Higher Immune Response Research Articles

Effect of Conjugation of Activated Glutaraldehyde-Nanochitosan with L-Asparaginase as an Anti Cancer Enzyme on its Stability and Physicochemical Properties

Introduction: The bacterial Asparaginase is used in the treatment of asparagine-dependent tumors, particularly lymphatic sarcoma and acute lymphoblastic leukemia. However, the instability of the enzyme increases the number of injections that are accompanied by high immune responses. The aim of this study was to investigate the conjugation of L-asparaginase with nanochitosan glutaraldehyde (NCG) derivative and its effect on the physichochemical properties of conjugated enzyme. Methods: In this experimental study, nanochitosan was synthesized using reduction method with acetic acid and its physicochemical properties were evaluated using transmission electron microscopy and particle size analyzer. Activated NCG derivative was produced using 3% acetic acid. The conjugation of NCG derivative to L-asparaginase was performed with different molar ratios of enzyme/nanochitosan (1:2, 1:5, 1:10 and 1:20) in the presence of sodium cyano borohydrate; and the ratio with the highest residual activity was used for physicochemical evaluation. The activity of enzyme at different temperatures and pH, its half-life and stability after freezing and resistance to proteolysis were analyzed through repeated measure analysis of variance using SPSS 18 software. Results: The results of this study showed that the conjugation of L-asparaginase with NCG derivative resulted in maintaining the 70% enzyme activity. The activity of conjugated enzyme was higher than native enzyme after freezing and trypsin treatment. The optimum pH and temperature of conjugated enzyme did not change, while it had higher activity in wide range of pH and temperature compared with native enzyme. Conclusion: Conjugation of L-asparaginase with NCG derivative improved physicochemical and stability of enzyme and this method can be used for production of improved L-asparaginase for clinical application.

Examination of the effect of niosome preparation methods in encapsulating model antigens on the vesicle characteristics and their ability to induce immune responses

Niosome nanoparticles can be prepared using different methods, each of which can affect the size and homogeneity of the prepared particles. The aim of this study was to establish if the method of preparation impacted on the prepared vesicles when loaded with a model protein and the type of immune responses induced to the vaccine antigen. Niosomes were prepared using both the traditional thin film hydration (TFH) technique and the microfluidic mixing (MM) technique. Influenza antigen was then entrapped in the niosomes and formulations tested for their ability to induce in vivo immune responses in immunised BALB/c mice. Niosomes prepared by MM had a mean size of 157 ± 1.8 nm and were significantly more uniform compared with the niosomes prepared using TFH (mean size 388 ± 10 nm). Niosomes play a key role as an adjuvant to help raise high antibody immune responses. This was confirmed in this study since animals treated with both types of niosomes and antigen were more responsive than unentrapped (free) antigen. Cytokine analysis showed that the TFH niosomes induced a Th1 immune response by raising IgG2a and high levels of IFN-ɣ, while the MM niosomes induced a Th2 immune response by inducing IgG1 (p < .05). These results confirmed that the method of preparation of the niosomes nanoparticles induced different immune responses and the average particle size of the niosomes differed depending on the method of manufacture. This indicates that particle size and uniformity are of importance and should be taken into consideration when designing an oral based delivery system for vaccine delivery.

O-antigen-deficient, live, attenuated Salmonella typhimurium confers efficient uptake, reduced cytotoxicity, and rapid clearance in chicken macrophages and lymphoid organs and induces significantly high protective immune responses that protect chickens against Salmonella infection

