Secondary Immunization Research Articles

Mitigating multiple stresses in Pangasianodon hypophthalmus with a novel dietary mixture of selenium nanoparticles and Omega-3-fatty acid

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg−1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L−1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p < 0.01) with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p < 0.01) with the supplementation of Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

The mRNA-1273 Vaccine Induces Cross-Variant Antibody Responses to SARS-CoV-2 With Distinct Profiles in Individuals With or Without Pre-Existing Immunity.

mRNA-based vaccines effectively induce protective neutralizing antibodies against SARS-CoV-2, the etiological agent of COVID-19. Yet, the kinetics and compositional patterns of vaccine-induced antibody responses to the original strain and emerging variants of concern remain largely unknown. Here we characterized serum antibody classes and subclasses targeting the spike receptor-binding domain of SARS-CoV-2 wild type and α, β, γ and δ variants in a longitudinal cohort of SARS-CoV-2 naïve and COVID-19 recovered individuals receiving the mRNA-1273 vaccine. We found that mRNA-1273 vaccine recipients developed a SARS-CoV-2-specific antibody response with a subclass profile comparable to that induced by natural infection. Importantly, these antibody responses targeted both wild type SARS-CoV-2 as well as its α, β, γ and δ variants. Following primary vaccination, individuals with pre-existing immunity showed higher induction of all antibodies but IgG3 compared to SARS-CoV-2-naïve subjects. Unlike naïve individuals, COVID-19 recovered subjects did not mount a recall antibody response upon the second vaccine dose. In these individuals, secondary immunization resulted in a slight reduction of IgG1 against the receptor-binding domain of β and γ variants. Despite the lack of recall humoral response, vaccinees with pre-existing immunity still showed higher titers of IgG1 and IgA to all variants analyzed compared to fully vaccinated naïve individuals. Our findings indicate that mRNA-1273 vaccine triggered cross-variant antibody responses with distinct profiles in vaccinees with or without pre-existing immunity and suggest that individuals with prior history of SARS-CoV-2 infection may not benefit from the second mRNA vaccine dose with the current standard regimen.

Comparison of the mucosal adjuvanticities of two Toll-like receptor ligands for recombinant adenovirus-delivered African swine fever virus fusion antigens

The mucosal immunity plays an important role against African swine fever virus (ASFV) infection and the efficacy of mucosal vaccination is highly dependent on the adjuvant. However, the mucosal adjuvant for ASFV vaccination is poorly studied. Toll-like receptor (TLR) ligands such as the FlaB flagellin from Vibrio vulnificus and the heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) hold a great promise as novel vaccine adjuvant. However, the mucosal adjuvanticities of such TLR ligands have not been studied in pigs. In this study, three recombinant Adenovirus (rAd) vectors, namely rAd-F1, rAd-FlaB-F1 and rAd-F1-Hsp70, were constructed by fusing the FlaB or mHsp70 to ASFV CD2v-p30-p54 fusion antigen. Western blotting showed that the three fusion proteins expressed in rAd-infected cells reacted positively with ASFV antibodies. After intranasal immunization of pigs with the three rAd vectors, the antigen-specific IgG antibodies were detectable from day 7 after primary immunization, which were significantly boosted by the secondary immunization. Strong Th1/Th2 cytokine responses were detected in the peripheral blood mononuclear cells. Compared to immunization with the control rAd-F1, significantly higher levels of the antigen-specific IgA antibodies were detected in the nasal fluids, tracheal washes and lung lavages.11of rAd-FlaB-F1- or rAd-F1-Hsp70-immunized pigs. Compared to immunization with rAd-Flab-F1, immunization with rAd-F1-Hsp70 induced significantly stronger mucosal IgA antibody response. Cytokine detection of the pig lung lavages showed that the elevated IgA antibody responses were correlated mainly with IL-4, IL-10 and IFN-α, which were confirmed by the significantly increased antigen-recall cytokine expression in the porcine alveolar macrophages. These data suggest that mHsp70 has potent mucosal adjuvanticity in pigs, and the fusion rAd vector can be used for ASFV mucosal vaccine development.

