Subunit Antigens Research Articles

Association of a vaccine adjuvant with endogenous HDL increases lymph uptake and dendritic cell activation

Vaccines are a powerful health intervention but there is still an unmet need for effective preventative and therapeutic vaccines for many diseases such as cancer and infections. Interstitial (e.g. subcutaneous (SC)) injection in nano-sized carriers such as high density lipoproteins (HDLs) can improve the access of vaccine subunit antigens or adjuvants to target immune cells in the lymphatics and potentiate vaccination responses such as cytotoxic T lymphocyte (CTL) responses (Kuai et al., 2016, 2018; Qian et al., 2016). Here we examined how cholesterol conjugation to the vaccine adjuvant CpG, and incorporation into HDL, changes lymphatic absorption and association with, and processing by, dendritic cells (DCs), ultimately influencing adjuvant efficacy. We investigated the lymphatic disposition of cholesterol conjugated CpG incorporated into HDL (HDL(Chol-CpG-Cy5)) relative to free cholesterol conjugated CpG (Chol-CpG-Cy5) and unconjugated CpG (free CpG-Cy5) after SC administration in rats and mice. HDL (Chol-CpG-Cy5) and Chol-CpG-Cy5 differentially altered CpG absorption into lymph vs. blood, but surprisingly resulted in similarly higher LN accumulation relative to free CpG. The mechanism of access of Chol-CpG-Cy5 into lymph might be partly due to association with endogenous HDL at the injection site followed by transport into lymph in association with the HDL. To measure CpG association with and processing by DCs and the strength of the immune response, mice were vaccinated with free ovalbumin (OVA) co-administered with the different CpG constructs. There were significant changes in DC activation that were reflective of the trend in LN accumulation at 24 h post-vaccination. However, T cell responses at 24 h and 7 days post-vaccination were not significantly different across the CpG groups although the response was less variable for Chol-CpG-Cy5 compared to free CpG Cy5 and also HDL(Chol-CpG-Cy5) – despite similar LN accumulation with the latter. Overall, our data indicate that cholesterol conjugation and incorporation into HDL increases adjuvant lymph disposition and DC activation.

Quantitative microcapillary electrophoresis immunoassay (mCE IA) for end-to-end analysis of pertactin within in-process samples and Quadracel® vaccine

Protein concentration is an important attribute in the production of subunit or component-based vaccine antigens. Rigorous monitoring of protein concentration is required to identify potential areas for yield improvement. The current GMP method for quantitation is the plate-based ELISA which requires numerous hands-on steps and has low sensitivity in comparison to new microfluidic systems. To address this issue, a sensitive automated microCapillary Electrophoresis ImmunoAssay (mCE IA) method was developed to accurately separate and quantitate pertactin (PRN), an important antigen of the modern acellular Pertussis (aP) vaccine. PRN is reported to be a low-yielding antigen; thus, it is critical to observe its concentration throughout its manufacturing process. First, a primary antibody for PRN was identified to establish suitable immunoprobing conditions for detection of PRN over a wide linear dynamic range that spans 3 orders of magnitude. Next, the pre-adsorbed PRN Drug Substance (DS) was used as a reference standard to quantitate PRN samples against a calibration curve with adequate accuracy and precision. Four representative samples including three in-process steps and final adjuvanted drug product: Quadracel®, were examined to demonstrate the capability of mCE IA to quantitate PRN with high sensitivity and specificity. The matrices of the selected samples contain additional components (e.g. other proteins, growth factors, cell culture media, residual ammonium sulfate, and aluminum adjuvant) often making the quantitation of PRN challenging. The specificity and method linearity were demonstrated by spiking pre-adsorbed PRN DS into the four representative samples. In addition, it was shown that reportable concentrations of PRN for nine downstream process steps as analyzed by our method is comparable to concentrations obtained with ELISA. Most importantly, this study demonstrated that our method’s quantitative accuracy is independent of matrix components, as each sample undergoes extensive dilution. This allows for seamless end-to-end analysis of PRN from fermenter harvest, through to complex downstream process samples to adjuvanted drug products. Finally, for the first time the developed and qualified mCE IA method was shown to quantify PRN throughout the entire manufacturing process to provide rapid feedback for process optimizations allowing for accurate yield and step-loss calculations.

