IgG2a Titers Research Articles

GFPBW1, a β-glucan from Grifola frondosa as vaccine adjuvant: APCs activation and maturation.

Adjuvants for vaccines with characteristics of improving adaptive immunity particularly via leverage of antigen presenting cells (APCs) are currently lacking. In a previous work we obtained a new soluble 300 kDa homogeneous β-glucan named GFPBW1 from the fruit bodies of Granola frondosa. GFPBW1 could activate macrophages by targeting dendritic cell associated C-type lectin 1 (Dectin-1)/Syk/NF-κB signaling to achieve antitumour effects. In this study the adjuvant effects of GFPBW1 were explored with OVA-antigen and B16-OVA tumor model. We showed that GFPBW1 (5, 50, 500 μg/mL) dose-dependently promoted activation and maturation of APCs in vitro by increasing CD80, CD86 and MHC II expression. We immunized female mice with OVA in combination with GFPBW1 (50 or 300 μg) twice with an interval of two weeks. GFPBW1 markedly and dose-dependently increased OVA-specific antibody titers of different subtypes including IgG1, IgG2a, IgG2b and IgG3, suggesting that it could serve as an adjuvant for both Th1 and Th2 type immune responses. Furthermore, GFPBW1 in combination with aluminum significantly increased the titers of OVA-specific IgG2a and IgG2b, but not those of IgG1, suggesting that GFPBW1 could be used as a co-adjuvant of aluminum to compensate for Th1 deficiency. For mice immunized with OVA plus GFPBW1, no obvious pathological injury was observed in either major organs or injection sites, and no abnormalities were noted for any of the hematological parameters. When GFPBW1 served as an adjuvant in the B16-OVA cancer vaccine models, it could accomplish entire tumor suppression with preventive vaccines, and enhance antitumour efficacy with therapeutic vaccines. Differentially expressed genes were found to be enriched in antigen processing process, specifically increased tumor infiltration of DCs, B1 cells and plasma cells in the OVA plus GFPBW1 group, in accordance with its activation and maturation function of APCs. Collectively, this study systematically describes the properties of GFPBW1 as a novel potent and safe adjuvant and highlights its great potential in vaccine development.

Role of muscle regenerative macrophages in adjuvant-mediated immune response skewing to seasonal influenza vaccine

Abstract MF59 (AddaVax-like) is a clinically approved nanoemulsion adjuvant with an unclear mechanism of action. Here we introduce a novel approach to investigating the role of muscle regeneration in adjuvanted vaccine response. We found that AddaVax adjuvanted quadrivalent inactivated flu vaccine (QIV) induced a Th2 immune response with IL-4, -5, and -13 expression in the lungs 5 days after H1N1 lethal challenge. In contrast, IMDQ-PC (TLR7/8 agonist) induced a Th1 lung response and higher IgG2a titers. In BMDM cultures, we found AddaVax unlike IMDQ-PC to require IFNγ co-incubation for the induction of iNOS+ proinflammatory M1 phenotype. Since IFNγ is crucial for muscle regeneration, we hypothesized that AddaVax triggers macrophage-dependent muscle regeneration which leads to an enhanced Th2 vaccine response. We found that AddaVax adjuvanted QIV led to an increase of Arg-I+ M2 macrophages in the muscle 4 days post-injection, similar to late-stage Th2 response in regeneration. Using IgG titers, Our hypothesis was further supported by finding AddaVax response to be diminished in aged mice where regeneration is impaired, but rescuable by depletion of circulating monocytes and reconstitution with young BMDMs. Using in-depth characterization and selective depletion, we will investigate the crosstalk between muscle regeneration and AddaVax response allowing a better understanding of the potential muscle role in inhibiting the Th1 response necessary in viral infections, and cancer vaccines.

