Th1-type Immune Response Research Articles

Novel Toll-Like Receptor 9 Agonist Derived from Cryptococcus neoformans Attenuates Allergic Inflammation Leading to Asthma Onset in Mice

Introduction: The enhanced type 2 helper (Th2) immune response is responsible for the pathogenesis of allergic asthma. To suppress the enhanced Th2 immune response, activation of the Th1 immune response has been an alternative strategy for anti-asthma therapy. In this context, effective Th1-inducing adjuvants that inhibit the development of allergic asthma but do not flare the side effects of the primary agent are required in clinical treatment and preventive medicine. Objective: In this study, we aimed to determine the regulation of the Th2 type immune response in asthma by a novel immunostimulatory oligodeoxynucleotide (ODN) derived from Cryptococcus neoformans, termed ODN112, which contains a cytosine-guanine (CG) sequence but not canonical CpG motifs. Methods: Using an ovalbumin-induced asthma mouse model, we assessed the effect of ODN112 on prototypical asthma-related features in the lung and on the Th1/Th2 profile in the lymph nodes and lung of mice treated with ODN112 during sensitization. Results and Conclusion: ODN112 treatment attenuated asthma features in mice. In the bronchial lymph nodes of the lungs and in the spleen, ODN112 increased interferon-γ production and attenuated Th2 recall responses. In dendritic cells (DCs) after allergen sensitization, ODN112 enhanced cluster of differentiation (CD) 40 and CD80 expression but did not alter CD86 expression. Interleukin-12p40 production from DCs was also increased in a Th2-polarizing condition. Our results suggest that ODN112 is a potential Th1-inducing adjuvant during Th2 cell differentiation in the sensitization phase.

Leishmania infantum pyridoxal kinase evaluated in a recombinant protein and DNA vaccine to protects against visceral leishmaniasis

Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.

Characterization of exosome-like vesicles derived from Taenia pisiformis cysticercus and their immunoregulatory role on macrophages

BackgroundTaenia pisiformis is one of the most common intestinal parasites in canines, and leads to serious economic losses in the rabbit breeding industry. Exosome-like vesicles from parasites play crucial roles in host-parasite interactions by transferring cargo from parasites to host cells and by modulating host immunological response through inducing production of host-derived cytokines. Nevertheless, the mechanism by which exosome-like vesicles from T. pisiformis cysticercus regulate the macrophage immune response remains unknown.MethodsUsing ultracentrifugation, we isolated exosome-like vesicles from excretory/secretory products (ESP) of T. pisiformis cysticercus. The morphology and size of purified vesicles were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The components of proteins and miRNAs within these vesicles were identified by proteomic analysis and high-throughput small RNA sequencing. The biological function of targets of exosomal miRNAs was predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, the expression of Th1- and Th2-type immune response associated cytokines in RAW264.7 macrophages were evaluated by qPCR and ELISA. We found that exosome-like vesicles were typical cup-shaped vesicles with diameters from 30 to 150 nm. A total of 87 proteins were identified by proteomic analysis, including proteins prominently associated with exosome-like vesicles biogenesis and vesicle trafficking. 41 known miRNAs and 18 novel miRNAs were identified in the exosome-like vesicles. Eleven selected miRNAs, including 7 known miRNAs (miR-71-5p, miR-10a-5p, miR-let-7-5p, miR-745-3p, miR-219-5p, miR-124-3p and miR-4989-3p) and 4 novel miRNAs (novel-mir-3, novel-mir-7, novel-mir-8 and novel-mir-11) were validated to exist in metacestiodes and exosome-like vesicles of T. pisiformis cysticercus by qPCR. The functions of most targets of exosomal miRNAs were mainly associated with signal transduction and the immune system. Additionally, T. pisiformis cysticercus-derived vesicles induced the production of IL-4, IL-6, IL-10, IL-13 and Arg-1, but downregulated the expression of IL-12, IFN-γ and iNOS in RAW264.7 macrophages.ConclusionsWe demonstrated that proteins and miRNAs enclosed within exosome-like vesicles from T. pisiformis cysticercus have immunomodulatory functions. Furthermore, exosome-like vesicles were shown to induce the macrophage Th2-type immune response in vitro. Our study suggests that exosome-like vesicles play an important role in the interaction between cysticerci and their hosts.

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis.

