Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments.

Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available in silico. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 µg (14-98 µg) after 5 days in 2 mL cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.

The frequency and function of nucleoprotein-specific CD8+ T cells are critical for heterosubtypic immunity against influenza virus infection.

Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.

Development of NP-Based Universal Vaccine for Influenza A Viruses.

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.

Redirecting antibody responses from egg-adapted epitopes following repeat vaccination with recombinant or cell culture-based versus egg-based influenza vaccines.

Repeat vaccination with egg-based influenza vaccines could preferentially boost antibodies targeting the egg-adapted epitopes and reduce immunogenicity to circulating viruses. In this randomized trial (Clinicaltrials.gov: NCT03722589), sera pre- and post-vaccination with quadrivalent inactivated egg-based (IIV4), cell culture-based (ccIIV4), and recombinant (RIV4) influenza vaccines were collected from healthcare personnel (18-64 years) in 2018-19 (N = 723) and 2019-20 (N = 684) influenza seasons. We performed an exploratory analysis. Vaccine egg-adapted changes had the most impact on A(H3N2) immunogenicity. In year 1, RIV4 induced higher neutralizing and total HA head binding antibodies to cell- A(H3N2) virus than ccIIV4 and IIV4. In year 2, among the 7 repeat vaccination arms (IIV4-IIV4, IIV4-ccIIV4, IIV4-RIV4, RIV4-ccIIV4, RIV4-RIV4, ccIIV4-ccIIV4 and ccIIV4-RIV4), repeat vaccination with either RIV4 or ccIIV4 further improved antibody responses to circulating viruses with decreased neutralizing antibody egg/cell ratio. RIV4 also had higher post-vaccination A(H1N1)pdm09 and A(H3N2) HA stalk antibodies in year 1, but there was no significant difference in HA stalk antibody fold rise among vaccine groups in either year 1 or year 2. Multiple seasons of non-egg-based vaccination may be needed to redirect antibody responses from immune memory to egg-adapted epitopes and re-focus the immune responses towards epitopes on the circulating viruses to improve vaccine effectiveness.

Enhancement of mucosal innate and adaptive immunity following intranasal immunization of mice with a bovine adenoviral vector.

Introduction: Nonhuman adenoviral (AdV) gene delivery platforms have significant value due to their ability to elude preexisting AdV vector immunity in most individuals. Previously, we have demonstrated that intranasal (IN) immunization of mice with BAd-H5HA, a bovine AdV type 3 (BAdV3) vector expressing H5N1 influenza virus hemagglutinin (HA), resulted in enhanced humoral and cell-mediated immune responses. The BAd-H5HA IN immunization resulted in complete protection following the challenge with an antigenically distinct H5N1 virus compared to the mouse group similarly immunized with HAd-H5HA, a human AdV type 5 (HAdV5) vector expressing HA. Methods: Here, we attempted to determine the activation of innate immune responses in the lungs of mice inoculated intranasally with BAd-H5HA compared to the HAd-H5HA-inoculated group. Results: RNA-Seq analyses of the lung tissues revealed differential expression (DE) of genes involved in innate and adaptive immunity in animals immunized with BAd-H5HA. The top ten enhanced genes were verified by RT-PCR. Consistently, there were transient increases in the levels of cytokines (IL-1α, IL-1ß, IL-5, TNF- α, LIF, IL-17, G-CSF, MIP-1ß, MCP-1, MIP-2, and GM-CSF) and toll-like receptors in the lungs of the group inoculated with BAdV vectors compared to that of the HAdV vector group. Conclusion: These results demonstrate that the BAdV vectors induce enhanced innate and adaptive immunity-related factors compared to HAdV vectors in mice. Thus, the BAdV vector platform could be an excellent gene delivery system for recombinant vaccines and cancer immunotherapy.

The limit of detection of the BioFire® FilmArray® gastrointestinal panel for the foodborne parasite Cyclospora cayetanensis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis. The BioFire® FilmArray® gastrointestinal (FilmArray GI) panel is a common method for diagnosing cyclosporiasis from clinical stool samples. The currently published limit of detection (LOD) of this panel is in genome equivalents; however, it is unclear how this relates to the number of C. cayetanensis oocysts in a clinical sample. In this study, we developed a technique to determine the LOD in terms of oocysts, using a cell sorter to sort 1 to 50 C. cayetanensis oocyst(s) previously purified from three human stool sources. We found the FilmArray GI panel detected samples with ≥20 C. cayetanensis oocysts in 100% of replicates, with varying detection among samples with 1, 5, or 10 C. cayetanensis oocysts. This method provides a parasitologically relevant LOD that should enable comparison among C. cayetanensis detection techniques, including the FilmArray GI panel.

