Antigen-specific Responses Research Articles

Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis.

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism largely dependent on the activating CD94/NKG2C receptor and its ligand HLA-E. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4β7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered individual HLA-E-restricted peptides that may constitute the dominant NK cell response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients that reduces efficacy and increases adverse reactions. Our laboratory has shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific Tcell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer, p(Man). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We find that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by T regulatory cells. We identify increased Tcell receptor signaling and increased apoptosis and exhaustion in Tcells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

A Pvs25 mRNA vaccine induces complete and durable transmission-blocking immunity to Plasmodium vivax

Plasmodium vivax (P. vivax) is the major malaria parasite outside of Africa and no vaccine is available against it. A vaccine that interrupts parasite transmission (transmission-blocking vaccine, TBV) is considered highly desirable to reduce the spread of P. vivax and to accelerate its elimination. However, the development of a TBV against this pathogen has been hampered by the inability to culture the parasite as well as the low immunogenicity of the vaccines developed to date. Pvs25 is the most advanced TBV antigen candidate for P. vivax. However, in previous phase I clinical trials, TBV vaccines based on Pvs25 yielded low antibody responses or had unacceptable safety profiles. As the nucleoside-modified mRNA–lipid nanoparticle (mRNA–LNP) vaccine platform proved to be safe and effective in humans, we generated and tested mRNA–LNP vaccines encoding several versions of Pvs25 in mice. We found that in a prime-boost vaccination schedule, all Pvs25 mRNA–LNP vaccines elicited robust antigen-specific antibody responses. Furthermore, when compared with a Pvs25 recombinant protein vaccine formulated with Montanide ISA-51 adjuvant, the full-length Pvs25 mRNA–LNP vaccine induced a stronger and longer-lasting functional immunity. Seven months after the second vaccination, vaccine-induced antibodies retained the ability to fully block P. vivax transmission in direct membrane feeding assays, whereas the blocking activity induced by the protein/ISA-51 vaccine dropped significantly. Taken together, we report on mRNA vaccines targeting P. vivax and demonstrate that Pvs25 mRNA–LNP outperformed an adjuvanted Pvs25 protein vaccine suggesting that it is a promising candidate for further testing in non-human primates.

Influenza immune imprinting synergizes PEI-HA/CpG nanoparticle vaccine protection against heterosubtypic infection in mice

The first influenza virus infection (imprinting) can lead to long-term immune memory and influence subsequent vaccinations and infections. Previously, we reported a polyethyleneimine (PEI)-Aichi hemagglutinin (HA)/CpG (PHC) nanoparticle with cross-protective potential against homologous and heterologous influenza strains. Here we studied how influenza immune imprinting influences the antibody responses to the PHC vaccination and the protection against heterosubtypic virus challenges. We found that pre-existing virus immunity can maintain or synergize the vaccine-induced antibody titers, depending on the imprinting virus HA phylogenetic group. The HA group 1 virus (PR8, H1N1)-imprinted mice displayed comparable antigen-specific antibody responses to those without imprinting post-PHC vaccination. In contrast, the group 2 virus (Phi, H3N2)-imprinted mice showed significantly more robust and balanced antibodies post-vaccination, conferring complete protection against body weight loss and lung inflammation upon heterosubtypic reassortant A/Shanghai/2/2013 (rSH, H7N9) virus challenge. Our findings suggest that influenza imprinting from the same HA phylogenetic group can synergize subsequent vaccination, conferring heterosubtypic protection.

Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates

Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. Macaques that developed LTBI (n=36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n=21; p<0.01, q<0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p<0.05) and increasing airway IgG responses to some proteins over time. Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity.

