Antigen-specific Cellular Immune Responses Research Articles

DnaJ, a promising vaccine candidate against Ureaplasma urealyticum infection.

Ureaplasma urealyticum (U. urealyticum, Uu) is a common sexually transmitted pathogen that is responsible for diseases such as non-gonococcal urethritis, chorioamnionitis, and neonatal respiratory diseases. The rapid emergence of multidrug-resistant bacteria threatens the effective treatment of Uu infections. Considering this, vaccination could be an efficacious medical intervention to prevent Uu infection and disease. As a highly conserved molecular chaperone, DnaJ is expressed and upregulated by pathogens soon after infection. Here, we assessed the vaccine potential of recombinant Uu-DnaJ in a mouse model and dendritic cells. Results showed that intramuscular administration of DnaJ induced robust humoral- and T helper (Th) 1 cell-mediated immune responses and protected against genital tract infection, inflammation, and the pathologic sequelae after Uu infection. Importantly, the DnaJ protein also induced the maturation of mouse bone marrow–derived dendritic cells (BMDCs), ultimately promoting naïve T cell differentiation toward the Th1 phenotype. In addition, adoptive immunization of DnaJ-pulsed BMDCs elicited antigen-specific Immunoglobulin G2 (IgG2) antibodies as well as a Th1-biased cellular response in mice. These results support DnaJ as a promising vaccine candidate to control Uu infections.Key points• A novel recombinant vaccine was constructed against U. urealyticum infection.• Antigen-specific humoral and cellular immune responses after DnaJ vaccination.• Dendritic cells are activated by Uu-DnaJ, which results in a Th1-biased immune response.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-12230-4.

A novel multi-epitope recombinant protein elicits an antigen-specific CD8+ T cells response in Trypanosoma cruzi-infected mice

About 6.5 million people worldwide are afflicted by Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. The development of a therapeutic vaccine to prevent the progression of Chagasic cardiomyopathy has been proposed as an alternative for antiparasitic chemotherapy. Bioinformatics tools can predict MHC class I CD8 + epitopes for inclusion in a single recombinant protein with the goal to develop a multivalent vaccine. We expressed a novel recombinant protein Tc24-C4.10E harboring ten nonameric CD8 + epitopes and using Tc24-C4 protein as scaffold to evaluate the therapeutic effect in acute T. cruzi infection. T. cruzi-infected mice were immunized with Tc24-C4.10E or Tc24-C4 in a 50-day model of acute infection. Tc24-C4.10E-treated mice showed a decreased parasitemia compared to the Tc24-C4 (non-adjuvant) immunized mice or control group. Moreover, Tc24-C4.10E induced a higher stimulation index of CD8 + T cells producing IFNγ and IL-4 cytokines. These results suggest that the addition of the MHC Class I epitopes to Tc24-C4 can synergize the antigen-specific cellular immune responses, providing proof-of-concept that this approach could lead to the development of a promising vaccine candidate for Chagas disease.

Not all T cell epitopes are equally desired: a review of in silico tools for the prediction of cytokine-inducing potential of T-cell epitopes.

Assessment of protective or harmful T cell response induced by any antigenic epitope is important in designing any immunotherapeutic molecule. The understanding of cytokine induction potential also helps us to monitor antigen-specific cellular immune responses and rational vaccine design. The classical immunoinformatics tools served well for prediction of B cell and T cell epitopes. However, in the last decade, the prediction algorithms for T cell epitope inducing specific cytokines have also been developed and appreciated in the scientific community. This review summarizes the current status of such tools, their applications, background algorithms, their use in experimental setup and functionalities available in the tools/web servers.

Smart combination of aluminum hydroxide and MF59 to induce strong cellular immune responses.

As two of the most widely used adjuvants, aluminum hydroxide and the oil-in-water emulsion MF59 have their intrinsic limitations: classical aluminum gel induces only weak cellular immune responses while MF59 cannot be used as an antigen delivery system due to its poor physical interaction with antigen molecules. Herein, we combined these two adjuvants and constructed a novel nano-vaccine delivery system by inserting aluminum hydroxide into the surface of a modified MF59 nano-emulsion (AlNEs). A model antigen ovalbumin (OVA) and an immune potentiator CpG were adsorbed on the surface of AlNEs (hereinafter AlNEs-OVA-CpG) through a facile mixing step. After subcutaneous injection, AlNEs-OVA-CpG effectively drained to lymph nodes, delivered both cargos into lymph node-resident antigen presenting cells (APCs), and escaped from lysosomes into the cytoplasm, resulting in enhanced antigen cross-presentation. Finally, AlNEs-OVA-CpG induced potent antigen-specific humoral and cellular immune responses, which significantly inhibited tumor growth and prolonged mice survival in a EG7-OVA tumor model. In sum, our results suggested that AlNEs have a great prospect to induce CD8+ T cell responses for subunit antigens.

