DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice

As of December 2022,2603 laboratory-identified Middle East respiratory syndrome coronavirus(MERS-CoV)infections and 935 associated deaths,with a mortality rate of 36%,had been reported to the World Health Organization(WHO).However,there are still no vaccines for MERS-CoV,which makes the prevention and control of MERS-CoV difficult.In this study,we generated two DNA vaccine candidates by integrating MERS-CoV Spike(S)gene into a replicating Vaccinia Tian Tan(VTT)vector.Compared to homologous immunization with either vaccine,mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses.The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012,England1 and KNIH strains of MERS-CoV.Prime-Boost immunization also induced strong MERS-S specific T cells responses,with high memory and poly-functional(CD107a-IFN-γ-TNF-α)effector CD8t T cells.In conclusion,the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S.This study not only provides a promising set of MERS-CoV vaccine candidates,but also proposes a heterologous sequential immunization strategy worthy of further development.

Expression patterns and action analysis of genes associated with physiological responses during rat liver regeneration:Cellular immune response

AIM： To study the cellular immune response during rat liver regeneration （LR） at a transcriptional level. METHODS： Genes associated with the cellular immune response were obtained by collecting the data from databases and retrieving articles. Gene expression changes during LR were detected by rat genome 230 2.0 array. RESULTS： A total of 127 genes were found to be associated with LR. The number of initially and totally expressing genes in the initial phase of LR [0.5-4 h after partial hepatectomy （PH）], transition from Go-G, （4-6 h after PH）, cell proliferation （6-66 h after PH）, cell differentiation and structure-function reconstruction （66-168 h after PH） was 54, 11, 34, 3 and 54, 49, 70, 49 respectively, illustrating that the associated genes were mainly triggered at the initiation of LR, and worked at different phases. According to their expression similarity, these genes were classified into 41 up-regulated, 21 predominantly up-regulated, 41 down-regulated, 14 predominantly down-regulated, 10 similarly up-regulated and down-regulated genes, respectively. The total up- and down-regulated expression times were 419 and 274, respectively, demonstrating that the expression of most genes was increased while the expression of a small number of genes was decreased. Their time relevance was classified into 14 groups, showing that the cellular physiological and biochemical activities were staggered during LR. According to the gene expression patterns, they were classified into 21 types, showing the activities were diverse and complicated during LR.CONCLUSION： Antigen processing and presentation are enhanced mainly in the forepart, prophase and anaphase of LR. T-cell activation and antigen elimination are enhanced mainly in the forepart and prophase of LR. A total of 127 genes associated with LR play an important role in cellular immunity.

Changes in cellular proliferation and plasma products are associated with liver failure

AIM To study the differences in immune response and cytokine profile between acute liver failure and selflimited acute hepatitis.METHODS Forty-six patients with self-limited acute hepatitis(AH), sixteen patients with acute liver failure(ALF), and twenty-two healthy subjects were involved in this study. The inflammatory and anti-inflammatory products in plasma samples were quantified using commercial enzyme-linked immunoassays and quantitative real-time PCR. The cellular immune responses were measured by proliferation assay using flow cytometry. The groups were divided into viral- and non-viral-induced selflimited AH and ALF. Thus, we worked with five groups: Hepatitis A virus(HAV)-induced self-limited acute hepatitis(HAV-AH), HAV-induced ALF(HAV-ALF), nonviral-induced self-limited acute hepatitis(non-viral AH), non-viral-induced acute liver failure(non-viral ALF), and healthy subjects(HC). Comparisons among HAV and non-viral-induced AH and ALF were performed.RESULTS The levels of mitochondrial DNA(mt DNA) and the cytokines investigated [interleukin(IL)-6, IL-8, IL-10, interferon gamma, and tumor necrosis factor] were significantly increased in ALF patients, independently of etiology(P < 0.05). High plasma mt DNA and IL-10 were the best markers associated with ALF [mt DNA: OR = 320.5(95%CI: 14.42-7123.33), P < 0.0001; and IL-10: OR = 18.8(95%CI: 1.38-257.94), P = 0.028] and death [mt DNA: OR = 12.1(95%CI: 2.57-57.07), P = 0.002; and IL-10: OR = 8.01(95%CI: 1.26-50.97), P = 0.027]. In the cellular proliferation assay, NK^(bright), NKT and regulatory T cells(TReg) predominated in virusspecific stimulation in HAV-induced ALF patients with an anergic behavior in the cellular response to mitotic stimulation. Therefore, in non-viral-induced ALF, anergic behavior of activated T cells was not observed after mitotic stimulation, as expected and as described by the literature. CONCLUSION mt DNA and IL-10 may be predictors of ALF and death. TReg cells are involved in immunological disturbance in HAV-induced ALF.