Tuberculosis (TB) is a major global health threat that claims more than one million lives annually. With a quarter of the global population harbouring latent TB, post-exposure vaccination aimed at high-risk populations that could develop active TB disease would be of great public health benefit. Mucosal vaccination is an attractive approach for a predominantly lung disease like TB because it elicits both local and systemic immunity. However, the immunological consequence of mucosal immunisation in the presence of existing lung immunity remains largely unexplored. Using a mycobacterial pre-exposure mouse model, we assessed whether pre-existing mucosal and systemic immune responses can be boosted and/or qualitatively altered by intranasal administration of spore- and nanoparticle-based subunit vaccines. Analysis of lung T cell responses revealed an increasing trend in the frequency of important CD4 and CD8 T cell subsets, and T effector memory cells with a Th1 cytokine (IFNγ and TNFα) signature among immunised mice. Additionally, significantly greater antigen specific Th1, Th17 and IL-10 responses, and antigen-induced T cell proliferation were seen from the spleens of immunised mice. Measurement of antigen-specific IgG and IgA from blood and bronchoalveolar lavage fluid also revealed enhanced systemic and local humoral immune responses among immunised animals. Lastly, peripheral blood mononuclear cells (PBMCs) obtained from the TB-endemic country of Mozambique show that individuals with LTBI showed significantly greater CD4 T cell reactivity to the vaccine candidate as compared to healthy controls. These results support further testing of Spore-FP1 and Nano-FP1 as post-exposure TB vaccines.

Production, purification and immunogenicity of Gag virus-like particles carrying SARS-CoV-2 components

The virus-like particle (VLP) platform is a robust inducer of humoral and cellular immune responses; hence, it has been used in vaccine development for several infectious diseases. In the current work, VLPs carrying SARS-CoV-2 Spike (S) protein (Wuhan strain) with an HIV-1 Gag core were produced using suspension HEK 293SF-3F6 cells by transient transfection. The Gag was fused with green fluorescent protein (GFP) for rapid quantification of the VLPs. Five different versions of Gag-Spike VLPs (Gag-S-VLPs) consisting of Gag-S alone or combined with other SARS-CoV-2 components, namely Gag-S-Nucleocapsid (N), Gag-S-Matrix (M), Gag-S-Envelope (E), Gag-S-MEN, along with Gag alone were produced and processed by clarification, nuclease treatment, concentration by tangential flow filtration (TFF) and diafiltration. A pilot mouse study was performed to evaluate the immunogenicity of the Gag-S-VLPs through the measurement of the humoral and/or cellular responses against all the mentioned SARS-CoV-2 components. Antibody response to Spike was observed in all variants. The highest number of Spike-specific IFN-γ + T cells was detected with Gag-S-VLPs. No induction of antigen-specific cellular responses to M, N or E proteins were detected with any of the Gag-S, M, E/or N VLPs tested. Therefore, the Gag-S-VLP, by reason of consistently eliciting strong antigen-specific cellular and antibody responses, was selected for further evaluation. The purification process was improved by replacing the conventional centrifugation by serial microfiltration in the clarification step, followed by Spike-affinity chromatography to get concentrated VLPs with higher purity. Three different doses of Gag-S-VLP in conjunction with two adjuvants (Quil-A or AddaVax) were used to assess the dose-dependent antigen-specific cellular and antibody responses in mice. The Gag-S-VLP adjuvanted with Quil-A resulted in a stronger Spike-specific cellular response compared to that adjuvanted with AddaVax. A strong spike neutralisation activity was observed for all doses, independent of the adjuvant combination.

Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses.

Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS-CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor-binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV-infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and CD4+ T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines.

Dual-Antigen-Displaying Nanovaccines Elicit Synergistic Immunoactivation for Treating Cancer and Preventing Infectious Complications.

As one of the most common complications, infection causes the majority of mortality in cancer patients. However, therapeutic strategies that can simultaneously suppress tumors and protect patients from infection have been rarely reported. Here, the use of dual-antigen-displaying nanovaccines (DADNs) is described to elicit synergistic immunoactivation for treating cancer and preventing infectious complications. DADNs are prepared by wrapping immunoadjuvant-loaded nanoparticles with a hybrid coating, which is fused from cell membranes that are separately genetically engineered to express tumor and infectious pathogenic antigens. Due to the presence of a dual-antigen combination, DADNs are able to promote the maturation of dendritic cells and more importantly to trigger cross-presentation of both combined antigens. During in vivo investigations, wefind that DADNs can reverse immunosuppression by stimulating tumor-associated antigen-specific T-cell responses, resulting in significantly delayed tumor growth in mice. These nanovaccines also elicit effective protective immunity against tumor challenges and induce robust production of pathogenic antigen-specific immunoglobulin G antibody in a prophylactic study. This work offers a unique approach to develop dual-mode vaccines, which are promising for synchronously treating cancer and preventing infection.

