Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

Correlates of protection for booster doses of the BNT162b2 vaccine

Variants of concern (VOC) of SARS-CoV2 and waning immunity pose a serious global problem. Overall, vaccination and prior infection appear to provide significant protection to the majority of individuals, but some remain susceptible to infection and severe disease. Rigorously identifying a broad spectrum of correlates of protection (COP) is necessary to identify these susceptible populations. The extent to which additional booster doses provide protection is also poorly understood. To address this need, we conducted a multicenter prospective study assessing the association between serological profiles and the risk for SARS-CoV-2 infection, comparing those vaccinated with three to four doses of Pfizer BNT162b2 vaccine. Of 608 healthy adults, 365 received three doses and 243 received four doses. During the first 90 days of followup, 239 (39%) were infected, of whom 165/365 (45%) received 3 doses and 74/243 (30%) four doses. We found that the fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple variants, and reduced the risk of symptomatic infection by 37% [95% I, 15% - 54%]. We identified several parameters based on IgG and IgA binding that were COPs. The strongest association with infection risk was reduced IgG levels to RBD mutants and IgA levels to VOCs, which was a COP in the three-dose group (HR=6.34, p=0.008) and in the four-dose group (HR=8.14, p=0.018). A combination of two commercially available ELISA assays were also associated with protection in both groups (HR = 1.84, p = 0.002; HR = 2.01, p = 0.025, respectively). Most importantly, we identified a subset of individuals with low antibody levels after three doses of vaccine that responded with a significant boost in neutralizing antibody titers after a fourth dose, but were still at significantly increased susceptibility to infection when compared to those who had pre-existing high levels of neutralizing antibodies. Thus, we identify a highly susceptible population that remains susceptible despite apparent responsiveness to vaccines. Further, we develop several specific and sensitive COPs that show dramatic effect sizes and may be utilized to identify individuals most at risk from future exposures.

SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8+ T cells.

Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7-CD45RA+ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.

Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity.

Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing variable influenza epitopes greatly outnumber antibodies reactive against conserved epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.

Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells.

Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence and breakthrough infections have changed vaccine policy to include booster immunizations. However, the effect of diverse and repeated antigen exposures on SARS-CoV-2 memory T cells is poorly understood. Here, we utilize DNA-barcoded MHC-multimers combined with scRNAseq and scTCRseq to capture the ex vivo profile of SARS-CoV-2-responsive T cells within a cohort of individuals with one, two, or three antigen exposures, including vaccination, primary infection, and breakthrough infection. We found that the order of exposure determined the relative distribution between spike- and non-spike-specific responses, with vaccination after infection leading to further expansion of spike-specific T cells and differentiation to a CCR7-CD45RA+ effector phenotype. In contrast, individuals experiencing a breakthrough infection mount vigorous non-spike-specific responses. In-depth analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all types of exposures elicit diverse repertoires characterized by shared, dominant TCR motifs, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and that current vaccination protocols continue to expand and differentiate spike-specific memory responses.

Host Predictors of Broadly Cross-Reactive Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants of Concern Differ Between Infection and Vaccination.

BACKGROUND: Following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination there is significant variability between individuals in protective antibody levels against SARS-CoV-2, and within individuals against different virus variants. However, host demographic or clinical characteristics that predict variability in cross-reactive antibody levels are not well-described. These data could inform clinicians, researchers, and policymakers on the populations most likely to require vaccine booster shots. METHODS: In an institutional review board-approved prospective observational cohort study of staff at St. Jude Children's Research Hospital, we identified participants with plasma samples collected after SARS-CoV-2 infection, after mRNA vaccination, and after vaccination following infection, and quantitated immunoglobulin G (IgG) levels by enzyme-linked immunosorbent assay to the spike receptor binding domain (RBD) from 5 important SARS-CoV-2 variants (Wuhan Hu-1, B.1.1.7, B.1.351, P.1, and B.1.617.2). We used regression models to identify factors that contributed to cross-reactive IgG against 1 or multiple viral variants. RESULTS: Following infection, a minority of the cohort generated cross-reactive antibodies, IgG antibodies that bound all tested variants. Those who did had increased disease severity, poor metabolic health, and were of a particular ancestry. Vaccination increased the levels of cross-reactive IgG levels in all populations, including immunocompromised, elderly, and persons with poor metabolic health. Younger people with a healthy weight mounted the highest responses. CONCLUSIONS: Our findings provide important new information on individual antibody responses to infection/vaccination that could inform clinicians on populations that may require follow-on immunization.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity.

BACKGROUND: Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host. METHODS: Luciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-ß and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity. RESULTS: Here we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity. CONCLUSIONS: Together our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

Cross-reactive Antibody Response to mRNA SARS-CoV-2 Vaccine After Recent COVID-19-Specific Monoclonal Antibody Therapy.

The efficacy of coronavirus disease 2019 (COVID-19) vaccines administered after COVID-19-specific monoclonal antibody is unknown, and "antibody interference" might hinder immune responses leading to vaccine failure. In an institutional review board-approved prospective study, we found that an individual who received mRNA COVID-19 vaccination <40 days after COVID-19-specific monoclonal antibody therapy for symptomatic COVID-19 had similar postvaccine antibody responses to SARS-CoV-2 receptor binding domain (RBD) for 4 important SARS-CoV-2 variants (B.1, B.1.1.7, B.1.351, and P.1) as other participants who were also vaccinated following COVID-19. Vaccination against COVID-19 shortly after COVID-19-specific monoclonal antibody can boost and expand antibody protection, questioning the need to delay vaccination in this setting. TRIAL REGISTRATION: The St. Jude Tracking of Viral and Host Factors Associated with COVID-19 study; NCT04362995; https://clinicaltrials.gov/ct2/show/NCT04362995.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Improving immunogenicity of influenza virus H7N9 recombinant hemagglutinin for vaccine development.