Mucosal vaccines - fortifying the frontiers.

Mucosal vaccines offer the potential to trigger robust protective immune responses at the predominant sites of pathogen infection. In principle, the induction of adaptive immunity at mucosal sites, involving secretory antibody responses and tissue-resident T cells, has the capacity to prevent an infection from becoming established in the first place, rather than only curtailing infection and protecting against the development of disease symptoms. Although numerous effective mucosal vaccines are in use, the major advances seen with injectable vaccines (including adjuvanted subunit antigens, RNA and DNA vaccines) have not yet been translated into licensed mucosal vaccines, which currently comprise solely live attenuated and inactivated whole-cell preparations. The identification of safe and effective mucosal adjuvants allied to innovative antigen discovery and delivery strategies is key to advancing mucosal vaccines. Significant progress has been made in resolving the mechanisms that regulate innate and adaptive mucosal immunity and in understanding the crosstalk between mucosal sites, and this provides valuable pointers to inform mucosal adjuvant design. In particular, increased knowledge on mucosal antigen-presenting cells, innate lymphoid cell populations and resident memory cells at mucosal sites highlights attractive targets for vaccine design. Exploiting these insights will allow new vaccine technologies to be leveraged to facilitate rational mucosal vaccine design for pathogens including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for cancer.

Abstract 1540: CARs and TCRs: Myth and reality

Abstract Here we address questions of comparative TCR and CAR function: How different are CARs from TCRs with regard to antigen sensitivity, selectivity, co-stimulation dependence, checkpoint control, cytokine sensitivity, and antigen-dependent exhaustion (defined as loss of cytotoxicity and proliferative capacity over time). We used optimized CARs and TCRs matched to the same target pMHCs, allowing direct comparison of these important parameters. We describe a large-scale, systematic comparison of 17 CARs and 5 TCRs directed at 5 different pMHC targets in both acute and chronic settings in vitro. Surprisingly, we find little fundamental difference between CARs and TCRs with respect to their relative sensitivity to known regulators of T cell activation, including co-stimulation, checkpoint, and cytokine control. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved, CARs can achieve levels of sensitivity and selectivity similar to their vaunted counterparts. Thus, other than signaling sensitivity (and perhaps selectivity), there may be little to distinguish CARs from TCRs in the context of engineered-cell therapy. CARs appear similar to TCRs with regard to immune control, undermining the notion that CARs can, of themselves, overcome regulators that limit T cell activity. Nevertheless, CARs tolerate much more sequence variability within their structural components and this, coupled with their capability to bind different antigen classes, smaller size and subunit simplicity, confers advantages that may foster a leading role for CARs in the next generation of cell therapeutics. We believe that the major conclusions reached here should instruct thinking about the boundaries around therapeutic designs for CARs, which appear to be very wide, and those around differentiation from TCRs, which appear to be much narrower. Citation Format: Xueyin Wang, Aaron D. Martin, Kathleen Negri, Michele McElvain, Julyun Oh, Ming-Lun Wu, Wen-Hua Lee, Agnes Hamburger, Han Xu, Alexander Kamb. CARs and TCRs: Myth and reality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1540.

Direct-Acting Small Molecule Activators of PP2A (SMAPs) in Myeloid Malignancies: Understanding Mechanisms of Cytotoxicity to Inform Rational Combinatorial Therapeutic Strategies

