STING agonists as promising vaccine adjuvants to boost immunogenicity against SARS-related coronavirus derived infection: possible role of autophagy.

As a major component of innate immunity and a positive regulator of interferons, the Stimulator of interferon gene (STING) has an immunotherapy potential to govern a variety of infectious diseases. Despite the recent advances regarding vaccines against COVID-19, nontoxic novel adjuvants with the potential to enhance vaccine efficacy are urgently desired. In this connection, it has been well-documented that STING agonists are applied to combat COVID-19. This approach is of major significance for boosting immune responses most likely through an autophagy-dependent manner in susceptible individuals against infection induced by severe acute respiratory syndrome Coronavirus (SARS­CoV­2). Given that STING agonists exert substantial immunomodulatory impacts under a wide array of pathologic conditions, these agents could be considered novel adjuvants for enhancing immunogenicity against the SARS-related coronavirus. Here, we intend to discuss the recent advances in STING agonists' recruitment to boost innate immune responses upon vaccination against SARS-related coronavirus infections. In light of the primordial role of autophagy modulation, the potential of being an antiviral vaccine adjuvant was also explored.

Adjuvants for cancer mRNA vaccines in the era of nanotechnology: strategies, applications, and future directions.

Research into mRNA vaccines is advancing rapidly, with proven efficacy against coronavirus disease 2019 and promising therapeutic potential against a variety of solid tumors. Adjuvants, critical components of mRNA vaccines, significantly enhance vaccine effectiveness and are integral to numerous mRNA vaccine formulations. However, the development and selection of adjuvant platforms are still in their nascent stages, and the mechanisms of many adjuvants remain poorly understood. Additionally, the immunostimulatory capabilities of certain novel drug delivery systems (DDS) challenge the traditional definition of adjuvants, suggesting that a revision of this concept is necessary. This review offers a comprehensive exploration of the mechanisms and applications of adjuvants and self-adjuvant DDS. It thoroughly addresses existing issues mentioned above and details three main challenges of immune-related adverse event, unclear mechanisms, and unsatisfactory outcomes in old age group in the design and practical application of cancer mRNA vaccine adjuvants. Ultimately, this review proposes three optimization strategies which consists of exploring the mechanisms of adjuvant, optimizing DDS, and improving route of administration to improve effectiveness and application of adjuvants and self-adjuvant DDS.

Comparative analysis of recurrence rates between intravesical gemcitabine and bacillus Calmette-Guérin induction therapy following transurethral resection of bladder tumors in patients with intermediate- and high-risk bladder cancer: A retrospective multicenter study.

PURPOSE: This study investigated the efficacy of intravesical gemcitabine as an alternative to bacillus Calmette-Guérin (BCG) therapy. MATERIALS AND METHODS: Data were retrospectively collected across seven institutions from February 1999 to May 2023. Inclusion criteria included patients with intermediate- or high-risk non-muscle invasive bladder cancer (NMIBC) who underwent transurethral resection of bladder tumors (TURBT) and received at least four sessions of intravesical gemcitabine or BCG induction therapy. Patient characteristics, complete remission (CR), occurrence, and progression rates were compared. RESULTS: In total, 149 patients were included in this study (gemcitabine, 63; BCG, 86). No differences were apparent between the two groups in baseline characteristics, except for the follow-up period (gemcitabine, 9.2±5.9 months vs. BCG, 43.9±41.4 months, p<0.001). There were no consistent significant differences observed between the two groups in the 3-month (gemcitabine, 98.4% vs. BCG, 95.3%; p=0.848), 6-month (94.9% vs. 90.0%, respectively; p=0.793) and 1-year CR rates (84.2% vs. 83.3%, respectively; p=0.950). Also, there was no significant statistical difference in progression-free survival between the two groups (p=0.953). The occurrence rates of adverse events were similar between the groups (22.2% vs. 22.1%; p=0.989); however, the rate of Clavien-Dindo grade 2 or higher was significantly higher in the BCG group (1.6% vs. 16.3%, respectively; p<0.001). CONCLUSIONS: Intravesical gemcitabine demonstrated efficacy comparable to BCG therapy for the first year in patients with intermediate- and high-risk NMIBC. However, long-term follow-up studies are warranted.

Cost-effectiveness of the adjuvanted quadrivalent influenza vaccine for older adults in South Korea.