In the present study, we developed an O-antigen-deficient, live, attenuated Salmonella Typhimurium (ST) strain (JOL2377) and assessed its safety, macrophage toxicity, invasion into lymphoid tissues, immunogenicity, and protection against Salmonella infection in chickens. The JOL2377 induced significantly lower cytotoxicity and higher level of cytokine response in IL-2, IL-10, IL-4, and IFN- γ than the WT strain upon macrophage uptake. It did not persist in macrophages or in chicken organs and rapidly cleared without systemic infection. None of the chicken were found to secrete Salmonella in feces into the environment exacerbating its attenuation. Interestingly JOL2377 successfully arrived in immunological hot-spots such as spleen, liver and bursa of Fabricius for an efficient antigen presentation and immune stimulation. Mucosal and parenteral immunization with JOL2377 significantly elicit antigen-specific humoral (IgY) and cell mediated responses marked by peripheral blood mononuclear cell proliferation, cytokine induction, increase in T-cell responses than non-immunized control. JOL2377 did not generate significant levels of LPS specific antibodies as compared to the WT strain due to the lack of immunogenic O-antigen component from its LPS structure. Upon virulent challenge, route dependent efficacy differences were leaving the intramuscular route is superior to the oral route on reducing splenic and liver colonization of the challenge ST. The least cytotoxicity, virulence, and superior immunogenicity of JL2377 that effectively engage both humoral and IFN- γ mediated CMI responses present an ideal scenario in host immune modulation to fight against intracellular pathogen Salmonella.

Identifying biomarkers to guide immunotherapy treatment of cancer of unknown primary.

e15252 Background: Cancer of unknown primary (CUP) is a metastatic cancer where the primary tissue of origin (ToO) evades detection despite extensive clinical investigation. Recent approaches to the management of CUP have involved gene-expression profiling to resolve the likely ToO and mutational profiling to identify targeted treatments. Current evidence is that both of these methods can have some clinical benefit but only in a minority of cases. Immunotherapy may also be effective in CUP, but clinical trials looking at this are yet to report and the best way to select patients who may benefit is uncertain. Methods: Through a national cohort study called Solving Unknown Primary Cancer (SUPER), 245 CUP patients had molecular profiling performed and clinical follow-up. Variant detection from DNA sequencing using a targeted panel of 386 genes (SureSelect Agilent) was used to calculate tumour mutation burden (TMB). Additionally, a custom gene expression assay including 225 genes associated with ToO and 35 immune genes (SUPERDx, nCounter NanoString) was used to classify the likely ToO and immune profile the tumours. We used the genomic data from 217 of these patients to explore interdependencies between immune gene-expression patterns and TMB. We also retrospectively reviewed the outcomes of patients treated with anti-PD1/PD-L1 immune checkpoint inhibition (ICI) therapy in whom genomic data was available. Results: TMB was weakly correlated with a gene-expression score for immune response (IR) based on averaged z-score values of six genes (CD8A, IFNG, PRF1, PD-L1, GZMA, GZMB) (Spearman rho = 0.21) with many outliers exhibiting low TMB but high IR scores. Among twenty patients treated with ICIs, with both molecular data and response data available, 6 had a partial response (PR), 5 had stable disease (SD) and 9 had progressive disease (PD). We found that an elevated IR score was more predictive of ICI response than TMB. Most patients with either PR or SD to ICI treatment had a high IR score (9/10), and most patients with PD had low IR scores. However, only 3/10 patients with TMB available and a PR or SD had a high TMB ( &gt; 10). Conclusions: Our results demonstrate that ICI therapy may benefit some CUP patients and that an immune response gene-expression scoremay predict ICI treatment response.

A microneedle vaccination with glycoprotein E of Varicella Zoster Virus elicits antibody production and polyfuctional T cells in mice

Abstract A microneedle has several benefits in that it is painless, easy to use and preventable from potential cross-contamination by using conventional needle. Besides these advantages, vaccination with microneedle may provide comparable or even higher immune response than vaccination through conventional intramuscular route. Here we have been developing herpes zoster vaccine using microneedle coated with recombinant glycoprotein E (gE) of VZV, which is essential in virus replication and cell-to-cell spread. Microneedles were coated by gE and three different adjuvants: double-mutant heat-labile toxin (dmLT), the combination of TLR1/2 and TLR3 ligands (Lpampo), or mutant cholera toxin A subunit (CTA). C57BL/6 mice were immunized with the coated microneedles two times at 2-week intervals. The overall increase in gE specific antibody titers was observed among adjuvanted groups compare to the non-adjuvanted group. Moreover, the humoral response was IgG1-dominated. Hence, a decreasing tendency of IgG1/IgG2c ratio was observed in gE adjuvanted with dmLT or Lpampo groups whereas relatively high ratio was detected in CTA group. This indicates that Th1 immune response complemented Th2 response in former groups. By conducting ELISPOT, we measured the amount of IFN-gamma secreting T cells as cell-mediated immune response is significant in immunogenicity of HZ vaccination. Moreover, polyfunctional CD4 T cells were increased after injection with gE +Lpampo coated microneedle. In consistent with humoral response, dmLT or Lpampo adjuvanted group exhibited enhanced cell-mediated immune response. Taken together, delivery of HZ subunit vaccine with microneedle may provide a potent immune response against reactivation of VZV.

