Human in vitro modeling of adjuvant formulations demonstrates enhancement of immune responses to SARS-CoV-2 antigen.

Adjuvants can enhance vaccine immunogenicity, but their mechanism of action is often incompletely understood, hampering rapid applicability for pandemic vaccines. Herein, we characterized the cellular and molecular activity of adjuvant formulations available for pre-clinical evaluation, including several developed for global open access. We applied four complementary human in vitro platforms to assess individual and combined adjuvants in unformulated, oil-in-water, and liposomal delivery platforms. Liposomal co-formulation of MPLA and QS-21 was most potent in promoting dendritic cell maturation, selective production of Th1-polarizing cytokines, and activation of SARS-CoV-2 Spike-specific CD4+ and CD8+ T cells in a co-culture assay. Select formulations also significantly enhanced Spike antigen-specific humoral immunity in vivo. This study confirms the utility of the cumulative use of human in vitro tools to predict adjuvanticity potential. Thus, human in vitro modeling may advance public health by accelerating the development of affordable and scalable adjuvants for vaccines tailored to vulnerable populations.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including coronavirus disease 2019. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines. OBJECTIVE: We sought to establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. METHODS: A murine model of diet-induced obesity and insulin resistance was used to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. RESULTS: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet, HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T-cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in mice fed a normal diet but not in HFD mice. CONCLUSIONS: The study demonstrated impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases.

A protocol for high-throughput screening for immunomodulatory compounds using human primary cells.

High-throughput screening is a powerful platform that can rapidly provide valuable cytotoxic, immunological, and phenotypical information for thousands of compounds. Human peripheral blood mononuclear cells (PBMCs) cultured in autologous plasma can model the human immune response. Here, we describe a protocol to stimulate PBMCs for 72 h and measure cytokine secretion via AlphaLISA assays and cell surface activation marker expression via flow cytometry. Cryopreserved PBMCs are incubated for 72 h with various small molecule libraries and the supernatants are harvested to rapidly measure secretion levels of key cytokines (tumor necrosis factor alpha, interferon gamma, interleukin 10) via the AlphaLISA assay. Almost simultaneously, the cells can be fixated and stained using antibodies against innate immune activation markers (CD80, CD86, HLA-DR, OX40) for analysis via flow cytometry. This multiplexed readout workflow can directly aid in the phenotypic identification and discovery of novel immunomodulators and potential vaccine adjuvant candidates. For complete details on the use and execution of this protocol, please refer to Chew et al.1.

Precision Vaccinology Approaches for the Development of Adjuvanted Vaccines Targeted to Distinct Vulnerable Populations.

Infection persists as one of the leading global causes of morbidity and mortality, with particular burden at the extremes of age and in populations who are immunocompromised or suffer chronic co-morbid diseases. By focusing discovery and innovation efforts to better understand the phenotypic and mechanistic differences in the immune systems of diverse vulnerable populations, emerging research in precision vaccine discovery and development has explored how to optimize immunizations across the lifespan. Here, we focus on two key elements of precision vaccinology, as applied to epidemic/pandemic response and preparedness, including (a) selecting robust combinations of adjuvants and antigens, and (b) coupling these platforms with appropriate formulation systems. In this context, several considerations exist, including the intended goals of immunization (e.g., achieving immunogenicity versus lessening transmission), reducing the likelihood of adverse reactogenicity, and optimizing the route of administration. Each of these considerations is accompanied by several key challenges. On-going innovation in precision vaccinology will expand and target the arsenal of vaccine components for protection of vulnerable populations.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

Background: Obesity and Type 2 Diabetes Mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines. Objective: To establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. Methods: We utilized a murine model of diet-induced obesity and insulin resistance to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. Results: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet (ND), HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8 + T cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in ND mice but not in HFD mice. Conclusion: We demonstrate impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases. Capsule summary: Obesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.

Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination.

Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized (a) the formulation delivery system, (b) structure, and (c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.

mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.

Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells.

Motivation: Vaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based on in vitro screening of human primary leukocytes. Summary: We describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ~2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNg and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers-an 8.6% hit rate-we downselected to five scaffolds that demonstrated robust efficacy and potency in vitro and evaluated the top hit, vinblastine sulfate, for adjuvanticity in vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCs in vitro that yielded a lead compound with in vivo adjuvanticity.

A Precision Adjuvant Approach to Enhance Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccines Optimized for Immunologically Distinct Vulnerable Populations.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused significant mortality, especially among older adults whose distinct immune system reflects immunosenescence. Multiple SARS-CoV-2 vaccines have received emergency use authorization and/or licensure from the US Food and Drug Administration and throughout the world. However, their deployment has heighted significant limitations, such by age-dependent immunogenicity, requirements for multiple vaccine doses, refrigeration infrastructure that is not universally available, as well as waning immunity. Thus, there was, and continues to be a need for continued innovation during the pandemic given the desire for dose-sparing, formulations stable at more readily achievable temperatures, need for robust immunogenicity in vulnerable populations, and development of safe and effective pediatric vaccines. In this context, optimal SARS-CoV-2 vaccines may ultimately rely on inclusion of adjuvants as they can potentially enhance protection of vulnerable populations and provide dose-sparing effects enabling single shot protection.

Acceptability of a Fentanyl Vaccine to Prevent Opioid Overdose and Need for Personalized Decision-Making.