Antigen-Specific T-Cell Repertoire Responses Following COVID-19 Vaccination Are Intact in Majority of Patients with B-Cell Lymphoid Malignancies on Active Therapy

Patients (Pts) with hematologic cancers are at increased risk for morbidity and mortality from COVID-19. Vaccination against COVID-19 has reduced morbidity and mortality, but the depth and breadth of immune responses to both vaccination and prior infection for patients with lymphoid malignancies, particularly in the context of active cancer treatment, remains incompletely understood. We previously observed impaired antibody response to a primary COVID-19 mRNA vaccination (2 dose series) in pts with lymphoma receiving B cell-depleting therapies (BCDT) (Ghione et. al., Blood 2021). These poor responses are expected to result from both intrinsic immune dysregulation from the hematologic malignancy, as well as treatment with BCDTs. However, a gap in knowledge relates to the extent that T cell responses to vaccination are intact in these patients. This question is important based on the established role of T cells in clearing virally infected cells through CTL function and driving cytokine responses. We investigated serological and cellular responses to COVID-19 vaccination (initial and subsequent doses). Cellular responses were quantified using the T-Detect COVID Test (Adaptive Biotechnologies, Seattle, WA), which combines multiplex PCR and next-generation sequencing (NGS) to quantify T-cell receptor beta (TCRβ) sequences that are known to react to COVID-19. We collected 205 serial blood samples from 97 pts with B-cell malignancies (NHL, n=67 and CLL, n=31). 75 had received/were receiving BCDT, and 22 were either on observation or were on a treatment not including BCDT. Only 26/75 (34%) pts on treatment/previously treated with BCDT mounted a significant IgG Ab response, as opposed to 20/22 (90.9%) pts who were not receiving BCDT (p= 0.007). Vaccination was associated with an increased proportion of TCRs associated with spike protein region of the COVID-19 genome after vaccination. 43 patients out of 97 patients from this cohort were T-Detect COVID positive at all the time points sampled, while 19 patients were negative at all time points. Overall, 73/97 (75%) patients demonstrated a T-cell response to spike protein consistent with vaccine induced immunity regardless of whether the patient received BCDT. Positive T-cell responses were seen irrespective of prior BCDT. In this cohort, 19 patients demonstrated T-cell responses outside of the spike region, which is suggestive of natural infection; 10 of these appeared consistent with breakthrough infections after vaccination as they were negative at an earlier time point. Pts with breakthrough infections did not suffer from severe COVID-19 infection and recovered despite being on BCDT. Taken together, we observed that patients with B-cell malignancies receiving BCDT have highly impaired antibody responses to COVID-19 vaccination while the majority have intact expansion of antigen-specific T cell populations. We observed no correlation between COVID-specific T-cells and COVID-specific IgG antibodies in this study population, but this may be impacted by the timing of the samples. In the setting of profound B-cell impairment, our results suggest that COVID-19 vaccination can induce potentially protective COVID-specific T-cell responses. Our findings provide rationale for additional studies of functional T cell responses to vaccination in these patients and delineating the extent that these T cell responses are associated with protection from severe COVID-19. We also note the broader application of our work to other vaccine-preventable respiratory viral infections, such as influenza and RSV.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Personal and Shared Tumor-Antigen Prioritization in Diffuse Large B Cell Lymphoma Patients Undergoing CD19 CAR T Cell Treatment