South Korea's National Immunization Program administers the quadrivalent influenza vaccine (QIV) to manage seasonal influenza, with a particular focus on the elderly. After reviewing the safety and immune response triggered by the adjuvanted QIV (aQIV) in individuals aged 65 and older, the Ministry of Food and Drug Safety in Korea approved its use. However, the extensive impact of aQIV on public health is yet to be fully understood. This study assessed the cost-effectiveness of replacing QIV with aQIV in South Korean adults aged 65 years and older. A dynamic transmission model, calibrated with national influenza data, was applied to compare the influence of aQIV and QIV on older adults and the broader population throughout a single influenza season. This study considered both the direct and indirect effects of vaccination on the elderly. We derived the incremental cost-effectiveness ratios (ICERs) from quality-adjusted life-years (QALYs) and costs incurred, validated through a probabilistic sensitivity analysis with 5,000 simulations. Findings suggest that transitioning to aQIV from QIV in the elderly would be cost-effective, particularly if aQIV's efficacy reaches or exceeds 56.1%. With an ICER of $29,267/QALY, considerably lower than the $34,998/QALY willingness-to-pay threshold, aQIV presents as a cost-effective option. Thus, implementing aQIV with at least 56.1% efficacy is beneficial from both financial and public health perspectives in mitigating seasonal influenza in South Korea.

Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

Development of novel Streptococcus equi vaccines with an assessment of their immunizing potentials and protective efficacies.

Strangles is a highly contagious disease of the equine upper respiratory tract caused by Streptococcus equi subspecies. Streptococcus equi subsp. equi (S. equi) and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was isolated, as local, hot, and field strains, from horses clinically suffering from respiratory distress. The isolated Streptococci were identified using bacteriological and molecular techniques. Four formulations of inactivated S. equi vaccines were developed and evaluated. The first formulation was prepared using the S. equi isolates, adjuvanted with MONTANIDE GEL adjuvant, while the second formulation was adjuvanted with MONTANIDE ISA-70 adjuvant. The other 2 formulations were inactivated combined vaccines prepared from both S. equi and S. zooepidemicus isolates. The 3rd formulation was the combined isolates adjuvanted with MONTANIDE GEL while the 4th formulation was the combined isolates adjuvanted with MONTANIDE ISA-70. The developed vaccines' physical properties, purity, sterility, safety, and potency were ensured. The immunizing efficacy was determined in isogenic BALB/c mice and white New Zealand rabbits using the passive hemagglutination test. Also, the antibodies' titer of the combined S. equi and S. zooepidemicus vaccine adjuvanted with MONTANIDE ISA-70 in foals was tracked using an indirect enzyme-linked immunosorbent assay. The protective efficacy of the developed vaccines was determined using a challenge test in both laboratory and field animal models, where a 75% protection rate was achieved. The combined vaccine proved to be more efficacious than the monovalent vaccine. Also, the MONTANIDE ISA-70 adjuvant provided significant protective efficacy than the MONTANIDE GEL. The current work is introducing a very promising mitigative and strategic controlling solution for strangles.

Immunity by AS03ation: The natural adjuvantage.

Humans do not respond equally to vaccination. To investigate why, Mulè et al. developed a multimodal framework and found that high responders after unadjuvanted influenza vaccination exist in a naturally adjuvanted state, mimicking innate immunophenotypes following AS03-adjuvanted vaccination. This highlights biological factors that set apart high-antibody responders and how adjuvants can boost innate immune cues to improve humoral immunity.

Hybrid response to SARS-CoV-2 and Neisseria meningitidis C after an OMV-adjuvanted immunization in mice and their offspring.

COVID-19, caused by SARS-CoV-2, and meningococcal disease, caused by Neisseria meningitidis, are relevant infectious diseases, preventable through vaccination. Outer membrane vesicles (OMVs), released from Gram-negative bacteria, such as N. meningitidis, present adjuvant characteristics and may confer protection against meningococcal disease. Here, we evaluated in mice the humoral and cellular immune response to different doses of receptor binding domain (RBD) of SARS-CoV-2 adjuvanted by N. meningitidis C:2a:P1.5 OMVs and aluminum hydroxide, as a combined preparation for these pathogens. The immunization induced IgG antibodies of high avidity for RBD and OMVs, besides IgG that recognized the Omicron BA.2 variant of SARS-CoV-2 with intermediary avidity. Cellular immunity showed IFN-γ and IL-4 secretion in response to RBD and OMV stimuli, demonstrating immunologic memory and a mixed Th1/Th2 response. Offspring presented transferred IgG of similar levels and avidity as their mothers. Humoral immunity did not point to the superiority of any RBD dose, but the group immunized with a lower antigenic dose (0.5 µg) had the better cellular response. Overall, OMVs enhanced RBD immunogenicity and conferred an immune response directed to N. meningitidis too.