A personal COVID-19 dendritic cell vaccine made at point-of-care: Feasibility, safety, and antigen-specific cellular immune responses

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a world-wide pandemic. Internationally, because of availability, accessibility, and distribution issues, there is a need for additional vaccines. This study aimed to: establish the feasibility of personal dendritic cell vaccines to the SARS-CoV-2 spike protein, establish the safety of a single subcutaneous vaccine injection, and determine the antigen-specific immune response following vaccination. In Phase 1, 31 subjects were assigned to one of nine formulations of autologous dendritic cells and lymphocytes (DCL) incubated with 0.10, 0.33, or 1.0 µg of recombinant SARS-CoV-2 spike protein, and admixed with saline or 250 or 500 µg of granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to injection, then assessed for safety and humoral response. In Phase 2, 145 subjects were randomized to one of three formulations defined by incubation with the same three quantities of spike protein without GM-CSF, then assessed for safety and cellular response. Vaccines were successfully manufactured for every subject at point-of-care. Approximately 46.4% of subjects had a grade 1 adverse event (AE); 6.5% had a grade 2 AE. Among 169 evaluable subjects, there were no acute allergic, grade 3 or 4, or serious AE. In Phase 1, anti-receptor binding domain antibodies were increased in 70% of subjects on day-28. In Phase 2, in the 127 subjects who did not have high levels of gamma interferon-producing cells at baseline, 94.4% had increased by day 14 and 96.8% by day 28. Point-of-care personal vaccine manufacturing was feasible. Further development of such subject-specific vaccines is warranted.

Protection Induced by Oral Vaccination with a Recombinant Yersinia pseudotuberculosis Delivering Yersinia pestis LcrV and F1 Antigens in Mice and Rats against Pneumonic Plague.

A newly attenuated Yersinia pseudotuberculosis strain (designated Yptb1) with triple mutation Δasd ΔyopK ΔyopJ and chromosomal insertion of the Y. pestis caf1R-caf1M-caf1A-caf1 operon was constructed as a live vaccine platform. Yptb1 tailored with an Asd+ plasmid (pYA5199) (designated Yptb1[pYA5199]) simultaneously delivers Y. pestis LcrV and F1. The attenuated Yptb1(pYA5199) localized in the Peyer's patches, lung, spleen, and liver for a few weeks after oral immunization without causing any disease symptoms in immunized rodents. An oral prime-boost Yptb1(pYA5199) immunization stimulated potent antibody responses to LcrV, F1, and Y. pestis whole-cell lysate (YPL) in Swiss Webster mice and Brown Norway rats. The prime-boost Yptb1(pYA5199) immunization induced higher antigen-specific humoral and cellular immune responses in mice than a single immunization did, and it provided complete short-term and long-term protection against a high dose of intranasal Y. pestis challenge in mice. Moreover, the prime-boost immunization afforded substantial protection for Brown Norway rats against an aerosolized Y. pestis challenge. Our study highlights that Yptb1(pYA5199) has high potential as an oral vaccine candidate against pneumonic plague.

Rational development of a combined mRNA vaccine against COVID-19 and influenza

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

Abstract 3571: Prevention of non-small cell lung cancer with kras mutation using a novel peptide vaccine

