Intravenous Immunization Research Articles

Arenavirus-Based Vectors Generate Robust SIV Immunity in Non-Human Primates.

Arenavirus-based vectors are being investigated as therapeutic vaccine candidates with the potential to elicit robust CD8 T-cell responses. We compared the immunogenicity of replicating (artPICV and artLCMV) and non-replicating (rPICV and rLCMV) arenavirus-based vectors expressing simian immunodeficiency virus (SIV) Gag and Envelope (Env) immunogens in treatment-naïve non-human primates. Heterologous regimens with non-replicating and replicating vectors elicited more robust SIV IFN-γ responses than a homologous regimen, and replicating vectors elicited significantly higher cellular immunogenicity than non-replicating vectors. The heterologous regimen elicited high anti-Env antibody titers when administered intravenously, with replicating vectors inducing significantly higher titers than non-replicating vectors. Intramuscular immunization resulted in more durable antibody responses than intravenous immunization for both vector platforms, with no difference between the replicating and non-replicating vectors. Overall, both replicating and non-replicating arenavirus vectors generated robust T- and B-cell-mediated immunity to SIV antigens in treatment-naïve non-human primates, supporting further evaluation of these vectors in a clinical setting for HIV therapy.

Intravenous Immunization with Triple Auxotrophs of Mycobacterium tuberculosis : A novel vaccine strategy against tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis ( Mtb ), remains a leading infectious cause of mortality worldwide despite widespread use of the BCG vaccine and the availability of sterilizing pharmacopoeia. Recent research indicates that the intravenous administration of BCG confers sterilizing immunity against Mtb pulmonary challenge in non-human primates. However, while BCG is relatively safe, complications such as disseminated BCGosis have been observed in immunocompromised individuals. Double auxotrophic mutants of Mtb lacking the ability to synthesize leucine and pantothenate are safe and sterilized in immunocompromised mice and SIV-infected Rhesus macaques. We examined how immunization with a Mtb triple auxotrophic strain, mc 2 7902, which cannot synthesize leucine, pantothenate, and arginine, protects immunocompetent mice from a virulent Mtb infection. The route of immunization was a crucial factor for protection with mc 2 7902 with intravenous immunization being 100 times more effective in protecting immunocompetent mice from Mtb challenge when compared to conventional subcutaneous vaccination with BCG. To further increase the safety of the attenuated auxotroph for vaccine purposes, the type VII secretion system Esx1 responsible for BCG attenuation was deleted in mc 2 7902. When tested by prime-boost immunization of immunocompetent mice followed by aerosol challenge with virulent Mtb , mc 2 7902 Δ esx1 provided similar protection to mc 2 7902. This robust protection against Mtb infection conferred by mc 2 7902 and mc 2 7902 Δ esx1 in a mouse model paves the way for new TB vaccine development using highly attenuated, auxotrophic Mtb strains.

Visualizing the early immune response to Mycobacterium tuberculosisinfection in macaques immunized with intravenous BCG

Abstract Tuberculosis (TB) causes 1.6 million deaths annually and the standard human vaccine, intradermal (ID) Bacille-Calmette Guérin (BCG), does not prevent pulmonary TB. Previously, we showed that intravenous (IV) BCG immunization elicited robust TB-specific lung-resident T cell responses compared to ID BCG in rhesus macaques (RMs). Following Mycobacterium tuberculosis (Mtb) challenge, 6 out of 10 RMs had no detectable disease as demonstrated by bacterial burden, granuloma formation, and primary Mtb-specific immune responses. Together, these data suggest that Mtb is cleared rapidly from the lungs of IV BCG-vaccinated RMs. To investigate the vaccine-induced lung environment and anamnestic response that might mediate protection against Mtb early after challenge, we developed a high-dose Mtb infection model in RMs that allowed us to locate and live-image areas of Mtb infection in the lungs using confocal microscopy. RMs immunized for 5 months with either IV or ID BCG were challenged with Mtb Erdman mCherry and euthanized 4 or 11 days later to characterize the lung microenvironment. We observed reductions in bacterial burdens in the lungs of RMs that received IV BCG compared to ID BCG-vaccinated and unvaccinated controls by CFU plating, flow cytometry, and confocal microscopy despite using a high challenge dose. Increased T cells were quantified in the lungs of both IV and ID BCG-vaccinated RMs compared to unvaccinated controls by flow cytometry and confocal microscopy. This research provides an opportunity to mine immune correlates of protection in a preclinical model, at the site of infection, and at a post-challenge time point that has been minimally characterized. NIH Intramural Program