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

1676-P: Immune Responses to Insulin B:9-23–Related Peptides Were Observed in Fulminant Type 1 Diabetes

Most type 1 diabetes (T1D) is considered to be a T helper (Th)-1 type anti-islet autoimmune disease. Recently, we reported that the immune responses to insulin B9-23-related peptides were strengthened in T1D. Meanwhile, fulminant T1D (FT1D), a novel subtype of T1D characterized by extremely rapid onset and complete deficiency of insulin due to destruction of pancreatic β-cells, has been believed to be caused by viral infection as the etiological mechanism; but, whether the etiology associates with anti-islet immune reactions or not remains unknown. This study aims to investigate whether the immune responses to insulin B9-23-related peptides associate with FT1D. Peripheral mononuclear cells (MNCs) were obtained from 72 patients with acute-onset T1D (AT1D; age, 46.2 ± 14.7 y/o; disease duration (DD), 8.8 ± 10.7 years), 15 patients with FT1D (age, 46.1 ± 14.2 y/o; DD, 4.7 ± 8.2 years) and 25 patients with type 2 diabetes (T2D; age, 63.3 ± 13.1 y/o; DD, 17.0 ± 10.2 years). The MNCs were stimulated by insulin B9-23, B10-24, B11-25 and B12-26 for 24 hours. Subsequently, ELISpot assay was performed for investigating a frequency of interferon (IFN)-gamma (g)-producing MNCs. The maximum number of IFN-g spots in the peptides-related data of each subject was used for analysis and expressed by the number of spot-forming cells (SFCs) per 2.5×105 cells. As a result, the spot number was significantly higher in AT1D (2.6 ± 3.0 SFCs) and FT1D (2.5 ± 2.6 SFCs) as compared to T2D groups (1.0 ± 1.1 SFCs) (P <0.05 by t-test), suggesting that Th1-type immune responses to insulin B9-23-related peptides were observed in FT1D, and that the degree of response was comparable in the two groups, suggesting the association between anti-islet autoimmunity and FT1D. This cross-sectional study could not reveal an association between the Th1-type immune responses and disease activity which may be inferred from the residual serum C-peptide levels or disease duration, which warrant further investigation by accumulating more subjects. Disclosure Y. Oikawa: None. A. Satomura: None. A. Haisa: None. S. Suzuki: None. A. Shimada: Advisory Panel; Self; Astellas Pharma Inc. Speaker’s Bureau; Self; Eli Lilly Japan K.K., Novo Nordisk Inc., Sanofi-Aventis.

Preventive as well as therapeutic significances of linoleic acid in the containment of Leishmania donovani infection

People suffering from malnutrition show compromised levels of ω-6 fatty acid and malnutrition is frequently observed among visceral leishmaniasis (VL) patients as disease inflicts primarily the socioeconomic destitute communities. Dietary linoleic acid (LA, 18:2; ω-6 fatty acid) is the principal source of essential fatty acid and its derivatives i.e. eicosanoids possess immune-modulatory activities. However, its role in VL is not yet established. LA was measured in VL human subjects (serum) as well as in Leishmania(L.)donovani infected hamsters (serum and visceral organs). Organ-specific mRNA expressions of various enzymes of the LA metabolic pathway were measured in visceral organs of infected hamsters. Our findings showed a decrease in the concentrations of LA in the serum samples of VL patients, suggesting malnutrition among these patients. However, in L. donovani infected hamsters, its level was not altered in the early infection (15 days) and then increased at late infection (60 days). Importantly, the supplementation of LA restored the Th-1 type of immune response and significantly reduced the parasite load within infected macrophages in vitro. This protective response of LA was mediated through 5-lipoxygenase pathway not via the cyclooxygenase pathway. Preventive usage of LA to mϕ followed by L. donovani infection also showed the strengthening of Th-1 immune response and significantly fewer parasite loads. Our findings demonstrate the protective role of LA in the containment of the parasite load. Incorporating LA rich oils in daily food habits across highly inflicted regions may be a significant advancement towards the eradication of the disease.