Abstract Non-small cell lung cancer (NSCLC) is a significant health issue associated with morbidity and mortality. One of the important findings to emerge in NSCLC is that specific genetic mutations that lead to cancer development occur early in the disease process. Unfortunately, identifying NSCLC at its earliest stages when it is curable has been a challenge even in high risk individuals. These gene mutations, especially those involving the Kras protein, can “drive” the development of NSCLC cancer as they alter the regulation of cell growth or block programmed cell death. Mutations in Kras have been reported in 30% of lung cancer cases and are associated with worse outcomes. This neo-antigen cannot generate effective anti-tumor immunity, suggesting lack of a cellular, cytotoxic immune response to cancer cells on mucosal/epithelial surfaces. No effective therapies specifically targeting mutant Kras have been developed. To overcome poor immunogenicity of neo-antigen, newer adjuvants (NA), biocompatible nanoparticles, were used to enhance immune response. It has been reported NA can produce cytotoxic and mucosal immunity safely in humans. Producing this type of immune response to mutated Kras through immunization could produce a significant anti-tumor effect. In the current study, we first tested NA combined with mutated and wild type (MHC II) Kras peptides vaccine for immunogenicity on the same genetic background as the animals in the inducible tumor model. Our data have demonstrated that NA in combination with multiple Kras peptides can increase CD4+ and CD8+ T cell numbers in the draining lymph node cells and enhance antigen specific TH1 and TH17 responses by luminex and ELISpot to increase the ability of the vaccine to kill tumor cells and elicit long-lasting immune memory. Furthermore, we evaluated anti-tumor efficacy of this vaccine in tetracycline-inducible mouse model with Kras mutation. In this challenge model, vaccinated mice showed higher IFNɤ and IL-17 levels and decreased the tumor incidence significantly compared to PBS or peptides alone group. In conclusion, we have demonstrated the antigen-specific cellular immune responses and mononuclear cell infiltrates correlate with tumor prevention. Our data suggest that this novel vaccine could prevent the development of NSCLC after validation in an appropriate animal model. Citation Format: Suhe Wang, Zhengyi Cao, Jesse Chen, Janczak Katarzyna, James Baker. Prevention of non-small cell lung cancer with kras mutation using a novel peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3571.

Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Poly-γ-glutamic acid/Alum adjuvanted pH1N1 vaccine-immunized aged mice exhibit a significant increase in vaccine efficacy with a decrease in age-associated CD8+ T cell proportion in splenocytes

BackgroundHighly contagious respiratory diseases caused by viral infections are a constantly emerging threat, particularly the elderly with the higher risk of developing serious complications. Vaccines are the best strategy for protection against influenza-related diseases. However, the elderly has lower vaccine efficacy than young population and the age-driven decline of the influenza vaccine efficacy remains unresolved.ObjectivesThis study investigates the effect of an adjuvant, poly-γ-glutamic acid and alum (PGA/Alum) on vaccine efficacy in aged mice (18-months) and its mechanism is investigated using ovalbumin as a model antigen and a commercial pandemic H1N1 (pH1N1) flu vaccine. Antigen trafficking, dendritic cell (DC) activation, and the DC-mediated T cell activation were analyzed via in vivo imaging and flow cytometry. Antigen-specific humoral and cellular immune responses were evaluated in sera and splenocytes from the vaccinated mice. Also, we analyzed gene expression profiles of splenocytes from the vaccinated mice via single-cell transcriptome sequencing and evaluated the protective efficacy against pH1N1 virus challenge.ResultsAged mice had lower antigen trafficking and DC activation than younger mice (6-weeks), which was ameliorated by PGA/Alum with increased antigen uptake and DC activation leading to improved antigen-specific IFN-γ+CD8+ T lymphocyte frequencies higher in the vaccinated aged mice, to a similar extent as PGA/Alum adjuvanted vaccine-immunized young mice. The results of single-cell transcriptome sequencing display that PGA/Alum also reduced the proportion of age-associated CD8+ T cell subsets and gene levels of inhibitory regulators in CD8+ T cells, which may play a role in the recovery of CD8+ T cell activation. Finally, PGA/Alum adjuvanted pH1N1 vaccine-immunized aged mice were completely protected (100% survival) compared to aged mice immunized with vaccine only (0% survival) after pH1N1 virus challenge, akin to the efficacy of the vaccinated young mice (100% survival).ConclusionsPGA/Alum adjuvanted pH1N1 vaccine-immunized aged mice showed a significant increase in vaccine efficacy compared to aged mice administered with vaccine only. The enhanced vaccine efficacy by PGA/Alum is associated with significant increases of activation of DCs and effector CD8+ T cells and a decrease in age-associated CD8+ T cell proportion of splenocytes. Collectively, PGA/Alum adjuvanted flu vaccine may be a promising vaccine candidate for the elderly.

A nanovaccine for enhancing cellular immunity via cytosolic co-delivery of antigen and polyIC RNA.