The protein phosphatase 2A (PP2A) functions as a tumor suppressor by counteracting oncogenic kinase signaling. Suppression of PP2A activity represents a critical process in the development and maintenance of many neoplasms, including myeloid malignancies; PP2A inhibition de-represses numerous proliferative and survival signaling pathways. However, complete loss of PP2A is lethal to cells. As such, indirect reactivation of PP2A has emerged as a promising therapeutic strategy for myeloid leukemias. Yet the broad applicability of such a strategy remains uncertain, as it relies on targeting endogenous PP2A inhibitors. In the present work we investigated the effects of direct acting small molecule activators of PP2A (SMAPs) (Sandokar et al. JCI 2017) on myeloid leukemia cells and uncovered a novel link between PP2A reactivation and NOXA-mediated apoptosis. This mechanism explains our observation of cooperativity between SMAPs and the BH3 mimetic ABT-199 (venetoclax). Additionally, we found that SMAPs elicit a protective stress response that can be overcome by inhibition ofprotein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).Given the known role of PP2A inactivation in both acute and chronic myeloid leukemia (AML and CML), we first tested the activity of SMAPs against cell lines derived from both diseases. Across all cell lines tested (CML- K562, Meg-01; AML- MOLM-14, HL-60, OCI-AML3, THP-1) SMAPs caused a dose-dependent decrease in cell viability. It is known that reactivation of PP2A induces apoptosis in myeloid leukemia cells, though this area has yet to be thoroughly investigated. Therefore, we first sought to clarify the mode of cell death in response to PP2A reactivation. Both by biochemical and flow cytometric analyses, SMAPs showed activation of the intrinsic apoptotic cascade. Furthermore, SMAP-mediated cell death was blocked by co-treatment with the caspase inhibitor z-VAD-FMK (50 μM). We previously demonstrated the target specificity of SMAPs for PP2A with methods including expression of SV40 small T antigen and PP2A alpha subunit drug-binding mutants (Sandokar et al. JCI 2017). Here, we confirmed that pre-treatment with the PP2A inhibitor okadaic acid at a dose that retains selectivity for PP2A (10 nM) attenuated SMAP-induced cell death and cleavage of PARP and caspase-3.Next, we investigated the effectors of SMAP-mediated apoptosis. To this end, we performed Western blots to detect changes in abundance and/or phosphorylation of a panel of Bcl-2 family member proteins. Interestingly, a strong upregulation of the pro-apoptotic protein NOXA occurred consistently across AML and CML cell lines. Further RT-PCR studies showed this increase to occur at the transcript level. Knockdown studies confirmed the importance of NOXA for SMAP-mediated apoptosis. These data represent the first reported link between PP2A and NOXA regulation. Since SMAPs target the NOXA-MCL1 axis of the Bcl-2 family, we next tested their efficacy in combination with the Bcl-2-selective BH3 mimetic ABT-199. We found co-treatment with SMAPs and ABT-199 to exhibit enhanced cell death compared to either agent alone.Concurrent with NOXA elevation, SMAP treatment modulated protein expression and phosphorylation levels for several known pathways downstream of PP2A. Interestingly, we additionally observed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) in SMAP-treated cells which correlated with upregulation of mRNA and protein for the downstream transcription factor ATF4. Blockade of eIF2α phosphorylation with PERK inhibitor GSK2656157 (350 nM) prevented ATF4 elevation and enhanced SMAP-induced apoptosis. This result supports the idea that the integrated stress response (ISR) via PERK-eIF2α-ATF4 acts to counter apoptosis in the context of PP2A reactivation in myeloid leukemia cells. Thus, the efficacy of SMAPs and other PP2A-activating therapeutics may be enhanced by combination with both BH3 mimetics and inhibitors of the ISR. DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

Defective HLA Class I Expression and Patterns of Lymphocyte Infiltration in Chordoma Tumors.