SARS-CoV-2 Spike Protein Expression In Vitro and Hematologic Effects in Mice Vaccinated With AZD1222 (ChAdOx1 nCoV-19).

Severe COVID-19 can be associated with a prothrombotic state, increasing risk of morbidity and mortality. The SARS-CoV-2 spike glycoprotein is purported to directly promote platelet activation via the S1 subunit and is cleaved from host cells during infection. High plasma concentrations of S1 subunit are associated with disease progression and respiratory failure during severe COVID-19. There is limited evidence on whether COVID-19 vaccine-induced spike protein is similarly cleaved and on the immediate effects of vaccination on host immune responses or hematology parameters. We investigated vaccine-induced S1 subunit cleavage and effects on hematology parameters using AZD1222 (ChAdOx1 nCoV-19), a simian, replication-deficient adenovirus-vectored COVID-19 vaccine. We observed S1 subunit cleavage in vitro following AZD1222 transduction of HEK293x cells. S1 subunit cleavage also occurred in vivo and was detectable in sera 12 hours post intramuscular immunization (1x1010 viral particles) in CD-1 mice. Soluble S1 protein levels decreased within 3 days and were no longer detectable 7–14 days post immunization. Intravenous immunization (1x109 viral particles) produced higher soluble S1 protein levels with similar expression kinetics. Spike protein was undetectable by immunohistochemistry 14 days post intramuscular immunization. Intramuscular immunization resulted in transiently lower platelet (12 hours) and white blood cell (12–24 hours) counts relative to vehicle. Similarly, intravenous immunization resulted in lower platelet (24–72 hours) and white blood cell (12–24 hours) counts, and increased neutrophil (2 hours) counts. The responses observed with either route of immunization represent transient hematologic changes and correspond to expected innate immune responses to adenoviral infection.

Production of hyperimmune serum against genotype VII Newcastle disease virus in rabbits with several applications.

Objective:This study aimed to produce hyperimmune serum against genotype VII Newcastle disease virus (NDV) with several applications.Materials and Methods:Production of hyperimmune serum against genotype VII NDV was performed on eight New Zealand white rabbits divided into four groups. Rabbits were immunized three times on the 1st day, the 14th day, and the 30th day. Blood sampling was carried out on the 8th day after the third immunization.Results:All groups showed the same pattern of hemagglutination inhibition (HI) titer results. HI titers would peak on the 5th or the 9th day after the second immunization, then decrease until the 3rd day after the third immunization, and increase again on the 5th day after the third immunization. Rabbits immunized intravenously showed higher HI titers than the other groups. These results indicate that the intravenous route for hyperimmune serum production against genotype VII Newcastle disease virus greatly affects the immune response result.Conclusions:The production of hyperimmune serum by intravenous immunization three times was able to produce the highest titer of 210 at 38 days. The agar gel precipitation test and the Western blot assay showed that the hyperimmune serum was specific for the Newcastle disease antigen.