Silk fibroin-based hydrogel as injectable carrier for prolonged immunization of plant-based COVID-19 subunit vaccine

Silk fibroin (SF)-based hydrogel was prepared to serve as an injectable carrier, aimed at prolonging the immunization effect of the plant-derived vaccine for the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). This vaccine consists of the Receptor-Binding Domain (RBD) linked with the Fragment Crystallizable (Fc) region of Human Immunoglobulin G 1 (IgG1), also referred to as the RBD-Fc vaccine. The formulation also includes Aluminium hydroxide gel (Alum). Ex-vivo experiments demonstrated that both the SARS-CoV-2 RBD-Fc-loaded SF hydrogel (RBD-Fc/SF hydrogel) and the Alum-adjuvanted SARS-CoV-2 RBD-Fc-loaded SF hydrogel (Alum-RBD-Fc/SF hydrogel) indicated localized retention. This resulted in a controlled and prolonged release of 75% and 25% over a period of 60 days, respectively. This facilitated continuous interaction between the immune system and the viral peptide, specifically the RBD, which could have significant implications for geometric mean titer (GMT). In immunization studies, the RBD-Fc/SF hydrogel and Alum-RBD-Fc/SF hydrogel had induced a more prolonged and stronger immunogenic response of mouse-RBD-specific IgG titer and IgG subtypes (IgG1 and IgG2a). The specific IgG1 and IgG2a titers of RBD-Fc/SF hydrogel (with GMTs of 67,559 and 1,056, respectively) and Alum-RBD-Fc/SF hydrogel (with GMTs of 155,209 and 1,838, respectively) were superior to the RBD-Fc vaccine without SF hydrogel (with GMTs of 3200 and 200, respectively). Furthermore, they were comparable to the responses elicited by commercial Alum-adjuvants currently in use (with GMTs of 51,200 and 4,850, respectively). Additionally, a concentration of 0.1%w/v SF exhibited significant adjuvant properties by augmenting the secretion of various cytokines. For instance, it enhanced the secretion of Interleukin-6 (IL-6) to 88.02 pg/mL, which is comparable to the secretion induced by commercial Alum adjuvants (39.48 pg/mL). These findings suggest that SF hydrogel-based vaccine formulations could enhance humoral immunity and induce long-lasting immune responses. However, further investigations are required to assess the neutralizing potential of the antibodies produced against SARS-CoV-2 and its variants. The prospects and outcomes of SF hydrogel-based vaccine formulations indicate their potential as a next-generation vaccine platform. Such innovative strategies could be pivotal in the fight against COVID-19 and other infectious diseases.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model. In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-β concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined. According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens. The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

1169. Unconjugated Multi-epitope Peptides Adjuvanted with ALFQ Induce Durable and Broadly Reactive Antibodies to Human and Avian Influenza Viruses

Abstract Background Traditional influenza vaccine production often leads to mismatch due to antigenic drift, thereby reducing protection. Alternate vaccine strategies that are peptide-based, nanoparticle-based or nucleic acid-based are being developed. Composite multi-epitope peptides provide a cost-effective and easily scalable strategy towards a universal influenza vaccine. In this study, we demonstrate that unconjugated, multi-epitope peptides formulated with the highly potent adjuvant Army Liposome Formulation with QS-21 (ALFQ) generate broadly reactive durable antibodies to human and avian influenza viruses when administered intramuscularly or intradermally. Methods ICR mice were immunized with an unconjugated composite influenza peptide vaccine comprising HA+NA (Flu Pep11) and Matrix (M1/M2/M2e)+T-cell epitope (Flu Pep5906) formulated with ALFQ. A low-dose (1 µg) and high-dose (20 µg) of vaccine was administered intramuscularly or intradermally. Isotype-specific IgG titers to composite peptides, individual epitopes, and multiple strains of influenza A (H1N1, H3N2, H5N1), and B (Yamagata, Victoria) were analyzed by ELISA. Neutralizing titers against Group 1 and Group 2 viruses were determined using microneutralization assay. Results A robust IgG1 and IgG2b antibody response was seen at day 21 post primary immunization and remained steady for all groups up to day 200 (duration of study). IgG2a and IgG3 titers were also generated. Antibodies recognized multiple strains each of human and avian influenza A and influenza B virus subtypes, 200 days post primary immunization, and were comparable between doses and routes. Strong neutralizing titers were shown against Group 1 and 2 influenza viruses. Conclusion An unconjugated peptide vaccine comprising multiple highly conserved epitopes of HA, NA and Matrix formulated with ALFQ adjuvant and administered intramuscularly or intradermally, generated broad and durable antibodies to human and avian influenza viruses. Induced antibodies neutralized seasonal and pandemic influenza strains. These composite peptides adjuvanted with ALFQ generate durable and broad immunity and provide a cost-effective and easily scalable strategy that moves us closer to a universal influenza vaccine. Disclosures All Authors: No reported disclosures

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

Boosting Mouse Defense against Lethal Toxoplasma gondii Infection with Full-Length and Soluble SAG1 Recombinant Protein.