Secretory IgA and course of COVID-19 in patients receiving a bacteria-based immunostimulant agent in addition to background therapy.

Mucosal immunity plays a major role not only in the prevention but probably also in the outcomes of COVID-19. An enhanced production of secretory immunoglobulin A (sIgA) might contribute to the activation of the immune response mechanisms. To assess the levels of sIgA produced by epithelial cells in the nasal and pharyngeal mucosa and those measured in salivary gland secretions and to study the course of COVID-19 following the combined scheme of intranasal and subcutaneous administration of a bacteria-based immunostimulant agent. This study included 69 patients, aged between 18 and 60, who had moderate COVID-19 infection. They were divided into two groups: Group 1 (control group) included 39 patients who received only background therapy, and Group 2 was made up of 30 patients who received background therapy in combination with the Immunovac VP4 vaccine, a bacteria-based immunostimulant agent, which was given for 11 days starting from the day of admission to hospital. The levels of sIgA were measured by ELISA in epithelial, nasal and pharyngeal swabs, and salivary gland secretions at baseline and on days 14 and 30. The combined scheme of intranasal and subcutaneous administration of the Immunovac VP4 vaccine in the complex therapy of patients with COVID-19 is accompanied by increased synthesis of sIgA in nasal and pharyngeal swabs, more intense decrease in the level of C-reactive protein (CRP) and reduction in the duration of fever and length of hospitalization compared to the control group. Prescribing a immunostimulant agent containing bacterial ligands in complex therapy for COVID-19 patients helps to enhance mucosal immunity and improves the course of the disease.

Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Adjuvanted Vaccine to Prevent Respiratory Syncytial Virus in Adults Ages 60 Years and Older.

Respiratory syncytial virus (RSV) is a prevalent cause of acute lower respiratory tract illness that disproportionately affects older adults, young children, and infants, which can lead to hospitalizations and death. The health impact on the elderly and infants accentuates the need for effective preventive strategies. Arexvy is the first approved vaccine to prevent lower respiratory tract illness caused by RSV in older adults ages 60 and older. It contains recombinant respiratory syncytial virus glycoprotein F stabilized in the prefusion conformation. Arexvy offers approximately 83% protection in adults and appears to maintain effectiveness for up to two RSV seasons. The vaccine was generally well tolerated in clinical trials, with the most frequently observed and reported adverse events being mild to moderate injection site pain, fatigue, myalgia, headache, and arthralgia. This article includes a description of Arexvy, the target population, contraindications, side effects, and clinical implications when considering the use of this vaccine.

Dimethyl-Dioctadecyl-Ammonium Bromide/Poly(lactic acid) Nanoadjuvant Enhances the Immunity and Cross-Protection of an NM2e-Based Universal Influenza Vaccine.

For most frequent respiratory viruses, there is an urgent need for a universal influenza vaccine to provide cross-protection against intra- and heterosubtypes. We previously developed an Escherichia coli fusion protein expressed extracellular domain of matrix 2 (M2e) and nucleoprotein, named NM2e, and then combined it with an aluminum adjuvant, forming a universal vaccine. Although NM2e has demonstrated a protective effect against the influenza virus in mice to some extent, further improvement is still needed for the induction of immune responses ensuring adequate cross-protection against influenza. Herein, we fabricated a cationic solid lipid nanoadjuvant using poly(lactic acid) (PLA) and dimethyl-dioctadecyl-ammonium bromide (DDAB) and loaded NM2e to generate an NM2e@DDAB/PLA nanovaccine (Nv). In vitro experiments suggested that bone marrow-derived dendritic cells incubated with Nv exhibited ∼4-fold higher antigen (Ag) uptake than NM2e at 16 h along with efficient activation by NM2e@DDAB/PLA Nv. In vivo experiments revealed that Ag of the Nv group stayed in lymph nodes (LNs) for more than 14 days after initial immunization and DCs in LNs were evidently activated and matured. Furthermore, the Nv primed T and B cells for robust humoral and cellular immune responses after immunization. It also induced a ratio of IgG2a/IgG1 higher than that of NM2e to a considerable extent. Moreover, NM2e@DDAB/PLA Nv quickly restored body weight and improved survival of homo- and heterosubtype influenza challenged mice, and the cross-protection efficiency was over 90%. Collectively, our study demonstrated that NM2e@DDAB/PLA Nv could offer notable protection against homo- and heterosubtype influenza virus challenges, offering the potential for the development of a universal influenza vaccine.