The IL-33/ST2 pathway suppresses murine colon cancer growth and metastasis by upregulating CD40 L signaling

Interleukin (IL)-33 is a member of the IL-1 family, participating in both helper T1 (Th1)- and Th2-type immune responses, but its ambiguous effects on tumor growth and related immune mechanisms remain unclear. Here, we report that recombinant mouse IL-33 (mIL-33) significantly inhibited colon cancer growth and metastasis to lung and liver in a murine CT26 or MC38 tumor-cell engraftment model. This effect could be associated with CD4+ T cells and CD40 L signaling, as depletion of CD4+ T cells or blocking CD40 L signaling in vivo partly abolished the antitumor function of IL-33. In addition, IL-33 treatment upregulated CD40 L expression on tumor-infiltrating lymphocytes, and promoted the activation of CD4+ T, CD8+ T and natural killer cells via CD40 L signaling. Furthermore, IL-33 was sufficient to induce the ST2 expression on CD4+ T cells, but not on CD8+ T and natural killer cells, indicating that IL-33 acted on CD4+ T cells via a positive-feedback loop. Our findings shed new light on the IL-33-mediated antitumor effects and mechanisms of Th1 action, and also suggest that IL-33 may serve as an activator to boost anticancer immune responses in singular or combinatory therapies.

Differential recruitment and activation of mucosal and systemic ILC for improved vaccine design

Abstract Innate lymphoid cells (ILC) have emerging roles in immune modulation, tissue homeostasis, and inflammation. ILC may function as innate counterparts of T cells, influencing early host responses against infection or vaccination, potentially modulating development of adaptive immune responses. Little is known, however, of their recruitment and activation following immunization. Here, we investigated the activity of ILC populations after immunization with poxvirus-based vaccine vectors. Our data suggest that fowlpox virus (FPV) and modified vaccinia Ankara strain (MVA) vectors can differentially polarize ILC responses at mucosae. Intranasal (IN) delivery of FPV vectors increased numbers of pulmonary ILC1 and ILC3 subsets expressing IL-17a and IL-22 through 72 hours post-immunization. In contrast, MVA vectors given IN enhanced pulmonary ILC2 numbers and their expression of IL-13. Our previous work indicated that mucosal immunization with these vectors induced high avidity T cell responses associated with transient inhibition of IL-4/13 expression by local ILC2. Since IL-25 has been shown to down regulate IL-12 and promote ILC2/IL-13 expression, we delivered FPV encoding an IL-25 binding protein (IL-25BP) in order to sequester host IL-25 at the site of immunization. While mucosal delivery of FPV-IL-25BP appeared to induce ILC2/IL-13 expression in the lungs, IM delivery reduced ILC2/IL-13 expression, increased IL-17a expression by local ILC1 and ILC3, and enhanced Th1-type adaptive immune responses against co-expressed vaccine antigens. Together, our findings suggest that manipulation of ILC responses by vaccine vectors, co-expressed immune modulators, and delivery route may be exploited for improved vaccine design.

Bcl3 dependent regulation of Dendritic Cell function in Toxoplasma gondii infection

Abstract The atypical IκB family member Bcl-3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in nuclei, thereby either positively or negatively modulating transcription in a context-dependent manner. Bcl-3 is critical for host resistance to Toxoplasma gondii and Bcl-3-deficient mice succumbed within 3–5 weeks after infection, correlating with an apparently impaired Th1-type adaptive immune response rather than a defect in the innate immune response. Surprisingly, mice lacking Bcl-3 in CD11c+ dendritic cells (DC) were as susceptible as mice globally deficient for Bcl-3, demonstrating that Bcl-3 is required in these cells to prime protective T-cell-mediated immunity to T. gondii. It remains to be determined however whether conventional or monocyte derived DCs are critical mediators of a successful T cell response against the invading pathogen. We generated mice specifically lacking Bcl-3 in conventional DCs (Zbtb46 cre knockout [KO]) or in monocyte derived cells, including monocyte derived DCs (LysM cre KO). Surprisingly, both conditional KO strains were as susceptible to T. gondii infection as CD11c+ conditional or total KO mice. Interestingly, we detected the presence of a unique subpopulation of conventional DCs that expressed the monocytic markers CD64 and Ly6C in infected spleen and various tissues including lung. Furthermore, this population was reduced in the absence of Bcl3. This hybrid DC population may have a crucial role in the T cell responses after infection, in particular in the generation of inflammatory cytokines such as IFNg, a cytokine which is indispensable for host protection.

Combining doxorubicin with stearylamine-bearing liposomes elicits Th1 cytokine responses and cures metastasis in a mouse model.