Toxoplasmosis is a major worldwide protozoan zoonosis. The surface antigen 1 (SAG1) of Toxoplasma gondii (T. gondii) has always been recognized as an ideal vaccine candidate antigen. However, the intact and soluble SAG1 protein is usually difficult to acquire in vitro, which is unfavorable for employing the recombinant protein as a vaccine candidate antigen. In the present study, we obtained the full-length SAG1 recombinant protein in soluble form by Escherichia coli Transetta (DE3) cells under optimized expression conditions. The immunogenicity and protective ability of this recombinant protein against T. gondii acute infection were evaluated in a mouse model. Monitoring changes in serum antibody levels and types, the presence of cytokines, and the rate of lymphocyte proliferation in vaccinated mice were used to assess humoral and cellular immune responses. Additional assessments were performed to determine the protective potency of the recombinant protein in combating T. gondii RH tachyzoites. It was found that the titers of both IgG2a and IgG2b were considerably greater in the immunized mice compared to the titers of IgG1 and IgG3. The levels of Th1-type cytokines (IFN-γ, IL-12p70, IL-2, and TNF-α) and Th2-type cytokines (IL-10) significantly increased when splenocytes from immunological group mice were treated with T. gondii lysate antigen. Compared to the control group, a recombinant protein substantially increased the longevity of infected mice, with an average death time prolonged by 14.50 ± 0.34 days (p < 0.0001). These findings suggest that the full-length and soluble SAG1 recombinant protein produced potent immune responses in mice and could be a preferred subunit vaccine candidate for T. gondii, offering a feasible option for vaccination against acute toxoplasmosis.

Structural characterization and adjuvant action of Paulownia tomentosa flower polysaccharide on the immune responses to classical swine fever vaccine in mice.

Paulownia tomentosa flower polysaccharide (PTFP) from dried cultured P. tomentosa flowers, is widely known for its immunomodulatory activities. Here, PTFP was extracted from Paulownia tomentosa flower using hot water extraction, followed by ethanol precipitation methods. Structural characterization of PTFP was revealed by scanning electron microscope, high-performance anion-exchange chromatography, gel chromatography, ultraviolet and infrared spectral. Meanwhile, adjuvant action of PTFT on the immune responses to classical swine fever vaccine in mice was evaluated to further proclaim the immune regulatory effect of PTFP. The results showed that PTFP was a type of heteropolysaccharide with a dense, rough surface and high molecular weight (667.02 kDa), mainly composed of glucose (30.93%), rhamnose (29.99%), galactose (15.66%), arabinose (6.95%), mannose (5.52%), and xylose (4.80%). The results of gel chromatography suggested that the molecular configuration of PTFP may be a spherical structure. The infrared spectrum results confirmed that the functional groups and chemical bond of PTFP contained -OH, O-H, C-H, C=O, C-O, etc. Moreover, PTFP exhibited obvious immune enhancement effect by improving concanavalin A (ConA), lipopolysaccharide (LPS), and CSFV E2-stimulated splenocyte growth and natural killer cell activity in CSFV-immunized mice. Similarly, the titers of CSFV E2-specific IgG, IgG1, IgG2a, and IgG2b antibodies and IFN-γ and IL-10 levels in CSFV-immunized mice were distinctly increased by PTFP treatment. Overall, PTFP was a macromolecular heteropolysaccharide primarily containing glucose and rhamnose, and possessed the auxiliary effect of immune enhancement on the immune responses to classical swine fever vaccine.