Resilience of High Immune Response (HIR) Genetics in the Context of Climate Change: Effects of Heat Stress on Cattle with Diverse Immune Response Genotypes

Abstract As the temperature-humidity index (THI) increases, animals are less able to regulate body temperature leading to decreases in production and immune function. The High Immune Response (HIR™) technology classifies animals based on estimated breeding values (EBVs) of their IR capacity as high (H), average (A), or low (L). H-responders with their unique IR genotype have less disease than A or L, but have not been evaluated for adaptation to heat stress. Therefore, rectal temperatures of beef &amp; dairy cows with known IR classification were recorded during THI&amp;lt; 74 (normal) and THI&amp;gt;74 (above normal). Results show that 50% of beef &amp; 65% of dairy cows had body temperature above 39.2 at THI&amp;gt;74. However, cows that ranked H for antibody response were significantly better at regulating temperature and respiration at THI&amp;gt;74, than A or L. In vitro experiments showed variability in PBMC function when cells were heat stressed (42□, 4hrs). PBMC were stimulated with ConA to measure proliferation or LPS to assess nitric oxide (NO) production. HSP70 was measured in control and heat treated cells. Dairy results indicated that H responders had significantly greater cell proliferation across all treatments and greater NO and HSP production with significance depending on the treatment. Beef results indicate that HSP70 from unstimulated PBMC increased after 1 or 2 heat treatments, and the magnitude of the change varied by IR class. Conversely, PBMCs challenged with LPS produced more NO before than after heat treatments. These results indicate that cattle with diverse immune response genotypes vary in their ability to withstand heat stress, and that it should be possible to identify resilient individuals with both greater immunity and adaptation to climate warming.

Immune Response, Antioxidant Biomarkers and Histology of Caecal Tonsils of quail Supplemented with Sodium Butyrate

This study aimed to evaluate the impacts of dietary supplementation of sodium butyrate (SB) on antioxidant biomarkers, immune response and caecal tonsils (CT) histomorphometry of quail. A total of 240 one-day-old quails were randomly allocated into four groups with three replicates each. The first group was fed a basal diet (BD) without SB (control, T1), the 2nd group fed 1 g SB/kg BD during the first period from 0-3 weeks, then BD until the sixth week as early short feeding SB (ESFSB, T2), the 3rd group fed 1 g SB/kg BD during the whole period from 0-6 weeks as long feeding SB (LFSB, T3), and the 4th group fed BD from 0-3 week then fed 1 g SB/kg BD during the second period from 3-6 weeks as late short feeding SB (LSFSB, T4). The SB supplementation in quail diet significantly increased (P<0.05) serum total antioxidant capacity and declined malondialdehyde level compared with the control group. The inclusion of SB had a higher immune response through the increase of SRBCs titer value (P<0.05) in ESFSB, LSFSB and LFSB groups than the control group. Histomorphometry parameters of CT were significantly improved in ESFSB and LFSB compared the control. The LFSB group fed a diet containing SB from 0-6 weeks had better antioxidant biomarkers, immune response and histomorphometry parameters of CT of quail. In conclusion, it is suggested feeding quail on diets containing SB through the whole growth period to display its positive influence on the antioxidant biomarkers and immunity of quail

The inflammatory response after an intestinal inflammatory challenge affects in an opposite manner to the female and male offspring gestated in hypothyroxinemia