Systems vaccinology of the BNT162b2 mRNA vaccine in humans.

The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) agreater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.

An Anti-Tumor Vaccine Against Marek's Disease Virus Induces Differential Activation and Memory Response of γδ T Cells and CD8 T Cells in Chickens.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. The most efficacious vaccine, CVI988/Rispens (CVI988), against MD has been used for several decades. However, the mechanisms leading to protective immunity following vaccination are not fully understood. In this study, employing multi-parameter flow cytometry, we performed a comprehensive analysis of T cell responses in CVI988-vaccinated chickens. CVI988 vaccination induced significant expansion of γδ T cells and CD8α+ T cells but not CD4+ T cells in spleen, lung and blood at early time-points. The expansion of these cells was CVI988-specific as infection with very virulent MDV RB1B did not elicit expansion of either γδ or CD8α+ T cells. Phenotypic analysis showed that CVI988 vaccination elicited preferential proliferation of CD8α+ γδ T cells and CD8αα co-receptor expression was upregulated on γδ T cells and CD8α+ T cells after immunization. Additionally, cell sorting and quantitative RT-PCR showed that CVI988 vaccination activated γδ T cells and CD8α+ T cells which exhibited differential expression of cytotoxic and T cell-related cytokines. Lastly, secondary immunization with CVI988 induced the expansion of CD8+ T cells but not γδ T cells at higher magnitude, compared to primary immunization, suggesting CVI988 did induce memory CD8+ T cells but not γδ T cells in chickens. Our results, for the first time, reveal a potential role of γδ T cells in CVI988-induced immune protection and provide new insights into the mechanism of immune protection against oncogenic MDV.

SIR1R2: Characterizing Malware Propagation in WSNs With Second Immunization

As an infrastructure of Internet of Things, WSNs (Wireless Sensor Networks) play a more and more important role. However, WSNs are vulnerable to malware and malware can destroy their data security and integrity, which motivates us to explore the role of malware propagation in WSNs. First, according to the actual propagation characteristics of malware in the WSNs and the process of the density change of all node types, we propose an epidemiology-based malware propagation model in consideration of a secondary immune mechanism. Then we set up differential equations to describe the propagation model. By solving differential equations, we can obtain two kinds of equilibrium points indicating that the density of all node types tends to be stable in the WSNs. One is to achieve an equilibrium where only susceptible nodes exist in the WSNs. The other is that malware always exists in the WSNs. Moreover, we prove the local and global stability of these two equilibrium points. Eventually, we analyze the influence and effect of the secondary immunity, forgetting mechanism and containment mechanism on malware propagation in the WSNs, and validate the proposed model through simulations.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI–40 13–am43 and L cII–101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

Size-Dependent Phagocytic Uptake and Immunogenicity of Gliadin Nanoparticles.

The main objective of the present study was to investigate the hemo and immune compatibility of gliadin nanoparticles as a function of particle size. Gliadin nanoparticles of different size were prepared using a modified antisolvent nanoprecipitation method. The hemolytic potential of gliadin nanoparticles was evaluated using in vitro hemolysis assay. Phagocytic uptake of gliadin nanoparticles was studied using rat polymorphonuclear (PMN) leukocytes and murine alveolar peritoneal macrophage (J774) cells. In vivo immunogenicity of gliadin nanoparticles was studied following subcutaneous administration in mice. Gliadin nanoparticles were non-hemolytic irrespective of particle size and hence compatible with blood components. In comparison to positive control zymosan, gliadin nanoparticles with a size greater than 406 ± 11 nm showed higher phagocytic uptake in PMN cells, while the uptake was minimal with smaller nanoparticles (127 ± 8 nm). Similar uptake of gliadin nanoparticles was observed in murine alveolar peritoneal macrophages. Anti-gliadin IgG antibody titers subsequent to primary and secondary immunization of gliadin nanoparticles in mice were in the increasing order of 406 ± 11 nm < 848 ± 20 nm < coarse suspension). On the other hand, gliadin nanoparticles of 127 ± 8 nm in size did not elicit immunogenic response. Phagocytosis and immunogenicity of gliadin nanoparticles are strongly influenced by particle size. The results of this study can provide useful information for rational design of protein-based nanomaterials in drug delivery applications.