Immunogenicity of High-Dose Egg-Based, Recombinant, and Cell Culture-Based Influenza Vaccines Compared With Standard-Dose Egg-Based Influenza Vaccine Among Health Care Personnel Aged 18-65 Years in 2019-2020.

Background: Emerging data suggest that second-generation influenza vaccines with higher hemagglutinin (HA) antigen content and/or different production methods may induce stronger antibody responses to HA than standard-dose egg-based influenza vaccines in adults. We compared antibody responses to high-dose egg-based inactivated (HD-IIV3), recombinant (RIV4), and cell culture-based (ccIIV4) vs standard-dose egg-based inactivated influenza vaccine (SD-IIV4) among health care personnel (HCP) aged 18-65 years in 2 influenza seasons (2018-2019, 2019-2020). Methods: In the second trial season, newly and re-enrolled HCPs who received SD-IIV4 in season 1 were randomized to receive RIV4, ccIIV4, or SD-IIV4 or were enrolled in an off-label, nonrandomized arm to receive HD-IIV3. Prevaccination and 1-month-postvaccination sera were tested by hemagglutination inhibition (HI) assay against 4 cell culture propagated vaccine reference viruses. Primary outcomes, adjusted for study site and baseline HI titer, were seroconversion rate (SCR), geometric mean titers (GMTs), mean fold rise (MFR), and GMT ratios that compared vaccine groups to SD-IIV4. Results: Among 390 HCP in the per-protocol population, 79 received HD-IIV3, 103 RIV4, 106 ccIIV4, and 102 SD-IIV4. HD-IIV3 recipients had similar postvaccination antibody titers compared with SD-IIV4 recipients, whereas RIV4 recipients had significantly higher 1-month-postvaccination antibody titers against vaccine reference viruses for all outcomes. Conclusions: HD-IIV3 did not induce higher antibody responses than SD-IIV4, but, consistent with previous studies, RIV4 was associated with higher postvaccination antibody titers. These findings suggest that recombinant vaccines rather than vaccines with higher egg-based antigen doses may provide improved antibody responses in highly vaccinated populations.

Antiviral Approaches against Influenza Virus.

Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.

Effect of Repeat Vaccination on Immunogenicity of Quadrivalent Cell-Culture and Recombinant Influenza Vaccines Among Healthcare Personnel Aged 18-64 Years: A Randomized, Open-Label Trial.

BACKGROUND: Antibody responses to non-egg-based standard-dose cell-culture influenza vaccine (containing 15 µg hemagglutinin [HA]/component) and recombinant vaccine (containing 45 µg HA/component) during consecutive seasons have not been studied in the United States. METHODS: In a randomized trial of immunogenicity of quadrivalent influenza vaccines among healthcare personnel (HCP) aged 18-64 years over 2 consecutive seasons, HCP who received recombinant-HA influenza vaccine (RIV) or cell culture-based inactivated influenza vaccine (ccIIV) during the first season (year 1) were re-randomized the second season of 2019-2020 (year 2 [Y2]) to receive ccIIV or RIV, resulting in 4 ccIIV/RIV combinations. In Y2, hemagglutination inhibition antibody titers against reference cell-grown vaccine viruses were compared in each ccIIV/RIV group with titers among HCP randomized both seasons to receive egg-based, standard-dose inactivated influenza vaccine (IIV) using geometric mean titer (GMT) ratios of Y2 post-vaccination titers. RESULTS: Y2 data from 414 HCP were analyzed per protocol. Compared with 60 IIV/IIV recipients, 74 RIV/RIV and 106 ccIIV/RIV recipients showed significantly elevated GMT ratios (Bonferroni corrected P < .007) against all components except A(H3N2). Post-vaccination GMT ratios for ccIIV/ccIIV and RIV/ccIIV were not significantly elevated compared with IIV/IIV except for RIV/ccIIV against A(H1N1)pdm09. CONCLUSIONS: In adult HCP, receipt of RIV in 2 consecutive seasons or the second season was more immunogenic than consecutive egg-based IIV for 3 of the 4 components of quadrivalent vaccine. Immunogenicity of ccIIV/ccIIV was similar to that of IIV/IIV. Differences in HA antigen content may play a role in immunogenicity of influenza vaccination in consecutive seasons. CLINICAL TRIALS REGISTRATION: NCT03722589.

Progress towards the Development of a Universal Influenza Vaccine.