Abstract Ulcerative colitis (UC) is a highly frequent inflammatory bowel disease (IBD), which causes are unknown. We have shown that the offspring gestated under thyroxine (T4) deficiency, condition named hypothyroxinemia (HTX), shows inflammatory features at the intestine. Moreover, the offspring gestated under HTX (HTX-offspring) suffers strong experimental autoimmune disease. Suggesting that gestational HTX could imprint their offspring with a high immune response after an inflammatory challenge. Thus, we think that the HTX-offspring will be more prone to suffer IBD. To analyze this hypothesis C57BL6 pregnant mice were induced with gestational HTX during E10 to E15 by giving them methimazole in the tap-drinking water. Then, UC was induced to the HTX-offspring or control at postnatal P55 by giving them dextran sodium sulphate (DSS) in the tap-drinking water for six days. Body weight, pathological score of IBD and hidden blood in stool were analyzed daily. Then, mice were sacrificed at day 10 to evaluate their inflammatory response. For that histological analysis were performed in the intestine; the content of cytokines in serum and intestine by ELISA and the populations of innate and adaptive immune cells at the intestine and spleen by flow cytometry. Our results shown that the male and female offspring developed an opposite immune response to UC. The female HTX-offspring were more resistant and the male HTX-offspring were more sensitive to UC compared to their respective female and male control groups. Thus, our results suggest that the immune response of an individual can be set up during gestation and that the normal level of maternal T4 during gestation is fundamental for the proper function of the offspring immune system.

Regulation of body temperature in Canadian beef cattle of various immune response phenotypes when the temperature humidity index is above normal

Abstract Climate change with increases in ambient temperature and humidity, affect animal life dramatically decreasing their production and reproduction potential, as well as making them more susceptible to different diseases. This leads to an increase in their core body temperature and respiration rate which in turn decreases their production, reproduction, and immune function. The High Immune Response (HIR™) technology which has been developed at the University of Guelph ranks animals using two arms of adaptive immune system to high (H), average (A), and low (L) immune responders. (H) immune responders have been reported to have fewer incidents of disease and robust immune response. (H) immune responders have been also reported to have better colostrum and milk quality making them an ideal model to examine the effects of climate change on health traits. The objective of this study was to check if (H) immune responder beef cattle are able to regulate their rectal temperature better. In this study, rectal temperatures of 36 beef cows were recorded during normal Temperature-Humidity Index (THI&amp;lt;74) and above normal THI (THI ≥ 74) once in the morning and once in the afternoon. Results indicated that 62.5% of cows showed increases in rectal temperature in THI ≥ 74. However, mean rectal temperatures of (H) antibody-mediated immune responders did not differ significantly during THI &amp;lt; 74 and THI ≥ 74; indicating that these high antibody responder beef cows were better able to regulate their body temperatures. This is the first time that genetic regulation of an immune response trait has been shown to influence the response to in vivo heat stress indicating that it may be possible to select cattle with both improved health and heat tolerance.

Immune Function Testing and Donor-Specific Antibody Production among Heart Transplant Patients

De novo donor-specific antibody (DSA) production has been correlated to the development of antibody-mediated rejection after heart transplantation. It is unclear whether the Cylex ImmuKnow (IK) assay - a rapid, quantitative assessment of T-cell-mediated immune function designed to monitor immunosuppression response - can be a useful tool in overall immunosuppression and identification of patients at risk for de novo DSA production. We hypothesize that, among heart transplant recipients, increased immune function activity as measured by the IK assay would correlate with de novo DSA production. We conducted a single center cohort study including all consecutive single organ heart transplant recipients from 2014 to 2018. The IK assay categorized subjects into low, moderate, or high immune response levels at 6, 12, and 24-month intervals. De novo DSA production based on human leukocyte antigen antibody testing was correlated to IK immune response levels at these time points. 122 single organ heart transplant recipients were included. High immune response levels were noted in 10%, 9%, and 12% of subjects at 6, 12, and 24-months, respectively. Compared to baseline, de novo DSA production was noted in 21%, 23%, and 25% of subjects at the respective time intervals. 6-month IK category did not correlate with DSA production at 6 (r = -0.03, p=0.9), 12 (r = -0.03, p=0.9), or 24 months (r = 0.15, p=0.3). Similar findings were noted at 12 and 24 months. Being in a high IK category at any interval was not associated with de novo DSA production at any time point (p>0.05). Univariate analysis of demographic factors, induction agents used, and co-morbidities (including EMV or CBV infection status) did not reveal significant associations with de novo DSA production. High T-cell-mediated immune response levels, a possible indicator of under-suppression, as measured by the IK assay were not associated with de novo DSA production after heart transplantation.

