氧基)乙基)氮杂二基)双(己烷-6,1-二基)双(2-己基癸酸酯)、1,2-二油酰-3-三甲基铵丙烷、1,2-二油酰-磷脂酰胆碱和甲氧基聚(乙二醇)-N-十四烷基十四酰胺-1-2k的分子量，分别在绝对乙醇（优级试剂）中溶解至20%、10%、20%和10%的分子浓度。所有四个组分以30.68:6.84:15.2:1的摩尔比在乙醇中混合，以准备LNP传递系统。mRNA根据请求比例在20 mM醋酸钠、2.5 mM KCl和0.1%海藻糖的缓冲液中稀释。通过NanoAssemblr Ignite+ LNPs（Precision Nanosystems）将空白LNPs与mRNA以1:3的重量比混合制备LNP-mRNA。脂质颗粒进一步通过Zetasizer Ultra光谱仪进行表征。 4.6.SARS-CoV-2 S1 mRNA的合成 通过PCR技术将SARS-CoV-2的S1序列扩增并插入到PVAX载体中。将黄热病病毒的5′-非翻译区（UTR）扩增并插入到Kozak序列之前，将人类线粒体核糖体的3′-UTR扩增并插入到S1序列之后，作为S1序列的5′-UTR和3′-UTR。构建的质粒用BamHI（目录号R0136V，NEB）在37℃下线性化3小时进行体外转录反应。通过体外转录反应（HiScribe T7 ARCA mRNA Kit，目录号E2060S，NEB）合成N1mψ修饰的S1 mRNA。反应混合物用DNase I处理，并使用氯化锂沉淀进行纯化。 4.7.VLS疫苗的制备 通过将LNP-mRNA和肽添加到缓冲液（0.1%海藻糖和3.5%葡萄糖）中，在室温下以30 r.p.m.混合30分钟，然后在4℃旋转混合10 r.p.m.过夜。VLS疫苗进一步通过Zetasizer Ultra光谱仪进行表征。 4.8.LNP转染的mRNA和VLS细胞 然后，将0.5、1、2、5和10μg的mRNA通过LNP传递系统转染到293T细胞中，并在转染后24小时进行西方印迹检测。同时，将mRNA和VLSs转染到16HBE、JASWII DCs、THP-1细胞和RAW264.7细胞中，在24小时后进行西方印迹分析和qRT-PCR测量。 4.9.共免疫沉淀 向LNP包裹的mRNA和VLS疫苗（VLS疫苗装载了BA.1 S1蛋白或N蛋白）中加入抗S1和抗N抗体。然后，加入50μl的磁性珠（BeaverBeads Protein A Matrix Antibody Purification Kit，目录号20102，苏州）并孵育30分钟，在室温下30 r.p.m.。接下来，在磁场中吸附含磁性珠的样品2-3分钟，室温下，上清液用于mRNA和蛋白含量测量。然后，用洗涤缓冲液洗涤珠，加入洗脱缓冲液进行洗脱，并通过qRT-PCR和ELISA测量mRNA和蛋白含量。 4.10.qRT-PCR 通过qRT-PCR测量VLS和mRNA疫苗中的mRNA含量。根据指南，引物的结合位点设计在SARS-CoV-2 S1基因区域，S1基因的一部分构建在pUC57质粒中。体外转录的mRNA用作标准样品。引物为F：TGGATCTGGAGGGAAAGCAGGCAACT和R：CCGATTGGCAGATCCACCAGAGGTTC，TaqMan探针（Sangon Biotech）的序列为5′-6FAM-ATGGCTACTTCAAGATCTATAGCAAGC-TAMRA-3′。通过绝对定量的qPCR确定组织中的病毒载量。根据世界卫生组织的指南，引物的结合位点设计在SARS-CoV-2 N基因区域，N基因构建在pUC57质粒上。引物为F：GAATGGCTGGCAATGGCGGTGATGCT和R：TTGTTGGCCTTTACCAGACATTTTGCT，TaqMan探针（Sangon Biotech）的序列为5′-6FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′。使用RNAiso Plus（T9108，Takara）从组织中提取病毒基因组RNA，使用一步PrimeScript RT-PCR试剂盒（Perfect Real Time）（TaKaRa，代码RR064A）进行反应。 组织和细胞中细胞因子的相对表达，使用RNAiso Plus（T9108，Takara）提取总RNA。基因表达以与LNP注射的小鼠或未感染病毒的细胞中的水平的倍数变化（2−∆∆Ct）表示，这些水平用于校准。使用一步SYBR PrimeScript PLUS RT-PCR试剂盒（TakaRa，代码RR096A）进行反应。使用的具体引物列在扩展数据表4和5中。 4.11.免疫荧光和共聚焦显微镜 收集肌肉组织并立即在液氮中冷冻。组织切片嵌入OCT（Tissue-Tek OCT Compound 4583，Sakura）中，并在冰晶切片机上切成5μm厚（CM1850，Leica）。组织切片固定和用5% BSA封闭。