Co-Immunization with DNA Vaccines Expressing SABP1 and SAG1 Proteins Effectively Enhanced Mice Resistance to Toxoplasma gondii Acute Infection.

Toxoplasma gondii (T. gondii) has many intermediate hosts, obligately invades nucleated cells, and seriously threatens human and animal health due to a lack of effective drugs and vaccines. Sialic acid-binding protein 1 (SABP1) is a novel invasion-related protein that, like surface antigen 1 (SAG1), is found on the plasma membrane of T. gondii. To investigate the immunogenicity and protective efficacy of DNA vaccines expressing SABP1 and SAG1 proteins against T. gondii acute infection, the recombinant plasmids pVAX1-SABP1 and pVAX1-SAG1 were produced and administered intramuscularly in Balb/c mice. Serum antibody levels and subtypes, lymphocyte proliferation, and cytokines were used to assess immunized mice's humoral and cellular immune responses. Furthermore, the ability of DNA vaccines to protect mice against T. gondii RH tachyzoites was tested. Immunized mice exhibited substantially higher IgG levels, with IgG2a titers higher than IgG1. When the immune group mice's splenocytes were stimulated with T. gondii lysate antigen, Th1-type cytokines (IL-12p70, IFN-γ, and IL-2) and Th2-type cytokine (IL-4) increased significantly. The combined DNA vaccine significantly increased the immunized mouse survival compared to the control group, with an average death time extended by 4.33 ± 0.6 days (p < 0.0001). These findings show that DNA vaccines based on the SABP1 and SAG1 genes induced robust humoral and cellular immunity in mice, effectively protecting against acute toxoplasmosis and potentially serving as a viable option for vaccination to prevent T. gondii infection.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Experimental allergic airway inflammation impacts gut homeostasis in mice

Background/Aims: Epidemiological data show that there is an important relationship between respiratory and intestinal diseases. To improve our understanding on the interconnectedness between the lung and intestinal mucosa and the overlap between respiratory and intestinal diseases, our aim was to investigate the influence of ovalbumin (OVA)-induced allergic airway inflammation on gut homeostasis. MethodsA/J mice were sensitized and challenged with OVA. The animals were euthanized 24 h after the last challenge, lung inflammation was determined by evaluating cells in Bronchoalveolar lavage fluid, serum anti-OVA IgG titers and colon morphology, inflammation and integrity of the intestinal mucosa were investigated. IL-4 and IL-13 levels and myeloperoxidase activity were determined in the colon samples. The expression of genes involved in inflammation and mucin production at the gut mucosa was also evaluated. ResultsOVA challenge resulted not only in lung inflammation but also in macroscopic alterations in the gut such as colon shortening, increased myeloperoxidase activity and loss of integrity in the colonic mucosal. Neutral mucin intensity was lower in the OVA group, which was followed by down-regulation of transcription of ATOH1 and up-regulation of TJP1 and MUC2. In addition, the OVA group had higher levels of IL-13 and IL-4 in the colon. Ova-specific IgG1 and OVA-specific IgG2a titers were higher in the serum of the OVA group than in controls. ConclusionsOur data using the OVA experimental model suggested that challenges in the respiratory system may result not only in allergic airway inflammation but also in the loss of gut homeostasis.

Adjuvant skewing of host immune response to vaccination and subsequent infection with influenza virus