Disruption of the Notch pathway aggravates airway inflammation by inhibiting regulatory T cell differentiation via regulation of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) regulate immunity and promote tolerance in asthma. Notch signalling is a highly conserved pathway that regulates the immune response; however, its role in pDC-mediated asthmatic airway inflammation is unclear. This study clarified the effects of Notch signalling on pDC-mediated airway inflammation using murine models of ovalbumin-sensitized allergic asthma. RBP-J-deficient pDCs (RBP-J-/- pDCs) were co-cultured with naïve CD4+ T cells and supernatants and T cell subtypes were analysed. RBP-J-/- pDCs were intranasally transferred to the airways of ovalbumin-sensitized recipient mice. Lung samples of all mice were subjected to tests for histopathology, cytokine profile of bronchoalveolar lavage fluid, airway hyperactivity and expression of T helper type 1 (Th1)/Th2 cells, regulatory T cells and type 2 innate lymphoid cells (ILC2s). The results showed that pDCs with and without RBP-J deficiency significantly differed in expression levels of cluster of differentiation 83 (CD83), but not CD80, CD86 and major histocompatibility complex class II. Co-culturing pDCs with naïve T cells revealed a poorer immunosuppressive effect of RBP-J-/- pDCs. This may be attributed to the lower expression levels of inducible co-stimulator ligand and lower production of interleukin 10 in RBP-J-/- pDCs than in control pDCs, which impeded T cell activation and Treg suppression. RBP-J-/- pDCs were associated with high ILC2 expression and severe Th2 immune responses and airway inflammation. Therefore, Notch signalling is critical for pDC-dependent immunoregulation, and RBP-J deficiency reduces pDC-based immunosuppression via T cell activation and Th cell differentiation. Thus, this pathway may be a therapeutic target for pDC-based anti-asthma immunotherapy.

Design, Construction and Immunogenicity Assessment of pEGFP-N1-KMP11-GP96 (Fusion) as a DNA Vaccine Candidate against Leishmania major Infection in BALB/c Mice

KMP-11 (Kinetoplastid membrane protein-Π) exists in all species of kinetoplastid family. It is fully conserved and the protein produced by this gene can induce a very high cellular immune response. We aimed to design a suitable construction for a Leishmania major DNA vaccine and evaluate the protective efficacy of it as a candidate for DNA vaccine against cutaneous leishmaniasis in BALB/c mice. This experimental study was conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. KMP-11 gene of L. major (MRHO/IR/75/ER, Iranian strain) and NT-GP96 of Xenopus GP96 DNA from a pBluescript-GP96 plasmid were amplified by PCR and the purified PCR products were cloned into the pJET1.2/blunt plasmid vector, then, subcloned into pEGFP-N1 plasmid as an expression vector. Finally, the KMP-11 gene was fused with GP96 and afterward the combination cloned in pEGFP-N1. All the cloned genes confirmed by enzyme digestions. Then, four groups of mice were immunized with PBS, pEGFP-N1, pEGFP-N1-KMP, and pEGFP-N1-fusion. Four weeks after immunization, all animals were challenged with L. major virulent promastigotes. The constructed fusion potentially showed an ability to elicit Th1 responses that led to cutaneous lesion healing. Interestingly, the group received KMP11-GP96 -GFP showed the highest ratio of IFN- γ /IL-4 and IgG2a/IgG1 compare to other groups. No side effect was observed after using the fusion in the mice. The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.

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.

Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

Programmable prodrug micelle with size-shrinkage and charge-reversal for chemotherapy-improved IDO immunotherapy

IDO blockade-based immunotherapy has been impeded by the activation of antitumor immune response and low delivery efficiency of immunotherapeutic, resulting from natural biological barriers and immune resistance. Herein, a programmable drug delivery nanosystem with enhanced tumor penetration and endocytosis is constructed for chemotherapy-enhanced immunotherapy by loading immune checkpoint IDO inhibitor NLG919 in pH/redox cascade-responsive prodrug micelle. The nanosystem shrinked micelles sizes and converted charge from negative to positive for enhanced tumor penetration and endocytosis in responding to the weakly acidic tumor microenvironment. The endocytosed nanosystem dramatically disassembled and released curcumin and NLG919 in redox-rich cytoplasm. In vitro and in vivo studies demonstrate that the nanosystem not only effectively overcame biological barriers, but also significantly boosted antitumor immune response and reduced immune resistance. It was realized by the combined effects of chemotherapy-enhanced immunogenicity, and NLG919-induced IDO-blockade immunotherapy, consequently inhibiting tumor growth, metastasis and recurrence with high efficiency in vivo. The study offers a nanoplatform with deep tumor penetration, high cellular uptake and effective antitumor immune response for the advance of chemo-immunotherapy.

Multivariate analysis of immune effect of hepatitis B vaccination in post-liver transplantation children aged under 5 years old

Objective To investigate the effect of hepatitis B vaccine immune response after liver transplantation in children under 5 years old and assess the necessity of multiple vaccination. Methods A retrospective collection of children who had liver transplantation from February 2014 to December 2018 in the same liver transplantation group in the Department of General Surgery, Beijing Friendship Hospital, Capital Medical University were analyzed. In accordance with the requirements of the doctor, 170 children under 5 years old injected with over 4 doses were enrolled, including 75 males and 95 females, aged from 4 months to 5 years old. After every dose of vaccination, the blood were collected to detect hepatitis B following regular review in liver transplantation clinic, observe the effect of HBsAb (hepatitis B surface antibody) after postoperative hepatitis B vaccination.Count data were represented by cases and percentage (%), and comparison between groups were analyzed by chi-square test. Results After the first course of treatment, in total children under 5 years old who completed the first course of treatment, 121 cases got successful immune responses(HBsAb≥100 IU/L) and 49 cases got the responses failure (HBsAb 100 IU/L anti-HBs titer had a higher immune response rate after the first course of postoperative vaccination (84.72%), and the immune response rate of children with anti-HBs titers <100 IU/L after the first course of hepatitis B vaccine was slightly lower(60.71%). Conclusions Most post-liver transplantation children can obtain protective antibodies to prevent re-infection of hepatitis B virus by inoculation with hepatitis B vaccine. Multiple inoculation can effectively induce hepatitis B antibody production and improve immune response level economically. The hepatitis B vaccination proved to be indispensable and is one of the current methods promoted and applied to post-liver transplantation. Key words: Liver transplantation; Hepatitis B vaccines; Child, preschool; Immunity

Abstract P4-06-11: High immune response identified as a good prognostic factor by proteomic SWATH-MS approach in 157 ER+/HER2- early breast cancer