Backgroun d: Durable response to Axi-cel CD19 CAR T cell therapy is associated with activation and clonotypic expansion of the host T cell repertoire (Cheloni et al Blood, ASH, 2022). The characterization of the tumor microenvironment (TME) and the identification of tumor antigens associated with therapeutic efficacy is a critical area of investigation. Aim: We sought to determine the association between the host immune activation in response to tumor-associated and tumor-specific antigens and the clinical response to CAR-T cell treatment in diffuse large B cell lymphoma patients. Methods: RNAseq was performed on FFPE lymphoma samples collected before CAR T cell infusion (N=16). Patients were divided into 3 distinct cohorts: stable progressive disease at 1 month post-CAR T (non-responders, n=2), disease relapse within 6 months post-CAR T (relapsed, n=3), and on-going response lasting greater than 1 year (long-term responders, n=11). For neoantigens, mutations were retrieved following the GATK best practices for RNAseq data and annotated with Variant Effect Predictor (VEP). Tumor-specific antigens and tumor-associated antigens were detected by prioritizing 9-11mer amino acid sequences, preferentially presented by class I molecules, observed solely or primarily in tumor samples, respectively, as compared to a pool of healthy controls derived from 250 RNAseq samples, corresponding to 29 tissues (GTEx Portal). Antigen prioritization was performed following weighted scoring considering RNA expression, translation potential, peptide-MHC binding affinity, predicted immunogenicity, physicochemical properties, and clonality, in the case of neoantigens. Using deconvolution methods and canonical marker expression, we characterized the major immune cell types within the TME. Results: Higher cell proportions of lymphocytes and innate immune cells, including NK, and activated dendritic cells were observed in long-term responders, suggesting that a higher infiltration of these cell types in the tumor bed is associated with a better response to CAR T cell therapy. Notably, a positive correlation between the mutational burden and the a/b clonotypic expansion of peripheral blood cytotoxic T cell populations, detected from single-cell immunoprofiling (Cheloni et al Blood, ASH, 2022) was observed in eight long-term responders, consistent with prior studies highlighting the significance of neoantigens in eliciting robust and durable immune response against cancer cells. A high occurrence of neoantigens originating from the Titin ( TTN) gene in two long-term responders with the highest mutational burden was observed, and from ACTB and ACTG1, mutated genes associated with lymphoid cancers. A substantial enrichment of shared tumor antigens originating from retroelements was observed in long-term responders, suggesting the presence of common epitopes that drive an antigen-specific response. Conclusion: These findings shed light on the potential immunogenicity of specific neoantigens and shared antigens derived from repetitive elements, which could serve as promising targets for immunotherapeutic strategies in lymphoma patients. Understanding the tumor immune landscape and antigenic features associated with long-term response to CAR T cell therapy may pave the way for the development of personalized and effective combinatorial treatments for lymphoid cancers.

Dual-Antigen Subunit Vaccine Nanoparticles for Scrub Typhus.

Orientia tsutsugamushi is the causative pathogen of scrub typhus, an acute febrile disease prevalent in the Asia-Pacific region that is spread to people through chigger bites. Despite the emerging threat, there is no currently available vaccine against O. tsutsugamushi. Here, we developed dual-antigen subunit vaccine nanoparticles using recombinant 47 kD and 56 kD proteins, which are immunogenic outer membrane antigens of O. tsutsugamushi. The biocompatible protein vaccine nanoparticles were formed via desolvation of r56 or r47E antigens with acetone, coating with an additional layer of the 56 kD protein, and stabilization with reducible homobifunctional DTSSP and heterobifunctional SDAD crosslinkers. The dual-antigen subunit vaccine nanoparticles significantly improved antigen-specific antibody responses in vaccinated mice. Most importantly, the dual-antigen nanoparticles coated with an additional layer of the 56 kD protein were markedly more immunogenic than soluble antigens or single-antigen nanoparticles in the context of cellular immune responses. Given the significance of cellular immune responses for protection against O. tsutsugamushi, these results demonstrate the potent immunogenicity of dual-layered antigen nanoparticles and their potential as a promising strategy for developing vaccines against scrub typhus.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice.

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

MRNAs encoding self-DNA reactive cGAS enhance the immunogenicity of lipid nanoparticle vaccines.