B Cell Subsets Differentially Contribute to the T Cell-Independent Memory Pool.

The roles distinct B cell subsets play in clonal expansion, isotype switching, and memory B cell differentiation in response to T cell-independent type 2 Ags (TI-2 Ags) has been understudied. Using sorted B cells from VHB1-8 knock-in mice, we evaluated B-1b, marginal zone, and follicular B cell responses to the TI-2 Ag, NP-Ficoll. All subsets extensively divided in response to NP-Ficoll. Nonetheless, B-1b cells exhibited significantly increased IgG switching and differentiation into Ab-secreting cells (ASC)-a finding that coincided with increased AgR signaling capacity and Blimp1 expression by B-1b cells. All subsets formed memory cells and expressed markers previously identified for T cell-dependent memory B cells, including CD80, PDL2, and CD73, although B-1b cells generated the greatest number of memory cells with higher frequencies of IgG- and CD80-expressing cells. Despite memory formation, secondary immunization 4 wk after primary immunization did not increase NP-specific IgG. However, boosting occurred in B-1b cell-recipient mice when IgG levels declined. CD80+ memory B-1b cells divided, class switched, and differentiated into ASC in response to Ag in vivo, but this was inhibited in the presence of NP-specific IgG. Furthermore, CD80 blockade significantly increased memory B-1b cell division and differentiation to ASC upon Ag restimulation. Collectively, these findings demonstrate B-1b, marginal zone B, and follicular B subsets significantly contribute to the TI-2 Ag-specific memory B cell pool. In particular, we show B-1b cells generate a functional CD80-regulated memory population that can be stimulated to divide and differentiate into ASC upon Ag re-encounter when Ag-specific IgG levels decline.

Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight ‘molecular budget’

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a ‘molecular budget’ medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

BTK Inhibitors in Cancer Patients with COVID-19: "The Winner Will be the One Who Controls That Chaos" (Napoleon Bonaparte).

As the SARS-CoV-2 (COVID-19) pandemic spreads and the number of Bruton's tyrosine kinase inhibitor (BTKi)-treated COVID-19-affected patients grows, we must consider the pros and cons of BTKi discontinuation for our patients. In favor of BTKi continuation, BTK plays an active role in macrophage polarization. By modulating key transcription factors, BTK may regulate macrophage polarization downstream of classic M1 and M2 polarizing stimuli and mitigate the hyperinflammatory state associated with COVID-19. In favor of BTKi discontinuation, we note a potentially increased risk of secondary infections and impaired humoral immunity. We hypothesize that the potential benefit of blunting a hyperinflammatory response to SARS-CoV-2 through attenuation of M1 polarization outweighs the potential risk of impaired humoral immunity, not to mention the risk of rapid progression of B-cell malignancy following BTKi interruption. On the basis of this, we suggest continuing BTKi in patients with COVID-19.

Cervical dystonia — improving the effectiveness of botulinum toxin therapy

Cervical dystonia is the most frequent form of focal dystonia. It is characterised by involuntary muscular contractions resulting in abnormal head/neck and shoulder movements and postures, which can be associated with tremor and pain. Local intramuscular injections of botulinum toxin type A (BoNT-A) is the treatment of choice, being both effective and well-tolerated. However, a considerable number (c. 30%) of patients discontinue this treatment. The aim of this review was to analyse the factors possibly responsible for treatment failures of cervical dystonia (CD), with special regard to the new classification known as the 'Col-Cap' concept and non-motor symptoms. Several factors analysed in this review are responsible for effective treatment: proper diagnosis of dystonia and exclusion of pseudodystonias, correct recognition of dystonia pattern and identification of new patterns according to the Col-Cap concept, muscle selection and precise injections under electromyography (EMG) and/or ultrasonography (US) guidance. Furthermore, concomitant diagnosis and treatment of non-motor symptoms such as depression, anxiety, fatigue, sleep problems, phobias and stigmatisation are crucial in obtaining the best overall effect of the treatment. Primary and secondary immunisation and non-responsiveness seem to be marginal problems nowadays due to a low potential of new BoNT-A formulations to produce neutralising antibodies. There is a need for new and relevant scales combining the Col-Cap concept patterns with non-motor symptoms and quality of life. There is also a lack of specific rehabilitation protocols which could enhance BoNT-A treatment results.