Abstract Background: Estrogen positive/HER2 negative (ER+/HER2-) early (e) breast carcinoma (BC) is an heterogeneous entity, on the prognostic and predictive plan. Several prognostic multigene tests have been developed to identify patients in whom chemotherapy could be safely avoided. Proteomic is a complementary approach as variations in mRNA expression only account for ≈40% of the tumor-encoded protein range and it is taking into account, as other OMICs the tumoral microenvironment Sequential Windowed Acquisition of All Theoretical fragment (SWATH-MS) proteomic approach let an accurate and reproducible label-free quantification of large proteome. To our knowledge, no study has been conducted in a large cohort of luminal BC by SWATH-MS. The aim of this study was to establish a proteomic cartography of ER+/HER2- eBC and identify prognostic biomarkers. Methods: Frozen primary tumors were collected from 157 ER+/HER2- eBC treated in the ICO cancer center between 2006 and 2009. Patients were included if they fulfilled the following criteria: 1) ductal carcinoma; 2) unilateral; 3) first occurrence; 4) have received adjuvant chemotherapy. Clinicopathologic characteristics as outcomes were collected. Each sample was analyzed using SWATH-MS acquisition method as previously described (Aebersold et al, 2012). Peak extraction of the SWATH data was performed using either the Spectronaut software (ver 8.0, Biognosys, Switzerland). Peptide identification results were filtered with a q-value of &amp;lt; 1%. We performed clustering analysis (fuzzy clustering method) based on the 15% of most variant proteins. Functional annotation of clusters based on GO biological process terms enrichment (GOEA) was performed by means of ToppGene and GORILLA web tools. Results: The median of follow-up was 8.34 years. Respectively 32, 4 and 7 patients presented a metastatic, locoregional and controlateral recurrence. 684 among 4555 proteins represented the 15% of most variants proteins.Two ER+/HER2- eBC clusters were identified (C1 [23%] and C2 [77%]) by means of fuzzy clustering and GOEA. Two significant clinicopathological differences were observed between the two subgroups: more unifocal tumors in C1 (P = 0.0415) and mostly a clear better outcome in term of Disease Free Survival (DFS), Distant DFS (DDFS) and Overall survival (OS) in patients belonging to C2 (cf table 1). Functional annotation found that C1 was characterized by mRNA processing and protein synthesis (GO:0006396: RNA processing; GO:0008380: RNA splicing; GO:0016071: mRNA metabolic process; GO:0022613: ribonucleoprotein complex biogenesis), and C2 by a high immune response (GO:0002757: immune response-activating signal transduction; GO:0050778: positive regulation of immune response; GO:0002253: activation of immune response; GO:0050776: regulation of immune response). Differential protein expression according to the C1-C2 clusters will be presented at the meeting. Conclusion: Proteomic cartography by SWATH-MS can clearly distinguish two ER+/HER2- eBC subgroups with clear different prognosis with a better outcome for C2 patients compared to C1 patients. High immune response observed in C2 could underlie this difference with results that must be confirmed on external cohort. Nevertheless this approach could be considered as a complementary approach, helpful for clinical decision for administration of adjuvant treatment. All (n=157)Cluster 1 (n = 36)Cluster 2 (n = 121)PDisease-free survival72%55.5%77.6%0.0016Distant disease-free survival79.6%66.6%83.4%0.0063Overall survival81%61%87%0.0009Specific overall survival86%69%91%0.0012 Citation Format: Anne Patsouris, Hamza Lasla, Pascal Jézéquel, Philippe Juin, Alice Boissard, Cécile Henry, Mario Campone, Catherine Guette. High immune response identified as a good prognostic factor by proteomic SWATH-MS approach in 157 ER+/HER2- early 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 P4-06-11.

Abstract P4-14-11: Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration

Abstract Background: Previously we reported a porous silicon micro-particle based cancer vaccine, which stimulated robust CD8+ T cell-dependent anti-tumor immunity in mice bearing HER2-positive breast cancer. In this study we evaluated the efficacy of the vaccine via single injection into reconstructed abdominal flap after primary tumor mastectomy. Method: To generate murine model of HER2 positive breast cancer murine, 1 × 105 TUBO/luciferase cells were injectedinto both mammary gland fat pad and left ventricle of each 6-8 weeks old female Balb/c mice. Primary and systemic metastatic tumor growth were monitored with a IVIS-200 imaging system. Four days after tumor inoculation, mammary gland fat pad with primary tumor was resected by mastectomy, and superficial inferior epigastric(SIE) vessels based abdominal flap was used for abdominal reconstruction. During the surgery, mice also received one of the three treatments below: 1) negative control without treatments; 2) single intra-flap vaccination of cancer vaccine; and 3) single injection of cancer vaccine via footpad injection. Therapeutic efficacy were monitored with IVIS system weekly. To analyze immune response, popliteal and inguinal lymph nodes were collected one day and seven days post vaccination, and activation of dendritic cells and T cells was evaluated with a flow cytometry. HER2 specific T cells in splenocytes which highly expressed interferon-gamma (IFNγ) were counted by ELISpot. CD3+ T cell infiltration in brain metastatic nodules were analyzed by immunohistochemical assay. Result: Prolonged survival of mice bearing HER2-positive breast cancer was achieved by both intra-flap and intra-dermal vaccination of cancer vaccine. Mice vaccinated via intra-flap injection extended median survival by 9 days (p=0.025), although not as long as intra-dermal treatment (15 days, p=0.034), comparing to control group. In lymph nodes, activated dendritic cells increased in both two vaccinated groups. Interestingly, intra-flap vaccination induced higher response in inguinal lymph node, which considered as the major draining lymph node of this route. Meanwhile, intra-dermal vaccination raised higher immune response in popliteal lymph node. In ELISpot assay, IFNγ secreted T cells were found abundant in splenocytes from both two vaccination groups. Furthermore, efficient infiltration of T cells inside the brain metastatic nodules was found in intra-dermal injection group, which provided potential protection from distal recurrence after mastectomy. Conclusion: Cancer vaccine administrated via intra-flap injection effectively stimulated systemic immune response, and slowed down tumor progression in murine HER2 positive breast cancer model. Citation Format: Xiaoling Liu, Junhua Mai, Chaoyang Meng, Wei Wei, Haifa Shen. Prolonged survival of mice bearing HER2 positive breast cancer with a therapeutic cancer vaccine via intra-reconstructed abdominal flap administration [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 P4-14-11.