Nucleic acid-based vaccines hold promise in preventing infections and treating cancer. The most common use of this technology is to encode antigenic proteins on mRNAs that are delivered to cells via lipid nanoparticle (LNP) formulations. In this study, we discovered that immunostimulatory proteins can also be encoded on mRNAs in LNPs. We found that an active mutant of the enzyme cGAS, referred to as cGAS∆N, acts as a catalytic adjuvant in LNP-encapsulated mRNA vaccines. The delivery of cGAS∆N mRNA via LNPs in combination with antigen mRNA-LNPs led to durable antigen-specific IFNγ-producing T cells that exceeded the efficiency of antigen-LNPs similar to those currently used in the clinic. This strategy did not compromise B cell responses; rather it induced Th1-biased antibody isotypes. This work unveils new vaccine design strategies using mRNA-encoded catalytic adjuvants that could be ideal for generating CD8+ T cell and B cell responses for immunotherapies.

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.

Hybrid mRNA Nano Vaccine Potentiates Antigenic Peptide Presentation and Dendritic Cell Maturation for Effective Cancer Vaccine Therapy and Enhances Response to Immune Checkpoint Blockade.

Cancer vaccines combined with immune checkpoint blockades (ICB) represent great potential application, yet the insufficient tumor antigen presentation and immature dendritic cells hinder improved efficacy. Here, a hybrid nano vaccine composed by hyper branched poly(beta-amino ester), modified iron oxide nano adjuvant and messenger RNA (mRNA) encoded with model antigen ovalbumin (OVA) is presented. The nano vaccine outperforms three commercialized reagents loaded with the same mRNA, including Lipofectamine MessengerMax, jetPRIME, and in vivo-jetRNA in promoting dendritic cells' transfection, maturation, and peptide presentation. In an OVA-expressing murine model, intratumoral administration of the nano vaccine significantly induced macrophages and dendritic cells' presenting peptides and expressing co-stimulatory CD86. The nano vaccine also elicited strong antigen-specific splenocyte response and promoted CD8+ T cell infiltration. In combination with ICB, the nano vaccine aroused robust tumor suppression in murine models with large tumor burdens (initial volume >300 mm3 ). The hybrid mRNA vaccine represents a versatile and readily transformable platform and augments response to ICB.

α-Glucosidase inhibitors boost gut immunity by inducing IgA responses in Peyer's patches.

Peyer's patches (PPs) are specialized gut-associated lymphoid tissues that initiate follicular helper T (Tfh)-mediated immunoglobulin A (IgA) response to luminal antigens derived from commensal symbionts, pathobionts, and dietary sources. IgA-producing B cells migrate from PPs to the small intestinal lamina propria and secrete IgA across the epithelium, modulating the ecological balance of the commensal microbiota and neutralizing pathogenic microorganisms. α-glucosidase inhibitors (α-GIs) are antidiabetic drugs that inhibit carbohydrate digestion in the small intestinal epithelium, leading to alterations in the commensal microbiota composition and metabolic activity. The commensal microbiota and IgA responses exhibit bidirectional interactions that modulate intestinal homeostasis and immunity. However, the effect of α-GIs on the intestinal IgA response remains unclear. We investigated whether α-GIs affect IgA responses by administering voglibose and acarbose to mice via drinking water. We analyzed Tfh cells, germinal center (GC) B cells, and IgA-producing B cells in PPs by flow cytometry. We also assessed pathogen-specific IgA responses. We discovered that voglibose and acarbose induced Tfh cells, GCB cells, and IgA-producing B cells in the PPs of the proximal small intestine in mice. This effect was attributed to the modification of the microbiota rather than a shortage of monosaccharides. Furthermore, voglibose enhanced secretory IgA (S-IgA) production against attenuated Salmonella Typhimurium. Our findings reveal a novel mechanism by which α-GIs augment antigen-specific IgA responses by stimulating Tfh-GCB responses in PPs, and suggest a potential therapeutic application as an adjuvant for augmenting mucosal vaccines.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1β, and IL-23. Invivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.