Abstract Despite being a vaccine-preventable disease, seasonal influenza is responsible for thousands of hospitalizations and deaths yearly. Currently licensed quadrivalent inactivated influenza virus vaccines (QIV) aim to induce neutralizing antibodies but are poor inducers of T-cell responses, which could be potentially overcome with adjuvants. We evaluated adjuvant skewing of host-immune responses after vaccination and its persistence after infection. Hereto, we compared two squalene-based adjuvants (AddaVax, and AddaS03) and IMDQ-PC, a lymph node-draining TLR7/8 agonist. All adjuvants resulted in enhanced antibody titers and protection against challenge with H1N1 influenza virus. Lung cytokine/chemokine profiling at 5 days post-infection showed that AddaVax results in higher levels of type 2 immunity-stimulating cytokines IL-4, -5, and -13 in the lungs, which is also reflected by a higher IgG1:IgG2a ratio. Meanwhile, IMDQ-PC showed higher serological IgG2a titers after vaccination and higher levels of IFNγ in the lungs after challenge. To investigate the role of the myeloid compartment in adjuvant skewing of host responses, we examined adjuvant-mediated skewing of bone-derived macrophages in-vitro using Arginase I and iNOS as M2 or M1 markers, respectively. We also pre-licensed macrophages with IL-4 and IFNγ to evaluate adjuvant-intrinsic skewing effects. Licensing was required to induce M1 or M2 phenotypes by squalene-based adjuvants, whereas IMDQ-PC skewed towards M1 phenotype irrespective of licensing. Correlating with the observed type I immune host response, IMDQ-PC induced iNOS more robustly with IFNγ than the other adjuvants. And, AddaS03 induced the highest levels of Arginase I in the IL-4 group. This study was also partly funded by CRIPT (Center for Research on Inuenza Pathogenesis and Transmission), a NIH NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014), by the NIAID funded SEM-CIVIC consortium to improve inuenza vaccines (contract number 75N93019C00051), to AG-S. By NIH/NIAID HHSN272201400008C (L.C.), and NIH/NIAID R21AI151229 and R01AI160706 (M.S.). B.G.D.G. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant N 817938).

Zika Vaccine Microparticles (MPs)-Loaded Dissolving Microneedles (MNs) Elicit a Significant Immune Response in a Pre-Clinical Murine Model

Although the global Zika epidemic in 2015-16 fueled vaccine development efforts, there is no approved Zika vaccine or treatment available to date. Current vaccine platforms in clinical trials are administered via either subcutaneous or intramuscular injections, which are painful and decrease compliance. Therefore, in the present study, we explored Zika vaccine microparticles (MPs)-loaded dissolving microneedles (MNs) with adjuvant MPs encapsulating Alhydrogel® and MPL-A® administered via the transdermal route as a pain-free vaccine strategy. We characterized the MNs for needle length, pore formation, and dissolvability when applied to murine skin. Further, we evaluated the in vivo efficacy of vaccine MPs-loaded MNs with or without adjuvants by measuring the immune response after transdermal immunization. The vaccine MPs-loaded dissolving MNs with adjuvants induced significant IgG, IgG1, and IgG2a titers in immunized mice compared to the untreated control group. After the dosing regimen, the animals were challenged with Zika virus, monitored for seven days, and sacrificed to collect spleen and lymph nodes. The lymphocytes and splenocytes from the immunized mice showed significant expressions of helper (CD4) and cytotoxic (CD8a) cell surface markers compared to the control group. Thus, this study puts forth a 'proof-of-concept' for a pain-free transdermal vaccine strategy against Zika.

Aluminum-based metal–organic framework nanoparticles as pulmonary vaccine adjuvants

The adoption of pulmonary vaccines to advantageously provide superior local mucosal protection against aerosolized pathogens has been faced with numerous logistical and practical challenges. One of these persistent challenges is the lack of effective vaccine adjuvants that could be well tolerated through the inhaled route of administration. Despite its widespread use as a vaccine adjuvant, aluminum salts (alum) are not well tolerated in the lung. To address this issue, we evaluated the use of porous aluminum (Al)-based metal–organic framework (MOF) nanoparticles (NPs) as inhalable adjuvants. We evaluate a suite of Al-based MOF NPs alongside alum including DUT-4, DUT-5, MIL-53 (Al), and MIL-101-NH2 (Al). As synthesized, MOF NPs ranged between ~ 200 nm and 1 µm in diameter, with the larger diameter MOFs matching those of commercial alum. In vitro examination of co-stimulatory markers revealed that the Al-based MOF NPs activated antigen presenting cells more effectively than alum. Similar results were found during in vivo immunizations utilizing ovalbumin (OVA) as a model antigen, resulting in robust mucosal humoral responses for all Al MOFs tested. In particular, DUT-5 was able to elicit mucosal OVA-specific IgA antibodies that were significantly higher than the other MOFs or alum dosed at the same NP mass. DUT-5 also was uniquely able to generate detectable IgG2a titers, indicative of a cellular immune response and also had superior performance relative to alum at equivalent Al dosed in a reduced dosage vaccination study. All MOF NPs tested were generally well-tolerated in the lung, with only acute levels of cellular infiltrates detected and no Al accumulation; Al content was largely cleared from the lung and other organs at 28 days despite the two-dose regime. Furthermore, all MOF NPs exhibited mass median aerodynamic diameters (MMADs) of ~ 1.5–2.5 µm when dispersed from a generic dry powder inhaler, ideal for efficient lung deposition. While further work is needed, these results demonstrate the great potential for use of Al-based MOFs for pulmonary vaccination as novel inhalable adjuvants.Graphical

RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine.

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)-PEG-Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant.Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

Chitosan-coated PLGA nanoparticles for transcutaneous immunization: Skin distribution in lysozyme-sensitized mice

The effect of transcutaneous immunization was studied using a combined system of poly(DL-lactide-co-glycolide) (PLGA) nanoparticles and iontophoresis (IP). Both hen egg-white lysozyme (HEL)-loaded PLGA nanoparticles coated with chitosan hydroxypropyltrimonium chloride and their fluorescent nanoparticles were prepared using an antisolvent diffusion method. Their mean volume diameters were 87.6 ± 38.9 nm and 84.9 ± 27.6 nm, respectively. It was suggested from the results of the ex vivo skin accumulation study using fluorescent nanoparticles that the HEL released from the nanoparticles to the skin surface was efficiently delivered to antigen-presenting cells. HEL-specific IgG1 and IgG2a titers were determined in an in vivo percutaneous immunoreactivity study using lysozyme-sensitized mice. As results, the group using nanoparticles and IP showed 1.33 times higher HEL-specific IgG1 titer than a sham treatment group. The HEL-specific IgG2a titer was 1.36 times higher in the nanoparticles and IP group than in the HEL solution and IP group. It was suggested from the quantification results of total IgE in serum that the combined use of PLGA nanoparticles and IP reduced the total IgE concentration. The level of cytokines may have decreased due to Th1 cell activation and relative suppression of Th2 cells. The cytokine level is presumed to be reduced by activation of Th1 cells and relative suppression of Th2 cells. The histamine amount in plasma and rectal temperature after the induction of anaphylactic shock using lysozyme-sensitized mice were also studied, which indicates that the combined use of PLGA nanoparticles and IP may provide the same therapeutic effect as an injection.

Immune enhancement of N-2-Hydroxypropyl trimethyl ammonium chloride chitosan/carboxymethyl chitosan nanoparticles vaccine

The immunogenicity and toxicity of N-2-Hydroxypropyl trimethyl ammonium chloride chitosan/N, O-carboxymethyl chitosan nanoparticles (N-2-HACC/CMCS NPs) as a universal vaccine adjuvant/delivery system remains unclear. The present study indicated that the positively charged N-2-HACC/CMCS NPs showed a regular spherical morphology, with a particle size of 219 ± 13.72 nm, zeta potential of 37.28 ± 4.58 mV, had hemocompatibility and biodegradation. Acute toxicity, repeated dose toxicity, abnormal toxicity, muscle stimulation, whole body allergic reaction evaluation in vitro, and cytotoxicity in vivo confirmed N-2-HACC/CMCS NPs is safe and non-toxic. N-2-HACC/OVA/CMCS NPs were prepared to evaluate the immunogenicity, which showed a particle size of 248.1 ± 15.53 nm, zeta potential of 17.24 ± 1.28 mV, encapsulation efficiency of 92.43 ± 0.96 %, and loading capacity of 42.97 ± 0.07 %. Oral or intramuscular route with the N-2-HACC/OVA/CMCS NPs in mice not only induced higher IgG, IgG1, IgG2a, and sIgA antibody titers, but also significantly produced higher levels of IL-6, IL-4, IFN-γ, and TNF-α, demonstrating that the N-2-HACC/OVA/CMCS NPs enhance humoral, cellular, and mucosal immune responses. Our results not only support the N-2-HACC/CMCS NPs to be a safe and potential universal nano adjuvant/delivery system in vaccine development, especially mucosal vaccines, but also rich the database knowledge of adjuvant/delivery systems, and provide new direction to introduce more licensed adjuvants.