There are no effective systemic therapies for chordoma. The recent successes of immunotherapeutic strategies in other cancers have resulted in a resurgence of interest in using immunotherapy in chordoma. These approaches rely on a functional interaction between the host's immune system and the expression of tumor peptides via the human leukocyte antigen (HLA) Class I antigen. It is not known whether chordoma cells express the HLA Class I antigen. (1) Do chordoma tumors exhibit defects in HLA Class I antigen expression? (2) What is the pattern of lymphocyte infiltration in chordoma tumors? Patients with chordoma treated at Massachusetts General Hospital between 1989 and 2009 were identified with permission from the institutional review board. Of the 75 patients who were identified, 24 human chordoma tumors were selected from 24 distinct patients based on tissue availability. Histology slides from these 24 formalin-fixed paraffin-embedded chordoma tissue samples were deparaffinized using xylene and ethanol and underwent heat-induced antigen retrieval in a citrate buffer. Samples were incubated with monoclonal antibodies directed against HLA Class I antigen processing machinery components. Antibody binding was detected via immunohistochemical staining. Staining intensity (negative, weakly positive, strongly positive) was assessed semiquantitatively and the percentage of chordoma cells stained for HLA Class I antigen subunits was assessed quantitatively. Hematoxylin and eosin-stained histology slides from the same 24 chordoma samples were assessed qualitatively for the presence of tumor-infiltrating lymphocytes and histologic location of these lymphocytes. Immunohistochemical staining with monoclonal antibodies directed against CD4 and CD8 was performed in a quantitative manner to identify the lymphocyte subtype present in chordoma tumors. All results were scored independently by two investigators and were confirmed by a senior bone and soft tissue pathologist. Seven of 24 chordoma samples exhibited no staining by the anti-HLA-A heavy chain monoclonal antibody HC-A2, two had weak staining intensity, and eight had a heterogeneous staining pattern, with fewer than 60% of chordoma cells exhibiting positive staining results. Four of 24 samples tested were not stained by the anti-HLA-B/C heavy chain monoclonal antibody HC-10, five had weak staining intensity, and 11 displayed a heterogeneous staining pattern. For the anti-β-2-microglobulin monoclonal antibody NAMB-1, staining was detected in all samples, but 11 had weak staining intensity and four displayed a heterogeneous staining pattern. Twenty-one of 24 samples tested had decreased expression in at least one subunit of HLA Class I antigens. No tumors were negative for all three subunits. Lymphocytic infiltration was found in 21 of 24 samples. Lymphocytes were primarily found in the fibrous septae between chordoma lobules but also within the tumor lobules and within the fibrous septae and tumor lobules. Twenty-one of 24 tumors had CD4+ T cells and 11 had CD8+ T cells. In chordoma tissue samples, HLA Class I antigen defects commonly were present, suggesting a mechanism for escape from host immunosurveillance. Additionally, nearly half of the tested samples had cytotoxic CD8+ T cells present in chordoma tumors, suggesting that the host may be capable of mounting an immune response against chordoma tumors. The resulting selective pressure imposed on chordoma tumors may lead to the outgrowth of chordoma cell subpopulations that can evade the host's immune system. These findings have implications in the design of immunotherapeutic strategies for chordoma treatment. T cell recognition of tumor cells requires HLA Class I antigen expression on the targeted tumor cells. Defects in HLA Class I expression may play a role in the clinical course of chordoma and may account for the limited or lack of efficacy of T cell-based immunity triggered by vaccines and/or checkpoint inhibitors.

A single subcutaneous or intranasal immunization with adenovirus-based SARS-CoV-2 vaccine induces robust humoral and cellular immune responses in mice.

Optimal vaccines are needed for sustained suppression of SARS‐CoV‐2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS‐CoV‐2 S1 subunit antigen (Ad5.SARS‐CoV‐2‐S1) for COVID‐19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS‐CoV‐2‐S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1‐specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS‐CoV‐2‐S1 produced S1‐specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen‐specific T‐cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus‐specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long‐term immunity. Thus, this Ad5‐vectored SARS‐CoV‐2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad‐based vaccines against COVID‐19 and other infectious diseases for sustainable global immunization programs.