BACKGROUND: The opioid epidemic worsened during the coronavirus disease 2019 (COVID-19) pandemic. Synthetic opioids (primarily fentanyl) comprise the most common drugs involved in overdose (OD) death. A vaccine that blocks fentanyl from reaching the brain to prevent OD is under development, and insight is needed into its acceptability. METHODS: Using a semi-structured interview guide, persons with opioid use disorder (OUD), family, professionals, and the public were interviewed about attitudes and concerns regarding a fentanyl vaccine. Reactions to fictional clinical vignettes of persons at risk of OUD because of pain and/or substance use histories were collected, analyzed, and quantified for favorability. Interviews were transcribed, coded, and analyzed thematically. RESULTS: Among N = 64 participants, (70.3% female, average age 32.4 years), attitudes were favorable toward a fentanyl vaccine, with preference for lifelong durability (76% of n = 55 asked). Perceived benefits centered on the potential for a life-saving intervention, suffering averted, healthcare dollars saved, and the utility of a passive harm reduction strategy. Concerns centered on uncertainty regarding vaccine safety, questions about efficacy, worry about implications for future pain management, stigma, and need for supportive counseling and guidance to personalize decision making. Reactions to vignettes revealed complex attitudes toward fentanyl vaccination when considering recipient age, health history, and future risks for addiction and pain. CONCLUSIONS: Positive responses to a fentanyl vaccine were found along with appreciation for the complexity of a vaccine strategy to prevent OD in the setting of pain and uncertain durability. Further research is needed to elucidate operational, ethical, and communications strategies to advance the model.

Precision Vaccine Adjuvants for Older Adults: A Scoping Review.

Older adults, defined as those ≥60 years of age, are a growing population vulnerable to infections including severe acute respiratory syndrome coronavirus 2. Although immunization is a key to protecting this population, immunosenescence can impair responses to vaccines. Adjuvants can increase the immunogenicity of vaccine antigens but have not been systematically compared in older adults. We conducted a scoping review to assess the comparative effectiveness of adjuvants in aged populations. Adjuvants AS01, MF59, AS03, and CpG-oligodeoxynucleotide, included in licensed vaccines, are effective in older human adults. A growing menu of investigational adjuvants, such as Matrix-M and CpG plus alum, showed promising results in early phase clinical trials and preclinical studies. Most studies assessed only 1 or 2 adjuvants and no study has directly compared >3 adjuvants among older adults. Enhanced preclinical approaches enabling direct comparison of multiple adjuvants including human in vitro modeling and age-specific animal models may derisk and accelerate vaccine development for older adults.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

The mRNA vaccine BNT162b2 demonstrates impaired TH1 immunogenicity in human elders in vitro and aged mice in vivo.

mRNA vaccines have been key to addressing the SARS-CoV-2 pandemic but have impaired immunogenicity and durability in vulnerable older populations. We evaluated the mRNA vaccine BNT162b2 in human in vitro whole blood assays with supernatants from adult (18-50 years) and elder (≥60 years) participants measured by mass spectrometry and proximity extension assay proteomics. BNT162b2 induced increased expression of soluble proteins in adult blood (e.g., C1S, PSMC6, CPN1), but demonstrated reduced proteins in elder blood (e.g., TPM4, APOF, APOC2, CPN1, and PI16), including 30-85% lower induction of TH1-polarizing cytokines and chemokines (e.g., IFNγ, and CXCL10). Elder TH1 impairment was validated in mice in vivo and associated with impaired humoral and cellular immunogenicity. Our study demonstrates the utility of a human in vitro platform to model age-specific mRNA vaccine activity, highlights impaired TH1 immunogenicity in older adults, and provides rationale for developing enhanced mRNA vaccines with greater immunogenicity in vulnerable populations.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Precision Vaccine Development: Cues From Natural Immunity.

Traditional vaccine development against infectious diseases has been guided by the overarching aim to generate efficacious vaccines normally indicated by an antibody and/or cellular response that correlates with protection. However, this approach has been shown to be only a partially effective measure, since vaccine- and pathogen-specific immunity may not perfectly overlap. Thus, some vaccine development strategies, normally focused on targeted generation of both antigen specific antibody and T cell responses, resulting in a long-lived heterogenous and stable pool of memory lymphocytes, may benefit from better mimicking the immune response of a natural infection. However, challenges to achieving this goal remain unattended, due to gaps in our understanding of human immunity and full elucidation of infectious pathogenesis. In this review, we describe recent advances in the development of effective vaccines, focusing on how understanding the differences in the immunizing and non-immunizing immune responses to natural infections and corresponding shifts in immune ontogeny are crucial to inform the next generation of infectious disease vaccines.

The sixth revolution in pediatric vaccinology: immunoengineering and delivery systems.

Infection is the predominant cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, very young infants fail to respond optimally to most vaccines currently in use, especially neonates. In 2005, Stanley Plotkin proposed that new delivery systems would spur a new revolution in pediatric vaccinology, just as attenuation, inactivation, cell culture of viruses, genetic engineering, and adjuvantation had done in preceding decades. Recent advances in the field of immunoengineering, which is evolving alongside vaccinology, have begun to increasingly influence vaccine formulation design. Historically, the particulate nature of materials used in many vaccine formulations was empiric, often because of the need to stabilize antigens or reduce endotoxin levels. However, present vaccine delivery systems are rationally engineered to mimic the size, shape, and surface chemistry of pathogens, and are therefore often referred to as "pathogen-like particles". More than a decade from his original assessment, we re-assess Plotkin's prediction. In addition, we highlight how immunoengineering and advanced delivery systems may be uniquely capable of enhancing vaccine responses in vulnerable populations, such as infants. IMPACT: Immunoengineering and advanced delivery systems are leading to new developments in pediatric vaccinology. Summarizes delivery systems currently in use and development, and prospects for the future. Broad overview of immunoengineering's impact on vaccinology, catering to Pediatric Clinicians and Immunologists.