Сучасні уявлення про патогенез міокардиту

Діагностика та прогнозування перебігу міокардиту – одна з найскладніших і невирішених проблем сучасної кардіології не тільки в Україні, а й в розвинених країнах світу. Загальновідомо, що для розроблення адекватних методів діагностики, лікування та попередження ускладнень необхідними є фундаментальні знання, які стосуються патогенетичних механізмів розвитку і прогресування певного захворювання. В патогенезі порушення скоротливої функції серця і його дилатації як у гостру, так і в хронічну стадію міокардиту першочергову роль відіграють імунопатологічні реакції, які виявляються автоімунізацією та гіперреактивністю щодо структурних елементів серцевого м’яза. В основі патогенетичних механізмів вірусного міокардиту лежить комплекс чинників – пряма цитотоксична дія вірусу на кардіоміоцити, активація процесів апоптозу, а також реакцій первинного і вторинного імунітету, ураження мікросудинного русла, ремоделювання скоротливого апарату серцевого м’яза. Основними прозапальними цитокінами, які виробляються імунними клітинами у вогнищі запалення, є γ-інтерферон, фактор некрозу пухлини α, інтерлейкін (ІЛ) 1β, ІЛ-2, ІЛ-6, ІЛ-17А, ІЛ-23. Інший механізм ушкодження міокарда пов’язаний з активацією імунопатологічних реакцій гуморального типу із синтезом кардіоспецифічних антитіл, зокрема до β1-адренорецептора, кардіального міозину, актину, ламініну, віментину та інших структур серцевого м’яза. Суттєва роль у патогенезі міокардиту нині відводиться стимуляції toll-подібних рецепторів 2-го та 4-го типу й активації матриксних металопротеїназ, що має безпосередній зв’язок із продукцією прозапальних цитокінів. Перспективним для уточнення деяких патогенетичних мезханізмів запального ураження серцевого м’яза на сьогодні вважається і вивчення ролі різних типів мікроРНК. На цей час світовою кардіологічною спільнотою визнана актуальність подальшого вивчення різних механізмів патогенезу міокардиту з метою визначення тих патогенетичних ланок, вплив на які може зменшити патологічний ефект імунозапального ураження серця та тяжкість перебігу захворювання й поліпшити прогноз для пацієнтів з міокардитом.

Viral vaccines improve PD-1 therapy against cancer

Abstract Programmed cell death-1 (PD-1) therapy is effective against various types of cancer. However, only 30% of cancers respond to PD-1 therapy, highlighting the need to further improve its efficacy. In this study, we interrogated whether one could exploit the immunostimulatory properties of viral vaccines to enhance PD-1 therapy in a B16 melanoma model that is poorly responsive to PD-1 therapy. Immunization of tumor-bearing mice with clinically approved viral vaccines, such as the smallpox virus vaccine and the yellow fever virus vaccine (YFV-17D), resulted in an unprecedented improvement of PD-1 therapy. This synergistic effect on PD-1 therapy was more potent upon a secondary immunization, suggesting a positive effect of recruiting pre-existent immune memory to the tumor microenvironment. Interestingly, co-administration of viral vaccines together with PD-1 therapy did not increase tumor-specific CD8 T cell responses relative to PD-1 therapy alone. Mechanistically, the adjuvant effect of viral vaccines on PD-1 therapy seems to be dependent on innate immune responses. These findings may be important for improving the efficacy of checkpoint-based immunotherapies by exploiting the immunogenicity of viral vaccines. Altogether, our data suggest that viral vaccines could be repurposed as immunotherapy adjuvants, and that the beneficial effects of viral vaccines on PD-1 therapy can be further improved by repetitive boosting of bystander immunity. R21 AI132848-01A1 (NIAID); IRG-15-173-21 (ACS), and the Lurie Cancer Center.