检测病毒抗原时，切片依次用小鼠抗SARS-CoV-2 S抗体（Sino Biological，目录号40150-V08B1）和Alexa Fluor 647偶联的山羊抗小鼠IgG二抗（Invitrogen）孵育。使用抗CD11c抗体（Abcam，目录号ab33483-N418）检测DCs，使用抗F4/80抗体（Zhengneng，目录号263101-31B1），Alexa Fluor 488偶联的山羊抗兔IgG二抗和647偶联的山羊抗小鼠IgG二抗（Invitrogen）检测巨噬细胞。所有细胞核均使用4,6-二氨二苯吲哚检测，并使用共聚焦显微镜（TCS SP2，Leica）分析。 4.12.病毒滴定 病毒滴度通过斑点测定法确定，按照标准协议进行，如前所述。简而言之，病毒经过梯度稀释并加入六孔板。羧甲基纤维素用作液体覆盖的基质，结晶紫用作染色以增强斑点可视化，细胞在37℃、5% CO2条件下培养7天。 4.13.中和试验 简而言之，血清在56℃下灭活30分钟，连续稀释1:4，并与病毒以100倍CCID50/100μl的滴度混合，在37℃下孵育2小时。混合物加入六孔板，并使用羧甲基纤维素作为基质，结晶紫用作染色以在37℃、5% CO2条件下培养后增强斑点可视化。同时，测量假病毒的血清中和滴度，50%抑制稀释（EC50）定义为血清稀释度，与病毒对照孔（病毒+细胞）相比，相对光单位减少了50%，减去只有细胞的对照组的背景相对光单位。简而言之，假病毒（Acro，目录号PSSO-HLC016）与测试样品的连续稀释1:64在37℃下孵育1小时，与病毒对照和细胞对照孔一起。然后，将293T-ACE2细胞加入每个孔中。在37℃、5% CO2环境中孵育48小时后，测量发光。 4.14.IFNγ特异性和IL-4特异性ELISpot检测 对于ELISpot检测，根据制造商的协议使用小鼠IFNγ和IL-4 ELISpot试剂盒（Mabtech）。简而言之，用脾淋巴细胞接种板，然后加入3μg的刺激物（XBB Spike RBD蛋白，目录号40592-V08H144，Sino Biological；B.1.1.529 Spike RBD蛋白，目录号SPD-C522e，Acro；BA.4/BA.5 Spike RBD蛋白，目录号SPD-C522R，Acro；BA.2.12.1 Spike RBD蛋白，目录号SPD-C522q，Acro；BQ.1.1 Spike RBD蛋白，目录号SPD-C5240，Acro；BA.2.75.2 Spike RBD蛋白，目录号SPD-C522z，Acro），然后将细胞加入并37℃孵育30小时。接下来，去除细胞和培养基，显色发展。使用ELISpot阅读器（Mabtech）计数有色斑点。 4.15.从小鼠淋巴结中分离B细胞、CD4+ T细胞和CD8a+ T细胞 在无菌条件下分离淋巴结并轻轻研磨，将淋巴细胞分离成悬浮液。使用MajoSort小鼠CD19纳米珠（目录号SPD-C522R，Acro 480002，BioLegend）富集B细胞，使用EasySep小鼠CD4+ T细胞分离试剂盒（目录号SPD-C522R，Acro 19852A，Stemcell）富集CD4+ T细胞，使用EasySep小鼠CD8a+选择试剂盒（目录号SPD-C522R，Acro 18953，Stemcell）富集CD8+ T细胞。 4.16.B细胞、CD4+ T细胞和CD8a+ T细胞的转录组分析 通过淋巴结分离B细胞、CD4+ T细胞和CD8a+ T细胞。使用TRIzol（目录号DP421，天根）提取总RNA。使用NanoDrop系统和Bioanalyzer评估RNA数量和完整性，并根据Illumina的说明准备样品并测序（Gene Denovo Biotechnology）。在京都基因与基因组百科全书分析中，选择表达变化2倍或更大的基因在P < 0.05，并按功能类别进行分组。所有转录组测序结果已上传至GSA；提交的指定访问号码为CRA010542。 4.17.酶联免疫吸附试验（ELISA） 使用SARS-CoV-2 Spike Protein ELISA Kit（Acro，目录号RAS-A039）定量S1抗原，并使用SARS-CoV-2 (2019-nCoV) Nucleoprotein ELISA Kit（Jiya Biotechnology）定量核蛋白。使用SARS-CoV-2 RBD (Wild-Type) Antibody (IgG) Detection Kits（Vazyme，目录号DD3201-01）、SARS-CoV-2 RBD (Omicron BA.4/5) Antibody (IgG) Detection Kits（Vazyme，目录号DD3214-01，中国）和Mouse Anti-2019-nCoV (S) IgA Elisa Kits（FineTest，目录号1906）进行S1-RBD IgG检测。