Surface exposed phosphatidylserine (PS) of cancer aids it to evade immune surveillance and thereby results in tumor progression. Earlier, we reported that PS targeting cationic liposomes, phosphatidylcholine-stearylamine (PC-SA), alone and in combination withdoxorubicin can result in complete remission of B16F10 melanoma in C57BL/6 mice without signs of toxicity. Inducing an immunogenic response is highly crucial for any cancer therapy as it is essential in improving the tumor microenvironment for any drug to act. Herein, we demonstrate that PC-SA, besides having tumor reducing ability, elicits a strong immune response. The combination therapy (PC-SA-DOX) is superior to free DOX in enhancing the anti-tumor immune effect on CD4-positive and CD8-positive T cells for IFN-γ, IL-2 and TNF-α production in sera and splenic culture supernatants of B16F10 tumor-induced mice. An upregulation of IL-12 and NO production is evidenced in spleen cultures of these mice, thereby showing a promising role of both Th1 type and innate immune response for host anti-tumor activity. Complete elimination of cancer is sometimes accomplished by surgery, but its effectiveness is often limited due to the propensity of cancers to spread to distant organs by metastasis. In our present study, we show that in PC-SA-DOX treated mice, the elevated Th1 cytokine levels create an immuno-protective environment which thereby facilitates in curing lung metastasis. Our results, therefore, warrant the need of effective immune stimulation by anticancer formulations for inhibition of solid tumors and metastasis, demonstrated by the liposomal DOX formulation.

Programmed cell death protein 1 and its ligands regulate immune balance in allergic rhinitis

Objective: To explore the expression and significance in regulating immune balance of programmed cell death protein 1 (PD-1) and its ligands PD-L1, PD-L2 in allergic rhinitis (AR). Methods: Eighty-two patients who received outpatient treatment due to high nasal reaction symptoms or were hospitalized due to nasal septum deviation and underwent nasal septum correction surgery in Department of Otorhinolaryngology Head and Neck Surgery of Renmin Hospital of Wuhan University from May 2018 to May 2019 were enrolled, including 42 males and 40 females, with the age ranging from 14 to 38 years old. Blood, inferior turbinate nasal mucosal tissue and relevant clinical data were collected. Patients were divided into AR group and control group due to clinical manifestation, skin prick test and detection of specific IgE (sIgE) in serum. Immunohistochemistry was used to detect the expression of PD-1 and its ligands in nasal mucosa of the two groups. Flow cytometry was used to detect the proportions of PD-1(+)CD4(+)T cells, PD-L1(+) myeloid dendritic cells (mDCs), PD-L2(+)mDCs and Th2 cells in peripheral blood of the two groups. The expression levels of total IgE, sPD-1, sPD-L1 and sPD-L2 in serum of the two groups were detected by ELISA. The measurement data of normal distribution or normal distribution after the logarithm conversion to Ln were compared by t test. Pearson correlation or Spearman correlation was used to analyze the correlation among the indicators. P<0.05 was considered statistically significant. Results: The expression of PD-1 and its ligands on the surface of immune cells in the nasal mucosa of the AR group was significantly higher than that of the control group. The ratio of PD-1(+)CD4(+)T cells, PD-L1(+)mDCs and Th2 cells in peripheral blood of AR group was significantly higher than that of the control group ((15.24±6.45)% vs (8.71±5.33)%, (8.79±2.01)% vs (5.74±2.90)%, (7.89±1.95)% vs (2.52±1.34)%, all P<0.05). There was no significant difference in the ratio of PD-L2(+)mDCs between the two groups. Correlation analysis found that the proportion of PD-1(+)CD4(+) T cells was positively correlated with the Visual Analogue Scale (VAS) score of AR, total IgE concentration and the serum sIgE concentration (r value was 0.501, 0.541, 0.608, respectively, all P<0.05). The proportion of PD-L1(+)mDCs was positively correlated with the VAS score of AR and the serum sIgE concentration (r value was 0.604, 0.563, respectively, all P<0.05). The proportion of Th2 cells in peripheral blood was positively correlated with the proportion of PD-L1(+)mDCs and PD-1(+)CD4(+)T cells (r value was 0.538, 0.623, respectively, all P<0.05). Serum total IgE, sPD-1 and sPD-L1 in the AR group were significantly higher than those in the control group ((6.34±1.38) ng/ml vs (4.89±1.10) ng/ml, (4.40±1.01) pg/ml vs (3.79±1.21) pg/ml, (3.88±0.25) pg/ml vs (3.57±0.23) pg/ml, all P<0.05), and there was no significant difference in sPD-L2 levels between the two groups. Correlation analysis showed that sPD-L1 was positively correlated with total IgE and sIgE concentration (r values was 0.32, 0.45, respectively, all P<0.05). Conclusions: PD-1 and PD-L1 are highly expressed on the surface of immune cells in peripheral blood and nasal mucosa of AR patients, and sPD-1 and sPD-L1 expression levels in peripheral blood of AR patients are increased. The PD-1/PD-L1 signaling pathway promote AR inflammatory response by inducing Th2 type immune response.