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

BackgroundVaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants.ResultsWe describe a holistic approach for the molecular design of recombinant protein antigens—considering both their manufacturability and antigenicity—informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii.ConclusionsThis study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

Robust Anti-SARS-CoV-2 Humoral and Cellular Immunity through cGAMP-loaded Microparticle Adjuvanted Subunit Vaccination

Abstract In less than one year, the COVID-19 pandemic has infected over 95 million people with greater than two million deaths globally. Vaccines against the SARS-CoV-2 virus offer the best chance at curbing the severity and spread of COVID-19. While initial COVID-19 vaccine candidates have demonstrated great promise and efficacy, it is unknown how broad and durable those responses will be. Furthermore, eliciting strong antiviral immune responses will prove critical against future emerging strains. Previously, we demonstrated that cyclic-AMP-GMP (cGAMP) encapsulated within acetalated dextran microparticles (MPs) adjuvants influenza subunit antigens to strongly protect against influenza viral challenge when used as an intramuscular vaccination in a mouse and ferret model. Based on these studies, we hypothesized that vaccination with the stabilized spike protein of SARS-CoV-2 (S2P) and cGAMP MPs would lead to protective anti-SARS-CoV-2 immune responses. We demonstrate that S2P vaccination with cGAMP MPs lead to increased total anti-SARS-CoV-2 IgG with a strong Th1 skewed IgG2c antibody response compared to other clinically relevant adjuvants. Vaccination using cGAMP MPs also increased the frequency of activated Th1 antigen-specific T cell responses compared to controls and other vaccine adjuvants, as determined by IL-2 and INFg ELISPOTs. Finally, vaccination using cGAMP MPs lead to comparable SARS-CoV-2 neutralizing antibody production and protection against mouse adapted SARS-CoV-2 infection. These studies demonstrate that vaccination with S2P and cGAMP MPs lead to a protective humoral response and improved antigen-specific T cell responses compared to other clinically relevant adjuvants.

Prevalence of SARS-CoV-2 Specific Antibodies in Asymptomatic Hemodialysis Patients

ABSTRACT Background Since the outbreak of the new coronavirus pandemic, the importance of carrying out an infection check to prevent acquisition and transmission among end-stage renal disease patients (ESRD) under maintenance hemodialysis (MHD) has become a major concern in the health care system. Applying serology screening tests could enlighten the view with regards to disease prevalence in dialysis wards. Methods We subjected 328 end-stage renal disease patients to maintenance hemodialysis. After dividing patients into suspicious and non-suspicious groups for COVID-19 infection based on their clinical manifestation, they were investigated for SARS-CoV-2 specific IgM and IgG screening against nucleoprotein (NP), spike protein (SP), and receptor-binding domain (RBD), utilizing our recently developed ELISA tests. Results We found that approximately 10.1% of asymptomatically tested cases were antibody positive. Although IgG positivity showed a higher prevalence than IgM across all three virus antigen subunits, there were no significant differences among mentioned immunoglobulins of the studied groups. The most prevalent antibody was from the IgG subtype against virus nucleoprotein (NP), while the lowest prevalence was attributed to receptor-binding domain (RBD) IgM. Conclusion High seropositive rate among asymptomatic end-stage renal disease patients, as a sample of high-risk population, reflected the importance of considering SARS-CoV-2 specific antibody screening for disease containment.

PepFun: Open Source Protocols for Peptide-Related Computational Analysis.

Peptide research has increased during the last years due to their applications as biomarkers, therapeutic alternatives or as antigenic sub-units in vaccines. The implementation of computational resources have facilitated the identification of novel sequences, the prediction of properties, and the modelling of structures. However, there is still a lack of open source protocols that enable their straightforward analysis. Here, we present PepFun, a compilation of bioinformatics and cheminformatics functionalities that are easy to implement and customize for studying peptides at different levels: sequence, structure and their interactions with proteins. PepFun enables calculating multiple characteristics for massive sets of peptide sequences, and obtaining different structural observables derived from protein-peptide complexes. In addition, random or guided library design of peptide sequences can be customized for screening campaigns. The package has been created under the python language based on built-in functions and methods available in the open source projects BioPython and RDKit. We present two tutorials where we tested peptide binders of the MHC class II and the Granzyme B protease.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Recombinant protein vaccines, a proven approach against coronavirus pandemics.