Assessment of experimental malaria vaccine induced protection in pre-exposed populations

Efforts to control and further reduce morbidity and mortality caused by Plasmodium falciparum infections in all age groups will be substantially strengthened by development of efficacious malaria vaccines in malaria pre-exposed populations. 1 Moorthy VS Newman RD Okwo-Bele J-M Malaria vaccine technology roadmap. Lancet. 2013; 382: 1700-1701 Summary Full Text Full Text PDF PubMed Scopus (1) Google Scholar Vaccine development against parasitic diseases represents a formidable challenge given the complexity of parasite life cycles and the intricate human host–parasite interactions. How to robustly assess vaccine-induced protection during clinical development is one of the key determinants driving successful vaccine development programmes. In the field of P falciparum malaria vaccine development, the availability of controlled human malaria infection (CHMI) model has helped to systematically select vaccine candidates, including the advanced subunit vaccines RTS,S 2 Sauerwein RW Roestenberg M Moorthy VS Experimental human challenge infections can accelerate clinical malaria vaccine development. Nat Rev Immunol. 2011; 11: 57-64 Crossref PubMed Scopus (193) Google Scholar (now recommended by WHO), R21, 3 Datoo MS Natama MH Somé A et al. Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial. Lancet. 2021; 397: 1809-1818 Summary Full Text Full Text PDF PubMed Scopus (43) Google Scholar and the whole, live organism-based vaccine, PfSPZ Vaccine, 4 Seder RA Chang L-J Enama ME et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science. 2013; 341: 1359-1365 Crossref PubMed Scopus (507) Google Scholar for further clinical development. The PfSPZ Vaccine contains irradiation-attenuated, purified, vialed, cryopreserved sporozoites of P falciparum (strain NF54) and numerous studies have shown the vaccine to be safe and tolerable in east and west African volunteers in a broad range of age groups. 5 Jongo SA Urbano V Church LWP et al. Immunogenicity and protective efficacy of radiation-attenuated and chemo-attenuated PfSPZ Vaccines in Equatoguinean adults. Am J Trop Med Hyg. 2021; 104: 283-293 Crossref PubMed Google Scholar , 6 Jongo SA Church LWP Mtoro AT et al. Safety and differential antibody and t-cell responses to the Plasmodium falciparum sporozoite malaria vaccine, PfSPZ Vaccine, by age in Tanzanian adults, adolescents, children, and infants. Am J Trop Med Hyg. 2019; 100: 1433-1444 Crossref PubMed Scopus (29) Google Scholar Safety and efficacy of a three-dose regimen of Plasmodium falciparum sporozoite vaccine in adults during an intense malaria transmission season in Mali: a randomised, controlled phase 1 trialA three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. Full-Text PDF

Chronic hepatitis B in pediatrics: To treat or not to treat, that is the question

Chronic hepatitis B virus infection is one of the most prevalent diseases worldwide. It may progress to cirrhosis and hepatocellular carcinoma. An early detection, not using intravenous drugs, sex education, and immunization are critical for prevention. An infection in the neonatal period and in the first year of life becomes chronic in more than 90 % of children. Vertical transmission from a mother with hepatitis B virus to the newborn infant is currently the most common mode of transmission. Detection, immunoglobulin administration, and immunization help to reduce it. Antiviral therapy may accelerate the transition from the active to the inactive phase of infection by two or three years, without affecting the recovery process. A timely treatment of some selected cases may prevent hepatitis B progression.

Current Understanding of Natural Antibodies and Exploring the Possibilities of Modulation Using Veterinary Models. A Review.

Natural antibodies (NAb) are defined as germline encoded immunoglobulins found in individuals without (known) prior antigenic experience. NAb bind exogenous (e.g., bacterial) and self-components and have been found in every vertebrate species tested. NAb likely act as a first-line immune defense against infections. A large part of NAb, so called natural autoantibodies (NAAb) bind to and clear (self) neo-epitopes, apoptotic, and necrotic cells. Such self-binding antibodies cannot, however, be considered as pathogenic autoantibodies in the classical sense. IgM and IgG NAb and NAAb and their implications in health and disease are relatively well-described in humans and mice. NAb are present in veterinary (and wildlife) species, but their relation with diseases and disorders in veterinary species are much less known. Also, there is little known of IgA NAb. IgA is the most abundant immunoglobulin with essential pro-inflammatory and homeostatic properties urging for more research on the importance of IgA NAb. Since NAb in humans were indicated to fulfill important functions in health and disease, their role in health of veterinary species should be investigated more often. Furthermore, it is unknown whether levels of NAb-isotypes and/or idiotypes can and should be modulated. Veterinary species as models of choice fill in a niche between mice and (non-human) primates, and the study of NAb in veterinary species may provide valuable new insights that will likely improve health management. Below, examples of the involvement of NAb in several diseases in mostly humans are shown. Possibilities of intravenous immunoglobulin administration, targeted immunotherapy, immunization, diet, and genetic modulation are discussed, all of which could be well-studied using animal models. Arguments are given why veterinary immunology should obtain inspiration from human studies and why human immunology would benefit from veterinary models. Within the One Health concept, findings from veterinary (and wildlife) studies can be related to human studies and vice versa so that both fields will mutually benefit. This will lead to a better understanding of NAb: their origin, activation mechanisms, and their implications in health and disease, and will lead to novel health management strategies for both human and veterinary species.