使用Mouse Anti-SARS-CoV-2 (B.1.1.529) Antibody IgG Titer Serologic Assay Kit (Spike S1)（Acro，目录号RAS-T061）、Mouse Anti-SARS-CoV-2 Antibody IgG Titer Serologic Assay Kit (Spike S1)（Acro，目录号RAS-T045）、Mouse Anti-SARS-CoV-2 (B.1.351) Antibody IgG Titer Serologic Assay Kit (Spike S1)（Acro，目录号RAS-T084）、Mouse Anti-SARS-CoV-2 (B.1.617.2) Antibody IgG Titer Serologic Assay Kit (Spike S1)（Acro，目录号RAS-T086）进行S1抗体检测。光密度值至少比阴性对照高2.1倍的血清样本被视为阳性。终点滴度定义为产生阳性光密度值的最高血清稀释度。几何平均滴度计算为每组阳性血清样本的终点滴度的几何平均值。 4.18.西方印迹法 蛋白质通过12% SDS-聚丙烯酰胺凝胶电泳（PAGE）分离，并转移到聚偏二氟乙烯（PVDF）膜上。膜用5%牛血清白蛋白-三羟甲基氨基甲烷缓冲液与Tween-20（Sigma-Aldrich）封闭，并与抗SARS-CoV-2 S1抗体（MHC0102，云南乐鹏科技）、抗ACE2抗体（Abcam，目录号ab15348）和抗DC-SIGN抗体（Santa Cruz Biotechnology，目录号sc-74589）孵育2小时；洗涤三次，然后用辣根过氧化物酶（HRP）标记的山羊抗小鼠IgG（H+L）（Sigma）孵育1小时。最后，PVDF膜洗涤三次，用ECL超敏化学发光试剂（NCM Biotech，目录号P10100）覆盖，并在Bio-Rad凝胶成像仪上曝光和显色。 4.19.SDS-PAGE的银染 用试剂盒（Fast Silver Stain Kit，目录号P0017S，Beyotime）对制备的SDS-PAGE凝胶进行银染。SDS-PAGE凝胶的银染分为十个步骤：固定、用30%乙醇洗涤、水洗、增感、水洗、银染、水洗、显色、终止和水洗。 4.20.流式细胞术分析 在疫苗初次和加强免疫后的第3天和第7天收集淋巴结和脾脏，并分离淋巴细胞。用于检测DC激活表面标记的流标抗体为PE/Cy5-CD45（目录号MA5-38732，Thermo Fisher）、PE/Cy7-CD11c（目录号A15849，Thermo Fisher）、FITC-CD80（目录号A14722，Thermo Fisher）、APC-CD83（目录号ab234119，Abcam）和PE-CD86（目录号12-0862-82，Thermo Fisher）。用于检测特定T细胞表面标记的流标抗体为BV421-CD44（目录号103019，BioLegend）、APC-CD25（目录号102011，BioLegend）、PE/Cy5-CD3（目录号100205，BioLegend）、FITC-CD4（目录号100405，BioLegend）、APC/Cy7-CD8（目录号100713，BioLegend）和PE-Tetramer（Helixgen COVID-19 MHC-I Tetramer）。用于检测活化B细胞表面标记的流标抗体为FITC-CD19（目录号115505，BioLegend）、PerCp/Cy5.5-GL7（目录号144609，BioLegend）和PE-S1（Expedeon，目录号336-005）。细胞在4℃下染色30分钟，流式细胞分析（LSR Fortessa，BD）前洗涤两次。 4.21.Luminex分析 根据制造商的说明进行Bio-Plex Mouse 23-Plex Panel试剂盒（目录号M60009RDPD）检测。简而言之，通过将重组标准品连续稀释，生成了从1.6到10,000 pg/ml的标准曲线。通过向每个孔中移液200μl的检测缓冲液来阻断滤板。10分钟后，通过真空吸引弃去检测缓冲液，向指定的样品孔中加入25μl的检测稀释液，向标准品孔中加入RPMI 1640 with GlutaMAX（Gibco）。然后，向相应的孔中加入标准品或样品，以及25μl的抗体包被的荧光珠子。生物素标记的二抗和链亲和素-荧光素标记的抗体随后通过交替孵育和洗涤步骤加入到孔中。然后，向孔中加入100μl的鞘液，立即使用Bio-Plex阵列阅读器在高和低RP1目标上使用五参数逻辑回归曲线进行读取。 4.22.统计分析和可重复性 对于西方印迹和电子显微镜、免疫荧光检测至少重复两次；对于ELISA，定量分析至少重复三次。所有数据表示为均值±标准误差。组间显著性差异通过GraphPad Prism进行分析。统计显著性设为P < 0.05。 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 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