With the COVID-19 pandemic now ongoing for close to a year, people all over the world are still waiting for a vaccine to become available. The initial focus of accelerated global research and development efforts to bring a vaccine to market as soon as possible was on novel platform technologies that promised speed but had limited history in the clinic. In contrast, recombinant protein vaccines, with numerous examples in the clinic for many years, missed out on the early wave of investments from government and industry. Emerging data are now surfacing suggesting that recombinant protein vaccines indeed might offer an advantage or complement to the nucleic acid or viral vector vaccines that will likely reach the clinic faster. Here, we summarize the current public information on the nature and on the development status of recombinant subunit antigens and adjuvants targeting SARS-CoV-2 infections.

Intranasal Subunit Vaccination Strategies Employing Nanomaterials and Biomaterials.

Intranasal vaccines offer key advantages over traditional needle-based vaccines. They are simple to administer and painless and establish local immunity at mucosal surfaces. Owing to these advantages, they are particularly attractive for use in resource-limited locations of the world. Subunit vaccines also have advantages for global distribution, as they can be engineered to be more stable to fluctuations in environmental conditions than live-attenuated or inactivated vaccines, but they tend to be poorly immunogenic intranasally. Toward realizing the potential of intranasal subunit vaccination, biomaterial-based technologies are emerging. This review provides an overview of recent progress in the preclinical development of biomaterial-based intranasal vaccines against subunit antigens and should serve as an effective introduction to the current state of this exciting field. We provide a brief overview of the obstacles facing intranasal vaccine development and identify key design criteria for consideration when designing biomaterials for intranasal subunit vaccine delivery. Promising strategies are discussed across a wide array of biomaterial classes, with a focus on selected exemplary works that highlight the considerable potential of intranasal vaccines and the biomaterial-based technologies that enable them.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Controlled Covalent Conjugation of a Tuberculosis Subunit Antigen (ID93) to Liposome Improved In Vitro Th1-Type Cytokine Recall Responses in Human Whole Blood.

Tuberculosis (TB) remains a foremost poverty-related disease with a high rate of mortality despite global immunization with Bacille Calmette–Guérin (BCG). Several adjuvanted recombinant proteins are in clinical development for TB to protect against the disease in infants and adults. Nevertheless, simple mixing of adjuvants with antigens may not be optimal for enhancing the immune response due to poor association. Hence, co-delivery of adjuvants with antigens has been advocated for improved immune response. This report, therefore, presents a strategy of using chemical conjugation to co-deliver an adjuvanted recombinant protein TB vaccine (ID93 + GLA-LSQ). Chemical conjugation involving glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used to associate the antigen (ID93) to the modified liposome (mGLA-LSQ). The physicochemical stability of the formulations was evaluated using high-performance liquid chromatography (HPLC) (adjuvant content), dynamic light scattering (DLS, particle size analysis), and sodium dodecyl sulfate-polyacrylamide gel (SDS) electrophoresis (protein analysis). The bioactivity was assessed by cytokine stimulation using fresh whole blood from 10 healthy donors. The conjugates of ID93 + mGLA_LSQ maintained liposomal and protein integrity with the two protein chemistries. The GLA and QS21 content of the vaccine were also stable for 3 months. However, only the glutaraldehyde conjugates provoked significant secretion of interleukin-2 (210.4 ± 11.45 vs 166.7 ± 9.15; p = 0.0059), interferon-gamma (210.5 ± 14.79 vs 144.1 ± 4.997; p = 0.0011), and tumor necrosis factor alpha (2075 ± 46.8 vs 1456 ± 144.8; p = 0.0082) compared to simple mixing. Conjugation of recombinant protein (ID93) to the liposome (mGLA_LSQ) through chemical conjugation resulted in a stable vaccine formulation, which could facilitate co-delivery of the subunit vaccine to promote a robust immune response.

Rational design of adjuvants for subunit vaccines: The format of cationic adjuvants affects the induction of antigen-specific antibody responses

A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.

Comparison of Immune Effects Between Brucella Recombinant Omp10-Omp28-L7/L12 Proteins Expressed in Eukaryotic and Prokaryotic Systems.