Heterologous Boosting With Listeria-Based Recombinant Strains in BCG-Primed Mice Improved Protection Against Pulmonary Mycobacterial Infection.

While Baccillus Calmette-Guerin (BCG) is used worldwide, tuberculosis (TB) is still a global concern due to the poor efficacy of BCG. Novel vaccine candidates are therefore urgently required. In this study, two attenuated recombinant Listeria strains, LMΔ-msv and LIΔ-msv were constructed by deletion of actA and plcB and expression of a fusion protein consisting of T cell epitopes from four Mycobacterium tuberculosis (Mtb) antigens (Rv2460c, Rv2660c, Rv3875, and Rv3804c). The safety and immunogenicity of the two recombinant strains were evaluated in C57BL/6J mice. After intravenous immunization individually, both recombinant strains entered liver and spleen but eventually were eliminated from these organs after several days. Simultaneously, they induced antigen-specific cell-mediated immunity, indicating that the recombinant Listeria strains were immunogenic and safe in vivo. LMΔ-msv immunization induced stronger cellular immune responses than LIΔ-msv immunization, and when boosted with LIΔ-msv, antigen-specific IFN-γ CD8+ T cell responses were notably magnified. Furthermore, we evaluated the protection conferred by the vaccine candidates against mycobacterial infection via challenging the mice with 1 × 107 CFU of BCG. Especially, we tested the feasibility of application of them as heterologous BCG supplement vaccine by immunization of mice with BCG firstly, and boosted with LMΔ-msv and LIΔ-msv sequentially before challenging. Combination immune strategy (LMΔ-msv prime-LIΔ-msv boost) conferred comparable protection efficacy as BCG alone. More importantly, BCG-vaccinated mice acquired stronger resistance to Mycobacterial challenge when boosted with LMΔ-msv and LIΔ-msv sequentially. Our results inferred that heterologous immunization with Listeria-based recombinant strains boosted BCG-primed protection against pulmonary mycobacterial infection.

125-LB: Beta-Cell Antigen-Specific Chimeric Antigen Receptor Tregs for Type 1 Diabetes Prevention and Treatment

Background: Autoimmunity is a consequence of low/unfit antigen-specific Tregs. If antigen-specific Tregs could be produced on demand against a desire autoimmune target, suppression of autoimmune diseases would be achievable. Here, we developed pancreatic beta cell-specific GAD65 CAR Tregs and explored their therapeutic potential against T1D. Methods: Two GAD65 B cell epitopes known to interact with two immunodominant regions in the N-terminal (CAR-N) and Middle (CAR-M) regions were selected for assembly onto T cell receptors (CD28 hinge-transmembrane-intracellular regions and CD3ζ intracellular domain) (1). These GAD65 CAR Tregs were used to prevent/treat diabetes in our humanized mouse model of T1D. Results: Adoptive transfer of CAR-N Tregs to the diabetic T1D mice (2) revealed that administration of beta-cell specific Tregs leads to enrichment of Treg cells at the pancreas level. Confocal images demonstrated the homing of CAR Tregs to peri-insular areas 48 hours after intravenous immunization. 30-day follow up, fasting blood glucoses and GTT/insulin secretion tests significantly deteriorated in control Groups as compared with CAR-N/M Tregs treated Groups. The pancreatic islet localization of GAD65 CAR Tregs was still demonstrable at 30 days. Conclusions: This is the first time a therapeutic approach is successful in abrogating the diabetes phenotype in T1D mice model. Conceivably, antigen-specific Treg redirection using antigen-specific CAR Tregs and consequent Teff downregulation will allow for recovery and reconstitution of beta cells in humans as well.