Circulating memory is sufficient for protective immunity to infection with Chlamydia muridarum

Abstract Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and yet no vaccine currently exists to protect the public from this reproductive tract pathogen. Recent reports suggest that CD4 tissue resident memory cells (TRM) are essential for vaccine-induced protection against Chlamydia trachomatis in a mouse infection model. Additionally, CD4 TRM in the female reproductive tract (FRT) have been shown to co-localize with APCs in memory lymphocyte clusters (MLCs) to enhance secondary adaptive immunity to reproductive tract infections. Here, we examine a requirement for CD4 TRM and MLCs in protective immunity against infection with Chlamydia muridarum (Cm). Using parabiosis of naïve and Chlamydia-immune mice, we demonstrate that although CD4 TRM form in the FRT after primary Cm infection, they are not required for protection. Furthermore, histological analysis of FRT tissue from parabionts revealed that circulating memory is sufficient for protection against intravaginal challenge with Cm in the absence of CD4 TRM or MLCs in the reproductive tract. Moreover, this protective circulating memory can be induced through intravaginal or intranasal challenge with Chlamydia, indicating that a strong mucosal immunization can generate circulating protection in a vaccine setting. Ongoing work using adoptive transfers and antibody depletions of CD4 T cells and B cells will reveal which memory lymphocytes are responsible for the observed circulating protection.

Inhibition of protein phosphatase-2A with LB-100 enhances antitumor immunity against glioblastoma.

Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

Primary and secondary anti-viral response captured by the dynamics and phenotype of individual T cell clones

The diverse repertoire of T-cell receptors (TCR) plays a key role in the adaptive immune response to infections. Using TCR alpha and beta repertoire sequencing for T-cell subsets, as well as single-cell RNAseq and TCRseq, we track the concentrations and phenotypes of individual T-cell clones in response to primary and secondary yellow fever immunization - the model for acute infection in humans - showing their large diversity. We confirm the secondary response is an order of magnitude weaker, albeit ∼10 days faster than the primary one. Estimating the fraction of the T-cell response directed against the single immunodominant epitope, we identify the sequence features of TCRs that define the high precursor frequency of the two major TCR motifs specific for this particular epitope. We also show the consistency of clonal expansion dynamics between bulk alpha and beta repertoires, using a new methodology to reconstruct alpha-beta pairings from clonal trajectories.

Abstract TMP24: Soluble St2 Links Innate Inflammation to Secondary Injury After Stroke and Subarachnoid Hemorrhage