Traditional approaches to cancer vaccines elicit weak CD8+ T cell responses and have largely failed to meet clinical expectations. This is in part due to inefficient antigen cross-presentation, inappropriate selection of adjuvant and its formulation, poor vaccine pharmacokinetics, and/or suboptimal coordination of antigen and adjuvant delivery. Here, we describe a nanoparticle vaccine platform for facile co-loading and dual-delivery of antigens and nucleic acid adjuvants that elicits robust antigen-specific cellular immune responses. The nanovaccine design is based on diblock copolymers comprising a poly(ethylene glycol)-rich first block that is functionalized with reactive moieties for covalent conjugation of antigen via disulfide linkages, and a pH-responsive second block for electrostatic packaging of nucleic acids that also facilitates endosomal escape of associated vaccine cargo to the cytosol. Using polyIC, a clinically-advanced nucleic acid adjuvant, we demonstrated that endosomolytic nanoparticles promoted the cytosolic co-delivery of polyIC and protein antigen, which acted synergistically to enhance antigen cross-presentation, co-stimulatory molecule expression, and cytokine production by dendritic cells. We also found that the vaccine platform increased the accumulation of antigen and polyIC in the local draining lymph nodes. Consequently, dual-delivery of antigen and polyIC with endsomolytic nanoparticles significantly enhanced the magnitude and functionality of CD8+ T cell responses relative to a mixture of antigen and polyIC, resulting in inhibition of tumor growth in a mouse tumor model. Collectively, this work provides a proof-of-principle for a new cancer vaccine platform that strongly augments anti-tumor cellular immunity via cytosolic co-delivery of antigen and nucleic acid adjuvant.

A Systematic Immuno-Informatic Approach to Design a Multiepitope-Based Vaccine Against Emerging Multiple Drug Resistant Serratia marcescens.

Serratia marcescens is now an important opportunistic pathogen that can cause serious infections in hospitalized or immunocompromised patients. Here, we used extensive bioinformatic analyses based on reverse vaccinology and subtractive proteomics-based approach to predict potential vaccine candidates against S. marcescens. We analyzed the complete proteome sequence of 49 isolate of Serratia marcescens and identified 5 that were conserved proteins, non-homologous from human and gut flora, extracellular or exported to the outer membrane, and antigenic. The identified proteins were used to select 5 CTL, 12 HTL, and 12 BCL epitopes antigenic, non-allergenic, conserved, hydrophilic, and non-toxic. In addition, HTL epitopes were able to induce interferon-gamma immune response. The selected peptides were used to design 4 multi-epitope vaccines constructs (SMV1, SMV2, SMV3 and SMV4) with immune-modulating adjuvants, PADRE sequence, and linkers. Peptide cleavage analysis showed that antigen vaccines are processed and presented via of MHC class molecule. Several physiochemical and immunological analyses revealed that all multiepitope vaccines were non-allergenic, stable, hydrophilic, and soluble and induced the immunity with high antigenicity. The secondary structure analysis revealed the designed vaccines contain mainly coil structure and alpha helix structures. 3D analyses showed high-quality structure. Molecular docking analyses revealed SMV4 as the best vaccine construct among the four constructed vaccines, demonstrating high affinity with the immune receptor. Molecular dynamics simulation confirmed the low deformability and stability of the vaccine candidate. Discontinuous epitope residues analyses of SMV4 revealed that they are flexible and can interact with antibodies. In silico immune simulation indicated that the designed SMV4 vaccine triggers an effective immune response. In silico codon optimization and cloning in expression vector indicate that SMV4 vaccine can be efficiently expressed in E. coli system. Overall, we showed that SMV4 multi-epitope vaccine successfully elicited antigen-specific humoral and cellular immune responses and may be a potential vaccine candidate against S. marcescens. Further experimental validations could confirm its exact efficacy, the safety and immunogenicity profile. Our findings bring a valuable addition to the development of new strategies to prevent and control the spread of multidrug-resistant Gram-negative bacteria with high clinical relevance.

Six-Month Follow-Up after Vaccination with BNT162b2: SARS-CoV-2 Antigen-Specific Cellular and Humoral Immune Responses in Hemodialysis Patients and Kidney Transplant Recipients.