Liposomal TLR9 Agonist Combined with TLR2 Agonist-Fused Antigen Can Modulate Tumor Microenvironment through Dendritic Cells.

Dendritic cells (DCs) are antigen-presenting cells involved in T cell activation and differentiation to regulate immune responses. Lipoimmunogens can be developed as pharmaceutical lipoproteins for cancer immunotherapy to target DCs via toll-like receptor 2 (TLR2) signaling. Previously, we constructed a lipoimmunogen, a lipidated human papillomavirus (HPV) E7 inactive mutant (rlipoE7m), to inhibit the growth of HPV16 E7-expressing tumor cells in a murine model. Moreover, this antitumor effect could be enhanced by a combinatory treatment with CpG oligodeoxynucleotides (ODN). To improve safety, we developed a rlipoE7m plus DOTAP liposome-encapsulated native phosphodiester CpG (POCpG/DOTAP) treatment to target DCs to enhance antitumor immunity. We optimized the formulation of rlipoE7m and POCpG/DOTAP liposomes to promote conventional DC and plasmacytoid DC maturation in vitro and in vivo. Combination of rlipoE7m plus POCpG/DOTAP could activate conventional DCs and plasmacytoid DCs to augment IL-12 production to promote antitumor responses by intravenous injection. In addition, the combination of rlipoE7m plus POCpG/DOTAP could elicit robust cytotoxic T lymphocytes (CTLs) by intravenous immunization. Interestingly, the combination of rlipoE7m plus POCpG/DOTAP could efficiently inhibit tumor growth via intravenous immunization. Moreover, rlipoE7m plus POCpG/DOTAP combined reduced the number of tumor-infiltrating regulatory T cells dramatically due to downregulation of IL-10 production by DCs. These results showed that the combination of rlipoE7m plus POCpG/DOTAP could target DCs via intravenous delivery to enhance antitumor immunity and reduce the number of immunosuppressive cells in the tumor microenvironment.

Intranasal vaccination with Listeria ivanovii as vector of Mycobacterium tuberculosis antigens promotes specific lung-localized cellular and humoral immune responses

We have previously demonstrated that a recombinant Listeria ivanovii (LI) strain expressing the ESAT-6 or Ag85C protein of Mycobacterium tuberculosis (Mtb) as a tuberculosis (TB) vaccine candidates induced antigen-specific cellular immune responses after intravenous immunization of mice. However, whether such recombinant strains could induce desired immune responses in the lung, where TB infection occurs, is not clear. In this paper, C57BL/6 J mice were intranasally vaccinated with attenuated LIΔactAplcB-Rv3875 (Δ refers to gene deletion in the bacterial genome) or LIΔactAplcB-Rv0129c, the two vaccine candidates that utilize LI as an antigen delivery vector. Bacterial load in the target organs, histological changes in the infected organs, the percentage of specific cytokine-secreting T cells in the lung and spleen, IgG levels in the serum and secretory IgA (SIgA) levles in bronchoalveolar lavage (BAL) fluid were determined at specific days post inoculation (dpi). The results showed that both strains were mainly confined to the lung and were eliminated at 10 dpi. The histological damage caused by the infection in the lung was slight and recovered by day 5. Intranasal vaccination of the mice twice at an interval of 4 weeks notably elicited TB antigen-specific CD4+ and CD8+ T cell responses in the lung and SIgA secretion in the pulmonary mucosa, and significantly enhanced the percentage of double-functional CD8+ T cells (IFN-γ+ TNF-α+ CD8+). To our knowledge, this is the first report regarding the used of LI vector vaccines to induce promising lung-localized cellular and humoral immune responses by intranasal vaccination. These data suggest that LI could be a novel and promising live vector to construct an intranasal vaccine against respiratory diseases.