Brucella, a genus of bacteria that causes brucellosis, infects and threatens domestic animals, and humans in endemic areas. Presently, some live attenuated vaccines of Brucella are used to immunize livestock; however, these vaccines are pathogenic to humans, can provoke abortion when administered to pregnant livestock, and induce antibodies in vaccinated livestock that affect the diagnosis of field infection. It is, therefore, very important for improving the safety and immune protection effects of Brucella vaccine. Currently, recombinant protein-based subunit vaccines are considered promising safe and effective alternatives against brucellosis. Here, we separately expressed the recombinant Omp10-Omp28-L7/L12 proteins of Brucella using eukaryotic and prokaryotic expression systems, which were then used as immunogens for evaluating their immune responses. Taishan Pinus massoniana pollen polysaccharides (TPPPS), an already verified natural adjuvant, was utilized to evaluate the immune conditioning effect on the recombinant proteins. Antibody levels, spleen lymphocyte proliferation, percentages of CD4+ and CD8+ T cells, and cytokine secretion in mice were examined after three successive immunizations. The protective effects against Brucella challenge were also evaluated in mice, and used a live vaccine as a positive control. The results indicated that the immune responses of the recombinant Omp10-Omp28-L7/L12 protein groups were significantly higher than those of the PBS control group. The recombinant Omp10-Omp28-L7/L12 protein expressed in Pichia pastoris (P. pastoris) exhibited a slightly higher expression level and immunogenicity than that expressed in Escherichia coli (E. coli), and the Omp10-Omp28-L7/L12 (P. pastoris) + TPPPS group provided the most pronounced immune effect. The protective results showed that the recombinant Omp10-Omp28-L7/L12 proteins expressed in the two expression systems had significantly better protective effects against Brucella melitensis challenge compared with the negative control, and the addition of TPPPS adjuvant could significantly improve the protective effects of subunit vaccines. However, we also noticed that all of the evaluated subunit vaccines induced less protection than the B. melitensis M5 live vaccine. These results indicate that the combination of recombinant Omp10-Omp28-L7/L12 antigen and TPPPS adjuvant shows potential as an effective brucellosis subunit vaccine, and P. pastoris is a preferred expression system to prepare this recombinant subunit antigen.

Dimerization of Dengue Virus E Subunits Impacts Antibody Function and Domain Focus.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

Temporal dynamics of innate and adaptive immune responses in broiler birds to oral delivered chitosan nanoparticle-based Salmonella subunit antigens

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) infection of poultry causes a significant risk to public health through contamination of meat and eggs. Current Salmonella vaccines have failed to provide strong mucosal immunity in the intestines to reduce Salmonella shedding and food contamination. Considering the short lifespan of broilers, an easy-to-deliver, safe and effective Salmonella vaccine is urgently needed. Our goal in this study was to demonstrate the ability of chitosan nanoparticle (CNP) vaccine delivery platform in activating immune response to Salmonella antigens in broilers inoculated orally. In an initial study, soluble whole antigen of SE entrapped in CNP was inoculated but the specific immune responses were poor. Therefore, the CNP entrapped immunogenic outer membrane proteins (OMP) and flagellin (FLA) of SE and surface conjugated with FLA [CNP-(OMP + FLA)] was developed. In broilers inoculated orally with CNP-(OMP + FLA) formulation once or twice, we monitored the temporal expression of innate immune molecules and antigen specific lymphocyte proliferation. In the cecal tonsils of CNP-(OMP + FLA) inoculated birds, we observed enhanced expression of mRNA coding Toll-like receptors (TLRs)- 1, 4, 5, and 7, especially at dpv 21. In addition, both OMP and FLA specific lymphocytes proliferation at dpv 7 and 21 by CNP-(OMP + FLA) were enhanced in the spleen. In conclusion, CNP-(OMP + FLA) formulation augmented both innate and lymphocyte responses in orally inoculated broilers. Further studies are needed to determine the candidate subunit CNP vaccine’s efficacy in a challenge trial.