PD-L1 plays a B cell-intrinsic role in suppressing the antibody response and anti-tumor immunity.

Abstract The discovery that PD-L1 inhibits PD-1 on CD8+ T cells has led to new mechanistic insights into the regulation of host immune responses. The contribution of PD-L1 expressed by different immune cell types remains poorly understood, partly due to the lack of cell type-specific Cd274 (encoding PD-L1) knockout mice. Here, to address the role of B cell-expressed PD-L1 in antibody response and tumor development, we have generated AicdacreCd274fl/fl mice, in which PD-L1 was ablated only in activated B cells. As compared to their wild-type littermates, AicdacreCd274fl/fl mice showed normal development and maintenance of B cells, T cells, DCs and macrophages, while mounting a stronger T-dependent high affinity NP-binding IgM, IgG1 and IgG2a titers upon immunization with NP-CGG plus alum. Such phenotype was copied by C57 mice treated with a blocking αPD-L1 antibody. Tumor growth from engrafted E0771 breast cancer cells in AicdacreCd274fl/fl mice was significantly delayed, in association with increased CD8+ T cell infiltration and activities. Moreover, B cells isolated from AicdacreCd274fl/fl mice or those treated with aPD-L1 displayed higher levels of proliferation and differentiation to plasma cells upon in vitro stimulation with the T-dependent B-cell stimulus CD154. This along with the altered gene expression in AicdacreCd274fl/fl mice and αPD-L1-treated cells suggests a B cell-intrinsic role of PD-L1 in regulating B cell differentiation. Furthermore, PD-L1 expressed on B cell surface was tightly regulated during differentiation and internalized upon αPD-L1 treatment, suggesting a possible new mechanism on how PD-L1+ B cells modulate host immune responses. Supported by grants from NIH: The Ruth L. Kirschstein NRSA Institutional Research Training Grant (to MK), NIH T32 Graduate Research in Immunology Program (to CC), R01 AI153506 and R21 AI135599 (to ZX). Mays Cancer Center Dew Foundation Pilot Grant (to ZX) and DoD BCRP BC170448 (to ZX)

Chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV) and resiquimod adjuvant (HPERC) as a novel booster vaccine for Mycobacterium tuberculosis.

This study attempted to explore the immunogenicity of chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV; HPE) and resiquimod adjuvant (HPERC) in BALB/c mice. HPE was initially expressed in E. coli BL21 cells. HPE and resiquimod adjuvant were then encapsulated in chitosan nanoparticles (HPERC). One group of mice were subcutaneously vaccinated on days 0, 14, and 28 with HPERC, and the other group was primed with bacilli Calmette-Guérin (BCG) on day 0 and then boosted with HPERC on days 14 and 28. Two weeks after the last injection, IFN-γ, IL-4, and IL-17 in spleen cell culture supernatants, and IgG2a and IgG1 titers in sera were measured. HPERC size was 130.84 ± 12.08nm (n = 5). Zeta potential of HPERC was 29 ± 4 mv. The highest IFN-γ concentration was detected in BCG-primed mice that were boosted with HPERC. In addition, IL-17 production was significantly increased in all groups compared with that of control, except in those that received nanoparticle (NP), adjuvant (ADJ), NP/ADJ, and fusion protein (Hspx-PPE44-EsxV) (HPE). Comparison of IFN-γ and IL-4 concentration determined that Th1 was activated in BCG-primed and HPERC-boosted group in comparison to the other groups. No significant difference in concentration of IL-4 was observed between groups receiving HPERC and BCG-primed and HPERC-boosted group in comparison to group BCG. Concentrations of IgG2a and IgG1 also increased compared to the control group and the rate of IgG2a was higher compared to IgG1. Chitosan containing HPERC vaccine could induce a high level of specific cytokines in mice. The group of mice which first received BCG and then HPERC as booster vaccine could produce significant amounts of IFN-γ, IL-17, and IgG2a.