Prevention of tuberculosis in macaques after intravenous BCG immunization

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette–Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography–computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

Induction of Activated T Follicular Helper Cells Is Critical for Anti-FVIII Inhibitor Development in Hemophilia a Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. While multiple lines of evidence indicate that the immune response against FVIII is CD4 T cell-dependent, the role of T follicular helper (TFH) cells, a newly identified subset of CD4 T cells, in FVIII inhibitor development is unknown. TFH cells are a critical player in germinal center formation, which provide cognate help to B cells and are fundamentally required for the generation of T cell-dependent B cell responses. Here we explored the activation and induction of TFH cells and their roles in anti-FVIII inhibitor production in FVIII-immunized mice. We first confirmed that inhibitor development depends on CD4 T cells as depletion of CD4 T cells during FVIII immunization in FVIIInull mice prevents inhibitor production. We then characterized the features of activated TFH cells in the spleen of immunized FVIIInull mice by flow cytometry analysis. Activated TFH cells were defined as CD4+Foxp3-CD44+CD62L-CXCR5+PD-1+. These cells express high levels of transcription factors BCL6, ICOS, and CD40L. We showed for the first time that intravenous FVIII immunization induced activation and accumulation and/or expansion of TFH cells in the spleen of FVIIInull mice. The percentage of CXCR5hiPD-1hi TFH cells within the effector CD4 population and the absolute numbers of activated TFH cells per spleen significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (19.6 ± 1.6 %, 8.1 ± 1.2%, and 7.6 ± 1.4%, respectively; 14.4 ± 2.0×104, 3.55 ± 0.8×104, and 3.5 ± 0.9×104, respectively). There was no TFH cell expansion observed in other secondary lymphoid organs (mesenteric and inguinal lymph nodes), suggesting that the spleen is the primary site for TFH activation when FVIII is administered intravenously. Our results showed that FVIII inhibitor-producing mice had increased GL-7+germinal center TFH cells together with increased germinal center formation in response to FVIII immunization and the emergence of TFH cells correlated with titers of anti-FVIII inhibitors. This indicates that a functional relationship exists between TFH cell activation and FVIII inhibitor production after FVIII immunization. To determine the extent to which the anti-FVIII inhibitor development is related to CXCR5+ TFH cells, we used a mixed BM chimera model consisting of a CD4 T cell-specific CXCR5 deficiency (CD4-CXCR5-/-). Compared with CD4-CXCR5+/+ control mice, there was a significant decrease in the inhibitor titer in CD4-CXCR5-/- mice after FVIII immunization (111.7 ± 46.2 vs 4.43 ± 3.53 BU/ml, respectively). Our results showed that CXCR5+/+ TFH cell-specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5+/+ TFH cells for the generation of FVIII-neutralizing antibodies. To examine specificity of TFH cell responses after FVIII immunization, we used an ex vivo T-cell proliferation assay. In vitro FVIII restimulation induced the antigen-specific proliferation of splenic CD4 T cells from FVIII-primed FVIIInull mice and those proliferating cells expressed the TFH hallmark transcription factor BCL6. These results demonstrated that antigen-specific TFH cells were induced after FVIII immunization and those cells could specifically respond to FVIII upon restimulation. To evaluate the FVIII-specific memory TFH cell responses in FVIIInull mice, matched pairs of FVIII-primed mice with similar inhibitor titers were rechallenged with FVIII antigen in vivo 2 months after the last immunization. The absolute numbers of TFH cells per spleen in FVIII rechallenged animals were significantly higher than those obtained from saline-injected primed mice (36.7 ± 11 ×104 vs 14.8 ± 3.8 ×104, respectively). These data suggest that memory FVIII-specific TFH cells are persistent which can induce efficient recall TFH responses after FVIII rechallenge. Together, our results demonstrate that the induction of activated TFH cells in FVIIInull mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH cell activation might be a promising strategy to prevent anti-FVIII inhibitor formation in hemophilia A patients. Disclosures No relevant conflicts of interest to declare.