Characteristic of K3 (CpG-ODN) as a Transcutaneous Vaccine Formulation Adjuvant.

Transcutaneous immunization (TCI) is easy to use, minimally invasive, and has excellent efficacy in vaccines against infections. We focused on toll-like receptor (TLR) ligands as applicable adjuvants for transcutaneous formulations and characterized immune responses. TCI was performed using poke-and-patch methods, in which puncture holes are formed with a polyglycolic acid microneedle on the back skin of mice. Various TLR ligands were applied to the puncture holes and covered with an ovalbumin-loaded hydrophilic gel patch. During the screening process, K3 (CpG-oligonucleotide) successfully produced more antigen-specific antibodies than other TLR ligands and induced T helper (Th) 1-type polarization. Transcutaneously administered K3 was detected in draining lymph nodes and was found to promote B cell activation and differentiation, suggesting a direct transcutaneous adjuvant activity on B cells. Furthermore, a human safety test of K3-loaded self-dissolving microneedles (sdMN) was performed. Although a local skin reaction was observed at the sdMN application site, there was no systemic side reaction. In summary, we report a K3-induced Th1-type immune response that is a promising adjuvant for transcutaneous vaccine formulations using MN and show that K3-loaded sdMN can be safely applied to human skin.

A novel TLR7 agonist as adjuvant to stimulate high quality HBsAg-specific immune responses in an HBV mouse model

BackgroundThe global burden of hepatitis B virus (HBV) infection in terms of morbidity and mortality is immense. Novel treatments that can induce a protective immune response are urgently needed to effectively control the HBV epidemic and eventually eradicate chronic HBV infection.MethodsWe designed and evaluated an HBV therapeutic vaccine consisting of a novel Toll-like receptor 7 (TLR7) agonist T7-EA, an Alum adjuvant and a recombinant HBsAg protein. We used RNA-seq, ELISA and hTLR7/8 reporting assays to characterize T7-EA in vitro and real-time PCR to evaluate the tissue-retention characteristics in vivo. To evaluate the adjuvant potential, we administrated T7-EA intraperitoneally in a formulation with an Alum adjuvant and HBsAg in normal and HBV mice, then, we evaluated the HBsAg-specific immune responses by ELISA and Elispot assays.ResultsT7-EA acted as an hTLR7-specific agonist and induced a similar gene expression pattern as an unmodified TLR7 ligand when Raw 264.7 cells were exposed to T7-EA; however, T7-EA was more potent than the unmodified TLR7 ligand. In vivo studies showed that T7-EA had tissue-retaining activity with stimulating local cytokine and chemokine expression for up to 7 days. T7-EA could induce Th1-type immune responses, as evidenced by an increased HBsAg-specific IgG2a titer and a T-cell response in normal mice compared to mice received traditional Alum-adjuvant HBV vaccine. Importantly, T7-EA could break immune tolerance and induce persistent HBsAg-specific antibody and T-cell responses in an HBV mouse model.ConclusionsT7-EA might be a candidate adjuvant in a prophylactic and therapeutic HBV vaccine.