Production, immunogenicity, stability, and safety of a vaccine against Clostridium perfringens beta toxins.

Background and Aim:The beta toxin is causing the most severe Clostridium perfringens-related diseases. This work was dedicated to developing a vaccine against beta toxin using C. perfringens type C (NCTC 3180).Materials and Methods:The crude toxoid harvest contained 710 limits of flocculation (Lf)/mL. The vaccine was formulated. Each 1 mL of the final vaccine product contained at least 50 Lf/mL of beta toxoids, 0.2 mL 3% aluminum hydroxide gel (equivalent to 5.18 mg of aluminum), <0.001% W/V thiomersal, formaldehyde <0.05% W/V, and ~0.7 mL phosphate-buffered saline (pH 7.2). The efficacy of the vaccine was evaluated by potency, stability, and safety tests.Results:The vaccine demonstrated 24.36 IU/mL (standard deviation, ±0.56) and 14.74 IU/mL (±0.36) of neutralizing antibodies in rabbits and cattle, respectively. Indeed, these levels were above the minimum recommended by international protocols since the obtained antibody levels had 2.43- and 1.47-fold increase in both rabbits and cattle, respectively, over the minimum antitoxin level suggested by the United States Department of Agriculture. Interestingly, our formulation was capable of inducing 1.65-fold higher immune responses in rabbits than that stimulated in cattle (65% increase) with a significant difference (p<0.0001). The vaccine was stable up to 30 months. The vaccinated rabbits were suffered from a temporarily slight increase in temperatures in the first 10 h without any significant difference (p>0.05).Conclusion:The research showed a procedure for the manufacturing process of the vaccine against C. perfringens beta toxins with a feasible quantity and the vaccine described here showed to be effective in eliciting levels of neutralizing antibodies higher than required by international standards. In addition, The vaccine was stable up to 30 months. Thus, it may represent an effective and safe for preventing C. perfringens-related diseases in rabbits and cattle, although further studies to prove its efficacy in the field on other farm animals are still needed.

Joint use of Ca – Modified Double-Strained RNA as an Immunostimulator with a Vaccine for the Prevention of Food andMouth Disease (FMD)

The purpose of this work is to study the effect of combined use of the immunostimulant preparation which is double-stranded Ca-modified RNA with inactivated FMD vaccine for cattle against A, O and Asia-1 subtypes of FMD. Preparation have been received from sodium nucleinate or allocated from a killer yeast of Saccharomices cerewisiae. The modification of Ca ions assayed by the addition of sterile solution of calcium chloride. Based on received data it can be concluded that the using of immunomodulator in cattle induces more early and high immune response. The FMD vaccine intramuscular administration together with the preparation provides high enough of an immune response and high titer of antibody maintained for a period of 90 days (time of observation).