159 Procedural Skills Needs for Canadian Paediatricians: A National Snapshot

Procedural practice by paediatricians in Canada is likely evolving. The availability of non- or minimally invasive technologies, practice type, geographical variation, and the expanding scope of multiprofessional providers are examples of factors that may contribute to such changes. There is, however, limited knowledge of the procedural competencies required of general paediatricians today. An accurate understanding of the skills required is vital for planning, preparing, and allocating limited educational resources both for current and future paediatricians. The primary objective of the study was to conduct a comprehensive analysis of the procedural practice of Canadian paediatricians, with the aim of building a needs assessment for independent general paediatric practice. A one-time survey was sent to paediatricians through the Canadian Paediatric Surveillance Program (CPSP) (www.cpsp.cps.ca/surveillance). In addition to demographic information about practice type and location, participants were asked to indicate the frequency with which they performed each of 32 pre-selected procedures. The procedure list was developed in consultation with CPSP members and a variety of paediatricians. Data was combined and summarized descriptively, with valid percentages calculated where appropriate. The survey response rate was 33% (938/2,822). Of the respondents, 589 (63%) participated either partly or entirely in general paediatrics practice, and 245 (42%) had practiced for > 20 years. All provinces were represented, and practice styles ranged from urban tertiary care to remote/rural. Of the respondents who practice general paediatrics, 71% reported performing procedures. Regularly performed procedures (at least monthly) included bag-mask ventilation for infants, lumbar puncture, ear curettage, peripheral intravenous line insertion and immunization (indicated by 54%, 48%, 39%, 27%, 25% of respondents, respectively). Rarely performed procedures (yearly or less) included chest compressions, intraosseous insertion, intubation, chest tube insertion and defibrillation (80%, 65%, 57%, 53%, 52%, respectively). Procedures reported as never performed included peripheral intravenous central catheter (PICC) line insertion, central venous line (CVL) insertion, arterial line insertion, tracheostomy tube change and thoracentesis (90%, 89%, 82%, 76%, 73%, respectively). This report of real-world experiences from Canadian general paediatricians suggests a wide variability in the frequency of procedural performance. It helps to establish priorities for continuing professional educational activities and postgraduate medical education curricula in the era of competency-based medical education. Additional investigation on importance and maintenance of proficiency of these procedures would be of added value to this needs assessment, and has been undertaken by the research group.

Hirudin ameliorates immunoglobulin A nephropathy by inhibition of fibrosis and inflammatory response

Immunoglobulin A nephropathy (IgAN) is characterized by mesangial IgA and IgG co-deposition. As the clinical course of IgAN is highly variable, a lot of patients will eventually develop to end-stage renal disease (ESRD) within years. Hirudin, a potent and specific thrombin inhibitor, has been reported to treat IgAN with hematuria, but the mechanism is unclear. Our study aims to explore the potential of hirudin and the underlying mechanism in the treatment of IgAN. The establishment of IgAN model was set up in rats through oral and intravenous immunization with bovine gamma-globulin (BGG). Results suggested that hirudin could reduce the increased level of proteinuria, serum creatinine and urea nitrogen in IgAN models. Besides that, hirudin ameliorated the elevated number of apoptotic bodies and expressions of apoptosis-related proteins (caspase-3 and caspase-9) in IgAN model. The fibrosis indexes (transforming growth factor β-1 (TGF-β1), Collagen-IV (CoI-IV) and Fibronectin-1) of kidney were remarkably suppressed in IgAN rats treated with hirudin compared with IgAN rats with no further treatment. IgAN rats exhibited remarkably increased inflammatory factors (IL-1β, IL-6, and IL-18), while hirudin treatment significantly alleviated these alterations. Moreover, the reduced levels of CD4+CD25+Foxp3+ Treg and CD4+IFN-γ+ Th1/CD4+IL-4+ Th2 could be reversed by hirudin in IgAN model. Furthermore, in the process of IgAN, hirudin could inactivate various pathways (IκBα, NF-κB, TNF-α, and VCAM-1) compared with IgAN model group. Taken together, our study indicated that hirudin could ameliorate IgAN through suppressing fibrosis and inflammatory response. These findings provide a new therapeutic method to treat IgAN.

