Fusion with extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 leads to enhancement of immunogenicity of Hantaan virus DNA vaccines in C57BL/6 mice

<p>Abstract</p> <p>Background</p> <p>Hantaan virus (HTNV) is the causative agent of the most severe form of a rodent-borne disease known as hemorrhagic fever with renal syndrome (HFRS). A safe and effective HTNV vaccine is needed. Vaccination with DNA constructs expressing fused antigen with bioactive factors, has shown promising improvement of immunogenicity for viral agents in animal models, but the effect of fusion strategy on HTNV DNA vaccine has not been investigated.</p> <p>Results</p> <p>DNA plasmids encoding the HTNV nucleocapsid protein (N) and glycoprotein (Gn and Gc) in fusion to the extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 (eCTLA-4) targeting to antigen presenting cells (APCs) were constructed. Intramuscular immunization of mice with plasmids expressing eCTLA-4-HTNV-N/GP fusion proteins leads to a significant enhancement of the specific antibody response as well as cytotoxic T-lymphocyte (CTL) response in C57BL/6 mice. Moreover, this effect could be further augmented when co-administered with CpG motifs.</p> <p>Conclusions</p> <p>Modification of viral antigen in fusion to bioactive factor will be promising to confer efficient antigen presentation and improve the potency of DNA vaccine in mice.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Comparison of serological assays to titrate Hantaan and Seoul hantavirus-specific antibodies

Abstract Background Hantaan and Seoul viruses, in the Hantavirus genus, are known to cause hemorrhagic fever with renal syndrome (HFRS). The plaque reduction neutralization test (PRNT), as conventional neutralization test for hantaviruses, is laborious and time-consuming. Alternatives to PRNT for hantaviruses are required. Methods In this study, the methods for Hantaan and Seoul viruses serological typing including microneutralization test (MNT), pseudoparticle neutralization test (PPNT) and immunofluorescence assay based on viral glycoproteins (IFA-GP) were developed and compared with PRNT using a panel of 74 sera including 44 convalescent sera of laboratory confirmed HFRS patients and 30 patients sera of non-hantavirus infection. Antibody titres and serotyping obtained with different methods above were analyzed by paired-t, linear correlation, McNemar χ2 and Kappa agreement tests. Results Antibody titres obtained with MNT50, PPNT50 and IFA-GP were significantly correlated with that obtained with PRNT50 (p  0.05) and less than (p  0.05). IFA-GP was less sensitive than PRNT50 and MNT50 for serotyping of hantaviruses infection (p < 0.05). However, for 79.5% (35/44) samples, serotyping determined by IFA-GP and PRNT50 were consistent. Conclusions MNT50 and PPNT50 both can be used as simple and rapid alternatives to PRNT50, and MNT50 is more specific while PPNT50 is more sensitive than other assays for neutralizing antibody determination. So far, this work has been the most comprehensive comparison of alternatives to PRNT

Pre-marketing immunogenicity and safety of a lyophilized purified human diploid cell rabies vaccine produced from microcarrier cultures: a randomized clinical trial

The large-scale production of a human diploid cell (HDC) vaccine (HDCV) for rabies is limited by several technical challenges. Kanghua Biological Products Co., Ltd., has successfully used microcarrier technology for the large-scale culture of HDCs in bioreactors to develop a lyophilized and purified HDCV. In this blinded, randomized, parallel-group study conducted between July and October 2014 in Mianzhu, Sichuan Province, China, we monitored the safety and immunogenicity of this vaccine in a healthy population vaccinated according to the Essen post-exposure immunization schedule. A hamster kidney cell vaccine was used as the control. Adverse reactions were monitored 0.5, 6, 24, 48, and 72 h post vaccination to assess safety. Neutralizing antibodies in venous blood were measured on day 7, 14, and 72 to evaluate the immunogenicity of the vaccine while follow-up monitoring continued for 1 month. No serious adverse reactions were observed in any volunteer. The incidence rates of systemic and local adverse reactions were, respectively, 10.6% and 2.9% in the test group and 20.0% and 13.6% in the control group. After the third injection, the positive conversion rates of antibodies in the test and control groups were 100% and 98.82%, respectively. In addition, the average antibody titers on day 7, 14, and 42, were respectively, 1.71, 2.72, and 1.29 times higher than those in the control group. These results indicate that HDCV had a better safety profile and higher immunogenicity than the hamster kidney cell rabies vaccine. Trial registration number: 20130602

Thyroid Dysfunction Induced by Fungicide Famoxadone Exposure Contributes to Nonalcoholic Fatty Liver Disease in Male Mice: <i>In Vivo</i>, <i>In Vitro</i>, and <i>In Silico</i> Studies

Thyroid dysfunction has become a serious public health problem, which is considered a trigger of nonalcoholic fatty liver disease (NAFLD). Pesticide exposure could contribute to thyroid dysfunction and NAFLD, but the relationship between these factors remains unclear. In this study, the effects of subchronic famoxadone exposure on thyroid and liver at no observed adverse effect level (NOEL) related concentrations were investigated using in vivo, in vitro, and in silico models. Famoxadone caused hepatic steatosis, lipid metabolism disorder, and liver oxidative stress and induced NAFLD in male mice. The suppression of hepatic fatty acid β-oxidation was the key factor of NAFLD, which was highly associated with hypothalamic–pituitary–thyroid (HPT) axis hormones disorder. Famoxadone disrupted thyroid hormone biosynthesis by causing thyroid follicle aberrations and abnormal HPT axis-related gene expression. In vitro studies confirmed that famoxadone inhibited the transport of thyroxine (T4) into hepatocytes and the conversion of T4 to triiodothyronine (T3). In silico studies verified that famoxadone interfered with the binding of thyroid hormones to proteins mediating thyroid hormone transport, conversion, and activation. This study comprehensively reported the association between NAFLD and thyroid dysfunction caused by famoxadone, providing new perspectives for the health risk evaluation of pesticides with a similar structure in mammals

Antigenic variations of recent street rabies virus

ABSTRACTThe genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened

Combining intramuscular and intranasal homologous prime-boost with a chimpanzee adenovirus-based COVID-19 vaccine elicits potent humoral and cellular immune responses in mice

The efficacy of many coronavirus disease 2019 (COVID-19) vaccines has been shown to decrease to varying extents against new severe acute respiratory syndrome coronavirus 2 variants, which are responsible for the continuing COVID-19 pandemic. Combining intramuscular and intranasal vaccination routes is a promising approach for achieving more potent immune responses. We evaluated the immunogenicity of prime-boost protocols with a chimpanzee adenovirus serotype 68 vector-based vaccine, ChAdTS-S, administered via both intranasal and intramuscular routes in BALB/c mice. Intramuscular priming followed by an intranasal booster elicited the highest levels of IgG, IgA, and pseudovirus neutralizing antibody titres among all the protocols tested at day 42 after prime immunization compared with the intranasal priming/intramuscular booster and prime-boost protocols using only one route. In addition, intramuscular priming followed by an intranasal booster induced high T-cell responses, measured using the IFN-γ ELISpot assay, that were similar to those observed upon intramuscular vaccination. All ChAdTS-S vaccination groups induced Th1-skewing of the T-cell response according to intracellular cytokine staining and Meso Scale Discovery cytokine profiling assays on day 56 after priming. This study provides reference data for assessing vaccination schemes of adenovirus-based COVID-19 vaccines with high immune efficacy