Protection against Trichinella spiralis in BALB/c mice via oral administration of recombinant Lactobacillus plantarum expressing murine interleukin-4

Interleukin-4 (IL-4), an immunomodulatory cytokine derived from activated T lymphocytes were shown to regulate Th2-type immune responses. It plays an important role in anti-parasitic infections. In this study, a recombinant plasmid was designed using murine IL-4 co-expressed with pgsA anchor system of Lactobacillus plantarum NC8 and was delivered by live Lactobacillus plantarum NC8, which exhibited an enhanced immunogenicity in protection of BALB/c mice from infection with Trichinella spiralis. The results showed that the levels of serum IgG1 and mucosal secretory IgA (sIgA) were both increased significantly in mice orally inoculated with NC8-pSIP409-pgsA-mIL-4, and the Th2 phenotype immune response was up-regulated. A 29.9 % reduction in adult worm burden at 7 days post-infection (dpi) and 83.3 % reduction in muscle larvae burden at 28 dpi were observed in immune-stimulated mice with NC8-pSIP409-pgsA-mIL-4. Moreover, weight loss and pathological changes were also improved in mice of NC8-pSIP409-pgsA-mIL-4 group. Taken together, it suggests that NC8-pSIP409-pgsA-mIL-4 could improve the intestinal mucosal immunity and promoted the elimination of the adult worm in Trichinella-infected mice. This study laid the foundation for the development of a novel vaccines against Trichinellosis.

Abstract P6-10-17: Breast MRI as a radiomic biomarker of immune response in HER2+ breast cancer

Abstract Rationale/Objectives: Tumor immune infiltration in HER2+ breast cancer is associated with improved outcomes and better responses to treatment with drugs such as trastuzumab. Breast MRI is a non-invasive technique that is part of the standard evaluation for localized breast cancer. Breast MRI dynamic-contrast enhanced (DCE) and diffusion-weighted imaging (DWI) sequences may detect immune responses in the tumor microenvironment. Apparent diffusion coefficient (ADC), which is derived from DWI, has been associated with tumor cellularity. In this pilot study, we hypothesized that patients with low ADC would be more immune responsive (i.e. have greater TIL infiltration) to trastuzumab. We also performed an exploratory analysis looking at differences in the expression of immune-relevant genes between “low” and “high” ADC patients. Methods: Patients with localized HER2+ breast cancers 10 mm or larger and planned for curative-intent therapy were eligible to enroll. After enrollment, patients received a single run-in dose of trastuzumab with paired breast MRI exams before and after treatment that included DCE and DWI sequences. Post-trastuzumab tissue was obtained from the patient’s surgical specimen or from a second biopsy (if planned for additional neoadjuvant treatment). MRI images were analyzed to measure the functional tumor volume and peak signal enhancement ratio from DCE and mean apparent diffusion coefficient (ADC) from DWI. TIL content was assessed by a breast pathologist in both the pre- and post-trastuzumab tissue specimens. The pre- and post-treatment samples were also analyzed using an immune-focused RNA-based multiplex gene expression assay. Results: Twelve women have enrolled to date with a median age of 53 years (range 37-70 years old). Five out of the twelve patients (42%) had estrogen-receptor negative tumors. For the 8 patients with pre- and post-treatment tissue currently available for analysis, the average pre-treatment TIL percentage was 22% (range 1-70%), the average post-treatment TIL percentage was 42% (range 1-90%), and the average absolute increase between pre- and post-treatment TILs was 20% (range 0%-58%). Pre- and post-treatment ADC showed a strong negative correlation with the increase in TIL content after trastuzumab treatment (r = -0.71 and r = -0.93, respectively). At submission, preliminary gene expression analysis of pre- and post-treatment tissue is available in 6 patients. The patients were binned into “high” and “low” ADC groups with the median pre-treatment ADC as the cut-off. The magnitude of change in gene expression after trastuzumab treatment (small vs large change) clustered by high or low pre-treatment ADC; this suggests that pre-treatment ADC may differentiate between two distinct immune phenotypes. Further analysis showed that the low ADC group had relatively higher pre-treatment gene expression of CD8, CD4, and CD19 (T and B cell markers) when compared to the high ADC group. The low ADC group also had a decrease in FoxP3 expression (regulatory T cell marker) versus the high ADC group, which had an increase in FoxP3 expression. The high ADC group also had a notable increase in CCR4 expression (associated with a TH2 type immune response), while the low ADC group did not have an increase. The combination of an increase in FoxP3 expression and an increase in CCR4 expression suggests that the high ADC group may have a more immune-inhibited microenvironment in response to trastuzumab. Conclusions: These initial analyses suggest that the pre-treatment ADC calculated from DWI may identify HER2+ breast cancers that will have an anti-tumor immune response to trastuzumab therapy. Final data from the full planned cohort will be presented at the meeting and include discussion of the DCE metrics. Based on this preliminary data, further study of breast MRI as an immune biomarker is warranted. Citation Format: Laura Carpin Kennedy, Grace Durenberger, Sasha E. Stanton, Shaveta Vinayak, Suzanne Dintzis, Savannah Partridge, VK Gadi. Breast MRI as a radiomic biomarker of immune response in HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-17.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Engineered hepatitis B core virus-like particle carrier for precise and personalized Alzheimer’s disease vaccine preparation via fixed-point coupling