Hemodialysis patients (HDP) and kidney transplant recipients (KTR) have a high risk of infection with SARS-CoV-2 with poor clinical outcomes. Because of this, vaccination of these groups of patients against SARS-CoV-2 is particularly important. However, immune responses may be impaired in immunosuppressed and chronically ill patients. Here, our aim was to compare the efficacy of an mRNA-based vaccine in HDP, KTR, and healthy subjects. Design: In this prospective observational cohort study, the humoral and cellular response of prevalent 192 HDP, 50 KTR, and 28 healthy controls (HC) was assessed 1, 2, and 6 months after the first immunization with the BNT162b2 mRNA vaccine. Results: After 6 months, 97.5% of HDP, 37.9% of KTR, and 100% of HC had an antibody response. Median antibody levels were 1539.7 (±3355.8), 178.5 (±369.5), and 2657.8 (±2965.8) AU/mL in HDP, KTR, and HC, respectively (p ≤ 0.05). A SARS-CoV-2 antigen-specific cell response to vaccination was found in 68.8% of HDP, 64.5% of KTR, and 90% of HC. Conclusion: The humoral response rates to mRNA-based vaccination of HDPs are comparable to HCs, but antibody titers are lower. Furthermore, HDPs have weaker T-cell response to vaccination than HCs. KTRs have very low humoral and antigen-specific cellular response rates and antibody titers, which requires other vaccination strategies in addition to booster vaccination.

Engineering mannosylated pickering emulsions for the targeted delivery of multicomponent vaccines

While research on cancer vaccines has made great strides in the field of immunotherapy, the targeted delivery of multiple effective components (rational-tailored antigens and adjuvants) remains a challenge. Here, we utilized the unique hierarchical structures of Pickering emulsions (particles, oil core, and water-oil interface) to develop mannosylated (M) Pickering emulsions (PE) that target antigen presenting cells and synergistically deliver antigenic peptides and the TLR9 agonist CpG (C) as an enhanced cancer vaccine (MPE-C). We chemically linked mannose residues to PLGA/PLAG-PEG nanoparticles and produced a dense array of mannose on the nanopatterned surface of Pickering emulsions, allowing for increased cellular targeting. Together with the inherent deformability of the oily core, MPE-C increased the droplet-cellular contact area and provoked the cellular recognition of mannose and CpG for enhanced immune activation. We found that MPE-C attracted a large number of APCs to the local site of administration, evidently increasing cellular uptake and activation. Additionally, we observed increased antigen-specific cellular immune responses, with potent anti-tumor effects against both E.G7-OVA and B16-MUCI tumors. Furthermore, MPE-C combined with PD-1 antibodies produced a significant tumor regression, resulting in synergistic increases in anti-tumor effects. Thus, through the strategic loading of mannose, antigens, and CpG, Pickering emulsions could serve as a targeted delivery platform for enhanced multicomponent cancer vaccines.

Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant

The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.

COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

Multifunctional Fructose 1,6-Bisphosphate Aldolase as a Therapeutic Target.

Fructose 1,6-bisphosphate aldolase is a ubiquitous cytosolic enzyme that catalyzes the fourth step of glycolysis. Aldolases are classified into three groups: Class-I, Class-IA, and Class-II; all classes share similar structural features but low amino acid identity. Apart from their conserved role in carbohydrate metabolism, aldolases have been reported to perform numerous non-enzymatic functions. Here we review the myriad “moonlighting” functions of this classical enzyme, many of which are centered on its ability to bind to an array of partner proteins that impact cellular scaffolding, signaling, transcription, and motility. In addition to the cytosolic location, aldolase has been found the extracellular surface of several pathogenic bacteria, fungi, protozoans, and metazoans. In the extracellular space, the enzyme has been reported to perform virulence-enhancing moonlighting functions e.g., plasminogen binding, host cell adhesion, and immunomodulation. Aldolase’s importance has made it both a drug target and vaccine candidate. In this review, we note the several inhibitors that have been synthesized with high specificity for the aldolases of pathogens and cancer cells and have been shown to inhibit classical enzyme activity and moonlighting functions. We also review the many trials in which recombinant aldolases have been used as vaccine targets against a wide variety of pathogenic organisms including bacteria, fungi, and metazoan parasites. Most of such trials generated significant protection from challenge infection, correlated with antigen-specific cellular and humoral immune responses. We argue that refinement of aldolase antigen preparations and expansion of immunization trials should be encouraged to promote the advancement of promising, protective aldolase vaccines.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Cellular Basis for the Enhanced Efficacy of the Fms-Like Tyrosine Kinase 3 Ligand (FL) Adjuvanted VCG-Based Chlamydia abortus Vaccine.

Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.

Chitin-derived polymer deacetylation regulates mitochondrial reactive oxygen species dependent cGAS-STING and NLRP3 inflammasome activation

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.