Influenza viruses are responsible for millions of cases globally and significantly threaten public health. Since pandemic and zoonotic influenza viruses have emerged in the last 20 years and some of the viruses have resulted in high mortality in humans, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. Current seasonal influenza vaccines provide strain-specific protection and are less effective against mismatched strains. The rapid antigenic drift and shift in influenza viruses resulted in time-consuming surveillance and uncertainty in the vaccine protection efficacy. Most recent universal influenza vaccine studies target the conserved antigen domains of the viral surface glycoproteins and internal proteins to provide broader protection. Following the development of advanced vaccine technologies, several innovative strategies and vaccine platforms are being explored to generate robust cross-protective immunity. This review provides the latest progress in the development of universal influenza vaccines.

Heterogeneous Ribonucleoprotein A1 (hnRNPA1) Interacts with the Nucleoprotein of the Influenza a Virus and Impedes Virus Replication.

Influenza A virus (IAV), like other viruses, depends on the host cellular machinery for replication and production of progeny. The relationship between a virus and a host is complex, shaped by many spatial and temporal interactions between viral and host proteome, ultimately dictating disease outcome. Therefore, it is imperative to identify host-virus interactions as crucial determinants of disease pathogenies. Heterogeneous ribonucleoprotein A1 (hnRNPA1) is an RNA binding protein involved in the life cycle of many DNA and RNA viruses; however, its role in IAV remains undiscovered. Here we report that human hnRNPA1 physically interacts with the nucleoprotein (NP) of IAV in mammalian cells at different time points of the viral replication cycle. Temporal distribution studies identify hnRNPA1 and NP co-localize in the same cellular milieu in both nucleus and mitochondria in NP-transfected and IAV-infected mammalian cells. Interestingly, hnRNPA1 influenced NP gene expression and affected viral replication. Most importantly, hnRNPA1 knockdown caused a significant increase in NP expression and enhanced viral replication (93.82%) in IAV infected A549 cells. Conversely, hnRNPA1 overexpression reduced NP expression at the mRNA and protein levels and impeded virus replication by (60.70%), suggesting antagonistic function. Taken together, results from this study demonstrate that cellular hnRNPA1 plays a protective role in the host hitherto unknown and may hold potential as an antiviral target to develop host-based therapeutics against IAV.

Impact of diabetes status on immunogenicity of trivalent inactivated influenza vaccine in older adults.

Individuals with type 2 diabetes mellitus experience high rates of influenza virus infection and complications. We compared the magnitude and duration of serologic response to trivalent influenza vaccine in adults aged 50-80 with and without type 2 diabetes mellitus. Serologic response to influenza vaccination was similar in both groups: greater fold-increases in antibody titer occurred among participants with lower pre-vaccination antibody titers. Waning of antibody titers was not influenced by diabetes status.

Adenoviral vector-based platforms for developing effective vaccines to combat respiratory viral infections.

Since the development of the first vaccine against smallpox over two centuries ago, vaccination strategies have been at the forefront of significantly impacting the incidences of infectious diseases globally. However, the increase in the human population, deforestation and climate change, and the rise in worldwide travel have favored the emergence of new viruses with the potential to cause pandemics. The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is a cruel reminder of the impact of novel pathogens and the suboptimal capabilities of conventional vaccines. Therefore, there is an urgent need to develop new vaccine strategies that allow the production of billions of doses in a short duration and are broadly protective against emerging and re-emerging infectious diseases. Extensive knowledge of the molecular biology and immunology of adenoviruses (Ad) has favored Ad vectors as platforms for vaccine design. The Ad-based vaccine platform represents an attractive strategy as it induces robust humoral and cell-mediated immune responses and can meet the global demand in a pandemic situation. This review describes the status of Ad vector-based vaccines in preclinical and clinical studies for current and emerging respiratory viruses, particularly coronaviruses, influenza viruses and respiratory syncytial viruses.

Innate lymphoid cells (ILC) in SARS-CoV-2 infection.

Innate Lymphoid Cells (ILCs) are a class of innate immune cells that form the first line of defense against internal or external abiotic and biotic challenges in the mammalian hosts. As they reside in both the lymphoid and non-lymphoid tissues, they are involved in clearing the pathogens through direct killing or by secretion of cytokines that modulate the adaptive immune responses. There is burgeoning evidence that these cells are important in clearing viral infections; therefore, it is critical to understand their role in the resolution or exacerbation of the disease caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this review, we summarize the recent findings related to ILCs in response to SARS-CoV-2 infections.

Comparison of the Immunogenicity of Cell Culture-Based and Recombinant Quadrivalent Influenza Vaccines to Conventional Egg-Based Quadrivalent Influenza Vaccines Among Healthcare Personnel Aged 18-64 Years: A Randomized Open-Label Trial.