Human infections of novel avian influenza A virus (H7N9) emerged in early 2013 and caused about 40% case-fatality through 2017. Therefore, development of influenza H7N9 vaccines is critical for pandemic preparedness. Currently, there are three means of production of commercial influenza vaccines: egg-based, mammalian cell-based, and insect cell-based platforms. The insect cell-based platform has the advantage of high speed in producing recombinant protein. In this study, we evaluate the stability and immunogenicity of two different influenza H7 HA expression constructs generated using the baculovirus system, including membrane-based full-length HA (mH7) and secreted ectodomain-based H7 (sH7). The mH7 construct could form an oligomer-rosette structure and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, the sH7 construct could not form an oligomer-rosette structure and did not have HA titer before cross-linking with anti-His antibody. Thermal stability tests showed that the sH7 and mH7 constructs were unstable at 43 °C and 52 °C, respectively. In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI and neutralizing antibody titers. In conclusion, further development of the mH7 vaccine candidate is desirable.

Assessment of pathogenicity and antigenicity of American lineage influenza H5N2 viruses in Taiwan.

During December 2003 and March 2004, large scale epidemics of low-pathogenic avian influenza (LPAI) H5N2 occurred in poultry farms in central and southern Taiwan. Based on genomic analysis, these H5N2 viruses contain HA and NA genes of American-lineage H5N2 viruses and six internal genes from avian influenza A/H6N1 viruses endemic in poultry in Taiwan. After disappearing for several years, these novel influenza H5N2 viruses caused outbreaks in poultry farms again in 2008, 2010 and 2012, and have evolved into high pathogenic AI (HPAI) since 2010. Moreover, asymptomatic infections of influenza H5N2 were detected serologically in poultry workers in 2012. Therefore, we evaluated antigenicity and pathogenicity of the novel H5N2 viruses in ferrets. We found that no significant antigenic difference was detected among the novel H5N2 viruses isolated from 2003 to 2014 and the novel H5N2 viruses could cause mild infections in ferrets. Monitoring zoonotic transmission of the novel H5N2 viruses is necessary.

TBK1-associated protein in endolysosomes (TAPE)/CC2D1A is a key regulator linking RIG-I-like receptors to antiviral immunity.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key RNA viral sensors for triggering antiviral immunity. The underlying mechanisms for RLRs to trigger antiviral immunity have yet to be explored. Here we report the identification of TAPE (TBK1-associated protein in endolysosomes) as a novel regulator of the RLR pathways. TAPE functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-ß promoter. TAPE knockdown impairs IFN-ß activation induced by RLRs but not IPS-1. TAPE-deficient cells are defective in cytokine production upon RLR ligand stimulation. During RNA virus infection, TAPE knockdown or deficiency diminishes cytokine production and antiviral responses. Our data demonstrate a critical role for TAPE in linking RLRs to antiviral immunity.

Relationship between different skin incisions and the injury of the infrapatellar branch of the saphenous nerve during anterior cruciate ligament reconstruction.

BACKGROUND: After anterior cruciate ligament (ACL) reconstruction, some patients suffered from sensory disturbance around the surgical incision of the leg. This research was aimed to investigate the relationship between the different skin incisions and the injury of the infrapatellar branch of the saphenous nerve (IPBSN) post ACL reconstruction. METHODS: ACL reconstructions were performed with quadruple hamstring tendon for 60 patients. Sensory disturbance around the skin incision was followed up at an average of 14.5 +/- 4.7 months post operation. Among the 60 patients, vertical incision for 35 patients and oblique incision for 25 patients were used for graft taking during ACL reconstruction. The lengths of the incisions were measured. The patients were asked to mark the sensory disturbance zone at follow up time, and then the marked area was measured. The IPBSN of 15 cadaver knees were anatomized. The distance between the IPBSN and the upper edge of the pes anserinus tendon at the middle point of the incision was measured. Independent-samples t-test, chi-square and Mann-Whitney tests were used for statistical analyses. RESULTS: The patients' age (P = 0.329), the follow-up time (P = 0.681), and the incision length (P = 0.732) between the two groups had no significant difference. Twenty-three patients (65.7%) in the vertical incision group had IPBSN injury compared with 6 patients (24.0%) in oblique incision group (P = 0.002). The average sensory disturbance area in vertical incision group ((48.0 +/- 75.3) cm(2)) was significantly larger (P = 0.004) than that in the oblique group ((8.4 +/- 19.4) cm(2)). The anatomy measurement showed the average distance between IPBSN and the upper edge of the pes anserinus tendon was 0.6 cm at the incision. CONCLUSIONS: Oblique incision with less risk of damage for IPBSN may be better for graft harvesting in ACL reconstruction. As the IPBSN is so near and parallel to the hamstring tendons, damage to the IPBSN is one of the potential complications for graft harvesting, regardless of the incision used. That's why even in the oblique incision group, 24% patients also had sensory disturbance complication.