Background: We recently found that soluble ST2 (sST2) is a plasma marker that predicts neurological outcome after ischemic stroke and aneurysmal subarachnoid hemorrhage. We sought to extend these findings by studying sST2 associations with markers of secondary brain injury and peripheral innate immunity. Methods: We studied 241 acute ischemic stroke (AIS) patients with sST2 level measured on admission (7.4 ± 3.7 hrs after stroke onset) and at least one follow-up brain imaging study, obtained at a mean of 2.2 ± 1.4 days after stroke onset. A subgroup (n=147) had a second sST2 measurement at a mean of 2.1 ± 1.7 days after stroke onset. A separate cohort of 110 subarachnoid hemorrhage (SAH) patients were studied who had plasma sST2 measured at approximately 3.5 ± 1.2 days after ictus. Markers of secondary brain injury included edema after AIS (midline shift), and delayed cerebral ischemia (DCI) or epileptiform discharges on EEG after SAH. The relationships between sST2 level and outcome measures were assessed. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry to phenotype the peripheral immune response. Results: In the AIS cohort, baseline plasma sST2 level was associated with the presence of midline shift (46.1 vs. 41.8 ng/mL, p=0.017), which further increased at 48 hr after stroke onset (50.6 vs. 38.4 ng/mL, p=0.006). In the SAH cohort, median sST2 in patients who developed new epileptiform abnormalities was higher compared to patients who did not (114.8 ng/ml vs. 74.7 ng/ml, p=0.024). Higher median sST2 concentration was also observed in those patients with DCI (90.8 vs 53.7ng/mL, p=0.003). In patients with high sST2, flow cytometry identified a peripheral monocyte population with decreased expression of CD14 (4.27x105 ± 2950 A.U. vs. 5.64x105 ± 1290 A.U., p&lt;0.001), and increased expression of CD16 (39,960 ± 272 A.U. vs. 34,869 ± 183 A.U., p&lt;0.001). Conclusion: Plasma sST2 independently predicts edema after stroke, and DCI and epileptiform abnormalities after subarachnoid hemorrhage. Elevated sST2 is also associated with changes in peripheral innate immune cells. These data suggest that sST2 links the innate immune response to secondary brain injury.

C5aR agonist enhances phagocytosis of fibrillar and non-fibrillar Aβ amyloid and preserves memory in a mouse model of familial Alzheimer's disease.

According to the amyloid hypothesis of Alzheimer's disease (AD) the deposition of prefibrillar and fibrillar Aβ peptide sets off the pathogenic cascades of neuroinflammation and neurodegeneration that lead to synaptic and neuronal loss resulting in cognitive decline. Various approaches to reduce amyloid load by reducing production of the Aβ peptide or enhancing amyloid clearance by primary or secondary immunization have not proven successful in clinical trials. Interfering with the normal function of secretases and suboptimal timing of Aβ peptide removal have been put forward as possible explanations. Complement, an innate component of the immune system, has been found to modulate disease pathology and in particular neuronal loss in the AD mouse model but its mechanism of action is complex. C1Q has been shown to facilitate phagocytosis of Aβ peptide but its Ablation attenuates neuroinflammation. Experiments in AD mouse models show that inhibition of complement component C5a reduces amyloid deposition and alleviates neuroinflammation. Phagocytes including microglia, monocytes and neutrophils carry C5a receptors. Here, a widely used mouse model of AD, 5XFAD, was intermittently treated with the oral C5a receptor agonist EP67 and several neuronal and neuroinflammatory markers as well as memory function were assessed. EP67 treatment enhanced phagocytosis, resulting in a significant reduction of both fibrillar and non-fibrillar Aβ, reduced astrocytosis and preserved synaptic and neuronal markers as well as memory function. Timely and phasic recruitment of the innate immune system offers a new therapeutic avenue of treating pre-symptomatic Alzheimer disease.

Restrained expansion of the recall germinal center response as biomarker of protection for influenza vaccination in mice.

Correlates of protection (CoP) are invaluable for iterative vaccine design studies, especially in pursuit of complex vaccines such as a universal influenza vaccine (UFV) where a single antigen is optimized to elicit broad protection against many viral antigenic variants. Since broadly protective antibodies against influenza virus often exhibit mutational evidence of prolonged diversification, we studied germinal center (GC) kinetics in hemagglutinin (HA) immunized mice. Here we report that as early as 4 days after secondary immunization, the expansion of HA-specific GC B cells inversely correlated to protection against influenza virus challenge, induced by the antigen. In contrast, follicular T helper (TFH) cells did not expand differently after boost vaccination, suggestive of a B-cell intrinsic difference in activation and differentiation inferred by protective antigen properties. Importantly, differences in antigen dose only affected GC B-cell frequencies after primary immunization. The absence of accompanying differences in total anti-HA or epitope-specific antibody levels induced by vaccines of different efficacy suggests that the GC B-cell response upon revaccination represents an early and unique marker of protection that may significantly accelerate the pre-clinical phase of vaccine development.