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Exploring the Structural Attributes of Yoda1 for the Development of New-Generation Piezo1 Agonist Yaddle1 as a Vaccine Adjuvant Targeting Optimal T Cell Activation.

Piezo1, a mechano-activated ion channel, has wide-ranging physiological and therapeutic implications, with the ongoing development of specific agonists unveiling cellular responses to mechanical stimuli. In our study, we systematically analyzed the chemical subunits in Piezo1 protein agonist Yoda1 to comprehend the structure-activity relationship and push forward next-generation agonist development. Preliminary screening assays for Piezo1 agonism were performed using the Piezo1-mCherry-transfected HEK293A cell line, keeping Yoda1 as a positive control. We introduce a novel Piezo1 agonist Yaddle1 (34, 0.40 µM), featuring a trifluoromethyl group, with further exploration through in vitro studies and density functional theory calculations, emphasizing its tetrel interactions, to act as an ambidextrous wedge between the domains of Piezo1. In contrast to the poor solubility of the established agonist Yoda1, our results showed that the kinetic solubility of Yaddle1 (26.72 ± 1.8 µM at pH 7.4) is 10-fold better than that of Yoda1 (1.22 ± 0.11 µM at pH 7.4). Yaddle1 (34) induces Ca2+ influx in human CD4+ T cell, suggesting its potential as a vaccine adjuvant for enhanced T cell activation.

Structure-Activity Relationships toward the Identification of a High-Potency Selective Human Toll-like Receptor-7 Agonist.

Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Exploring structural engineering approach to formulate and characterize next-generation adjuvants.

It is critical to emphasize the importance of vaccination as it protects us against harmful pathogens. Despite significant progress in vaccine development, there is an ongoing need to develop vaccines that are not only safe but also highly effective in protecting against severe infections. Subunit vaccines are generally safe, but they frequently fail to elicit strong immune responses. As a result, there is a need to improve vaccine effectiveness by combining them with adjuvants, which have the potential to boost the immune system many folds. The process of developing these adjuvants requires searching for molecules capable of activating the immune system, combining these promising compounds with an antigen, and then testing this combination using animal models before approving it for clinical use. Liposomal adjuvants work as delivery adjuvants and its activity depends on certain parameters such as surface charge, vesicle size, surface modification and route of administration. Self-assembly property of peptide adjuvants and discovery of hybrid peptides have widened the scope of peptides in vaccine formulations. Since most pathogenic molecules are not peptide based, phage display technique allows for screening peptide mimics for such pathogens that have potential as adjuvants. This chapter discusses about peptide and liposome-based adjuvants focusing on their properties imparting adjuvanticity along with the methods of formulating them. Methods of adjuvant characterization important for an adjuvant to be approved for clinical trials are also discussed. These include assays for cytotoxicity, T-lymphocyte proliferation, dendritic cell maturation, cytokine and antibody production, toll-like receptor dependent signaling and adjuvant half-life.

Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.

The synthesis and preliminary immunological evaluation of a dual-adjuvant SARS-CoV-2 RBD vaccine: Covalent integration of TLR7/8 and iNKT cell agonists.

Covalently linking an adjuvant to an antigenic protein enhances its immunogenicity by ensuring a synergistic delivery to the immune system, fostering a more robust and targeted immune response. Most adjuvant-protein conjugate vaccines incorporate only one adjuvant due to the difficulties in its synthesis. However, there is a growing interest in developing vaccines with multiple adjuvants designed to elicit a more robust and targeted immune response by engaging different aspects of the immune system for complex diseases where traditional vaccines fall short. Here, we pioneer the synthesis of a dual-adjuvants protein conjugate Vaccine 1 by assembling a toll-like receptor 7/8 (TLR7/8) agonist, an invariant natural killer T cell (iNKT) agonist with a clickable bicyclononyne (BCN). The BCN group can bio-orthogonally react with azide-modified severe acute respiratory syndrome coronavirus-2 receptor-binding domain (SARS-CoV-2 RBD) trimer antigen to give the three-component Vaccine 1. Notably, with a mere 3 µg antigen, it elicited a balanced subclass of IgG titers and 20-fold more IgG2a than control vaccines, highlighting its potential for enhancing antibody-dependent cellular cytotoxicity. This strategy provides a practicable way to synthesize covalently linked dual immunostimulants. It expands the fully synthetic self-adjuvant protein vaccine that uses a single adjuvant to include two different types of adjuvants.