Heterogenous aggregates of amyloid β (Aβ) and tau protein play a key role in Alzheimer’s disease (AD) progression. As the epitope profiles of Aβ and abnormally phosphorylated tau change over the course of the disease, tailor immunotherapy to specific patient groups according to the disease stage by targeting corresponding Aβ and tau species or both of tau and Aβ might improve treatment efficacy. However, epitope peptides have low immunogenicity and peptide vaccine preparation is laborious and time-consuming. We here first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of truncated Hepatitis B core protein (HBc)(1-149). The resulted recombinant protein HBc-SpyCatcher (HBc-S) could assemble into uniform and stable virus-like particles (VLPs), and readily bind to the SpyTag conjugated epitope peptides. Several series of peptides such as linear, cyclic and phosphorylated peptides including Aβ(1-6), Aβ(1-15), cAβ(1-7), cEP1, cEP2 from β-amyloid monomer or oligomers, T294, pTau396-404, pTau422 from tau proteins, were glued onto HBc-S VLPs, forming HBc-S-peptide (HBc-S-P) VLPs and efficiently elicited specific Th2-type immune responses. When applied to AD transgenic mice, HBc-S-pTau422 alleviated cognition deficits and neuropathology progression in Tau.P301S transgenic mice. The present study provides an easy, quick, convenient and universal platform for high-throughput peptide epitope screening and personalized peptide vaccine preparation.

Vaccination with a novel multi-epitope ROP8 DNA vaccine against acute Toxoplasma gondii infection induces strong B and T cell responses in mice

Rhoptry proteins (ROPs) are involved in the cell invasion and parasitophorous vacuole (PV) formation and also vital for survival of Toxoplasma gondii (T. gondii) within host cells. ROP8 have a main role during the early phase of infection and can express in tachyzoite and bradyzoite forms. In the present study, we designed a novel multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein to evaluate the immunity and protective efficacy against acute T. gondii infection in BALB/c mice. For this purpose, several bioinformatics online servers were used. At first, the potential epitopes were selected for T and B cells using immune epitope database (IEDB) and BCPREDS online services. Then, the selected epitopes were fused together by SAPGTP linker. Finally, the physico-chemical features, secondary and tertiary structures, antigenicity, and allergenicity of the peptide were evaluated through different bioinformatics tools. Lastly, the multi-epitope peptide was successfully cloned into pcDNA3.1 expression vector. The DNA vaccine was subcutaneously injected into BALB/c mice and the immune responses of the vaccinated mice and controls were determined. The obtained results revealed that the multi-epitope ROP8 peptide has 183 amino acid residues with average molecular weight (MW) of 18.974 kDa. More than 98 % residues of the peptide were incorporated in favored and allowed regions of the Ramachandran plot. The antigenicity of multi-epitope peptide were estimated 0.8751 and 0.7649 by ANTIGENpro and VaxiJen servers, respectively. BALB/c mice immunized with DNA vaccine showed significantly increased the level of specific anti-T. gondii antibodies (P < 0.05), and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a TH1-type cellular immune response with production of IFN-γ and prolonged survival time, compared with the control groups (P < 0.05). The findings revealed that the multi-epitope ROP8 DNA vaccine induced strong humoral and cellular responses and prolonged the survival time in BALB/c mice, suggesting selection of potential epitopes may be a promising strategy for the design of multi-epitope-based vaccines.

Lactococcus lactis expressing sand fly PpSP15 salivary protein confers long-term protection against Leishmania major in BALB/c mice.

Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months.

&lt;p&gt;Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax&lt;/p&gt;

IntroductionAluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis.Methods and ResultsHerein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150–200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel.ConclusionThese results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.