Generation of IL-3 secreting T helper cells is associated with microbial challenge at barriers and IL-1 family of cytokines

Abstract Naive CD4+ T cells differentiate into distinct T helper subsets upon T cell receptor stimulation in a unique cytokine environment. The cytokine environment leads to the expression of subset-specific master transcription factor which enables secretion of cytokines characteristic of that subset and suppression of cytokines associated with other T helper subsets. We recently described that cutaneous Mycobacterium bovis BCG vaccination in mice induces a subset of T helper cells characterized by the secretion of IL-3. Here, we report that the induction of this subset of T helper cells is route dependent; cutaneous but not intravenous immunization with M. bovis BCG induced these cells. Similar results were obtained with a model of cutaneous Herpes Simplex Virus II infection. Infection with Herpes Simplex Virus by vaginal route or with M. tuberculosis by aerosol route induced these cells suggesting mucosal sites are conducive to their generation. In these scenarios, IL-3 secreting cells co-expressed GM-CSF, IFNγ, TNF and IL-2. In vitro polarization experiments showed that IL-3 secretion by CD4+ T cells was facilitated by IL-1 family of cytokines and suppressed by cytokines that induce Th1 and Th2 helper cells. The characteristic cytokine expression pattern and the dependence on route and cytokine environment of these cells support the idea that IL-3 secreting CD4+ T cells may represent a unique T helper subset.

Protection from experimental cerebral malaria with a single intravenous or subcutaneous whole-parasite immunization

Cerebral malaria is a life-threatening complication of Plasmodia infection and a major cause of child mortality in Sub-Saharan Africa. We report that protection from experimental cerebral malaria in the rodent model is obtained by a single intravenous or subcutaneous whole-parasite immunization. Whole-parasite immunization with radiation-attenuated sporozoites was equally protective as immunization with non-attenuated sporozoites under chemoprophylaxis. Both immunization regimens delayed the development of blood-stage parasites, but differences in cellular and humoral immune mechanisms were observed. Single-dose whole-parasite vaccination might serve as a relatively simple and feasible immunization approach to prevent life-threatening cerebral malaria.

A Promising Listeria-Vectored Vaccine Induces Th1-Type Immune Responses and Confers Protection Against Tuberculosis.

Deaths associated with tuberculosis (TB) is rising and accounted for 1.4 million deaths in 2015 many of which were due to drug-resistant bacteria. Vaccines represent an important medical intervention, but the current Bacilli Calmette-Guerin (BCG) vaccine is not ideal for the protection of teenagers and adults. Therefore, a safe and effective vaccine is urgently needed. In this study, we designed a novel vaccine using an attenuated Listeria monocytogenes strain carrying fusion antigen FbpB-ESAT-6 (rLM) and characterized its safety and protective efficacy against Mycobacterium tuberculosis (M.tb) infection in mice. Compared to the wild type strain yzuLM4 and parental strain LMΔactA/plcB (LM1-2), the virulence of rLM was significantly reduced as judged by its infectious kinetics and LD50 dose. Further characterization of intravenous immunization showed that prime-boost vaccination significantly increased the levels of Th1 cytokines (IFN-γ, IL-17, and IL-6), and enhanced cytotoxic T lymphocyte (CTL) CTLs activity, suggesting that rLM could elicit potent Th1/Th17 responses. More importantly, rLM significantly conferred the protection against M.tb H37Rv challenge. Collectively, our findings indicated that rLM is a novel and useful tool to prevent M.tb infection, and can be potentially be used to boost BCG-primed immunity.

Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53–68%) than IV-I (93–95%). Unprotected mice developed less sporozoite-specific CD8+ and CD4+ effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8+ dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.