BACKGROUND: RIV4 and cell-culture based inactivated influenza vaccine (ccIIV4) have not been compared to egg-based IIV4 in healthcare personnel, a population with frequent influenza vaccination that may blunt vaccine immune responses over time. We conducted a randomized trial among healthcare personnel (HCP) aged 18-64 years to compare humoral immune responses to ccIIV4 and RIV4 to IIV4. METHODS: During the 2018-2019 season, participants were randomized to receive ccIIV4, RIV4, or IIV4 and had serum samples collected prevaccination, 1 and 6 months postvaccination. Serum samples were tested by hemagglutination inhibition (HI) for influenza A/H1N1, B/Yamagata, and B/Victoria and microneutralization (MN) for A/H3N2 against cell-grown vaccine reference viruses. Primary outcomes at 1 month were seroconversion rate (SCR), geometric mean titers (GMT), GMT ratio, and mean fold rise (MFR) in the intention-to-treat population. RESULTS: In total, 727 participants were included (283 ccIIV4, 202 RIV4, and 242 IIV4). At 1 month, responses to ccIIV4 were similar to IIV4 by SCR, GMT, GMT ratio, and MFR. RIV4 induced higher SCRs, GMTs, and MFRs than IIV4 against A/H1N1, A/H3N2, and B/Yamagata. The GMT ratio of RIV4 to egg-based vaccines was 1.5 (95% confidence interval [CI] 1.2-1.9) for A/H1N1, 3.0 (95% CI: 2.4-3.7) for A/H3N2, 1.1 (95% CI: .9-1.4) for B/Yamagata, and 1.1 (95% CI: .9-1.3) for B/Victoria. At 6 months, ccIIV4 recipients had similar GMTs to IIV4, whereas RIV4 recipients had higher GMTs against A/H3N2 and B/Yamagata. CONCLUSIONS: RIV4 resulted in improved antibody responses by HI and MN compared to egg-based vaccines against 3 of 4 cell-grown vaccine strains 1 month postvaccination, suggesting a possible additional benefit from RIV4. CLINICAL TRIALS REGISTRATION: NCT03722589.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Influenza Virus Infects and Depletes Activated Adaptive Immune Responders.

Influenza infections cause several million cases of severe respiratory illness, hospitalizations, and hundreds of thousands of deaths globally. Secondary infections are a leading cause of influenza's high morbidity and mortality, and significantly factored into the severity of the 1918, 1968, and 2009 pandemics. Furthermore, there is an increased incidence of other respiratory infections even in vaccinated individuals during influenza season. Putative mechanisms responsible for vaccine failures against influenza as well as other respiratory infections during influenza season are investigated. Peripheral blood mononuclear cells (PBMCs) are used from influenza vaccinated individuals to assess antigen-specific responses to influenza, measles, and varicella. The observations made in humans to a mouse model to unravel the mechanism is confirmed and extended. Infection with influenza virus suppresses an ongoing adaptive response to vaccination against influenza as well as other respiratory pathogens, i.e., Adenovirus and Streptococcus pneumoniae by preferentially infecting and killing activated lymphocytes which express elevated levels of sialic acid receptors. These findings propose a new mechanism for the high incidence of secondary respiratory infections due to bacteria and other viruses as well as vaccine failures to influenza and other respiratory pathogens even in immune individuals due to influenza viral infections.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Immunogenicity of standard, high-dose, MF59-adjuvanted, and recombinant-HA seasonal influenza vaccination in older adults.

The vaccine efficacy of standard-dose seasonal inactivated influenza vaccines (S-IIV) can be improved by the use of vaccines with higher antigen content or adjuvants. We conducted a randomized controlled trial in older adults to compare cellular and antibody responses of S-IIV versus enhanced vaccines (eIIV): MF59-adjuvanted (A-eIIV), high-dose (H-eIIV), and recombinant-hemagglutinin (HA) (R-eIIV). All vaccines induced comparable H3-HA-specific IgG and elevated antibody-dependent cellular cytotoxicity (ADCC) activity at day 30 post vaccination. H3-HA-specific ADCC responses were greatest following H-eIIV. Only A-eIIV increased H3-HA-IgG avidity, HA-stalk IgG and ADCC activity. eIIVs also increased polyfunctional CD4+ and CD8+ T cell responses, while cellular immune responses were skewed toward single-cytokine-producing T cells among S-IIV subjects. Our study provides further immunological evidence for the preferential use of eIIVs in older adults as each vaccine platform had an advantage over the standard-dose vaccine in terms of NK cell activation, HA-stalk antibodies, and T cell responses.