Development of hepatitis C virus vaccine using hepatitis B core antigen as immuno-carrier

AIM: To develop hepatitis C virus (HCV) vaccine using HBcAg as the immuno-carrier to express HCV T epitope and to investigate its immunogenicity in mice. METHODS: We constructed the plasmid pTrc-coreNheI using gene engineering technique, constructed the pcDNA3.1-coreNheI-GFP plasmid with GFP as the reporter gene, and transfected them into Hela cells. The expression of GFP was observed under confocal microscopy and the feasibility of using HBcAg as an immuno-carrier vaccine was studied. pTrc-core gene with a synthetic T epitope antigen gene of HCV (35-44aa) was fused and expressed in the plasmid pTrc- core-HCV (T). For the fusion of the HBcAg-T protein, sucrose, density gradient centrifugation was used, and its molecular weight and purity were analyzed by SDS- PAGE. Then balb/c mice were immunized by the plasmid with the HBcAg (expressed by pTrc-core) protein as control. The tumor regression potential was investigated in mice and evaluated at appropriate time. After three times of immunization, the peripheral blood and spleen of vaccinated mice were collected. HBcAb was detected by ELISA, and nonspecific T lymphocyte proliferation and response of splenocytes were respectively examined by MTT assay. T cell subset of blood and spleen were detected by FACS. RESULTS: GFP was successfully expressed. Tumor regression trial showed that no tumor formation was found in the group receiving immunization, while tumor xenograft progression was not changed in the control group. Strong nonspecific lymphocyte proliferation response was induced. FACS also showed that the ratio of CD8+ T cells in the experimental group was higher than the controls, but the serum HBcAb in experimental group was similar to the control. CONCLUSION: HBcAg can be used as an immuno-carrier of vaccine, the fusion of HBcAg-T protein could induce stronger cellular immune responses and it might be a candidate for therapeutic vaccines specific for HCV.

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egglaying stage.Venom proteins play various roles in the host physiology,development,immunity,and behavior manipulation and regulation.In this study,we identified a venom protein,MmRhol,a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone,egg production,and cocoons formation in the female wasps.We demonstrated that MmRho1 entered the cotton bollworm's(host)hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining.Furthermore,wasp MmRhol interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down.In conclusion,this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.

The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection

This review describes the woodchuck and the woodchuck hepatitis virus (WHV) as an animal model for pathogenesis and therapy of chronic hepatitis B virus (HBV) infection and disease in humans. The establishment of woodchuck breeding colonies, and use of laboratory-reared woodchucks infected with defined WHV inocula, have enhanced our understanding of the virology and immunology of HBV infection and disease pathogenesis, including major sequelae like chronic hepatitis and hepatocellular carcinoma. The role of persistent WHV infection and of viral load on the natural history of infection and disease progression has been firmly established along the way. More recently, the model has shed new light on the role of host immune responses in these natural processes, and on how the immune system of the chronic carrier can be manipulated therapeutically to reduce or delay serious disease sequelae through induction of the recovery phenotype. The woodchuck is an outbred species and is not well defined immunologically due to a limitation of available host markers. However, the recent development of several key host response assays for woodchucks provides experimental opportunities for further mechanistic studies of outcome predictors in neonatal- and adult-acquired infections. Understanding the virological and immunological mechanisms responsible for resolution of self-limited infection, andfor the onset and maintenance of chronic infection, will greatly facilitate the development of successful strategies for the therapeutic eradication of established chronic HBV infection. Likewise, the results of drug efficacy and toxicity studies in the chronic carrier woodchucks are predictive for responses of patients chronically infected with HBV. Therefore, chronic WHV carrier woodchucks provide a well-characterized mammalian model for preclinical evaluation of the safety and efficacy of drug candidates, experimental therapeutic vaccines, and immunomodulators for the treatment and prevention of HBV disease sequelae.

Immunogenicity of the Spike Glycoprotein of Bat SARS-like Coronavirus

A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

Influence of Photoperiod over Morphometric and Hematological Parameters of Juvenile Piracanjubas （Brycon orbygnianus）

The aim of this study was to evaluate morphometric and hematological parameters of juveniles Piracanjubas treated with two distinct photoperiod. Twenty nine fish with 100 g, in a factorial design were performed in 18 h light：6 h dark （18L：6D） and 6L：lSD two different photoperiod, and morphometric and hematological parameters were evaluated at 45 d and 90 d after photoperiod treatment. Benzocaine 1% was used for euthanasia. Fish were weighed and measured, so as visceral fat, liver and gonads. Hematocrit, total count of white cells, lymphocytes and neutrophils were counted. Results showed that there was no significant relationship between bodyweight and standard length. Significant interaction （P = 0.00018） had been found between the fat index （FI） and gonado-somatic index （GSI） and the photoperiod and time. At 90 d, the fish kept in 18L：6D had a lower rate of fat than the fish kept in 6L：18D （P 〈 0.05）. The GSI was higher in the group 6L.＇18D compared with the 18L：6D （P 〈 0.005）. There was no interaction between photoperiod and period of treatment for total leukocyte count, however with 90 d, the total leukocyte count was decreased （P 〈 0.05）. Moreover, decrease of the hematocrit from 58.1% to 46.9% and an increase of lymphocytes from 31.5% to 43.5% had been found in fish kept in 18L：6D after 90 d （P 〈 0.05）. Fish kept in 6L：ISD presented an increase in segmented neutrophils and lymphocytes （P 〈 0.05）. It is concluded that photoperiod maintained at 6L： 18D for Piracanjubas permits a greater gonadal development and an amount of immune cells over the photoperiod maintained in 18L： 6D.

Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th1-type immune response and CTL effects following intranasal immunization

The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses.Thus,we constructed an attenuated Salmonella strain SL5928(fliC/esat)expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi.The chimeric flagellin functioned normally,as demonstrated using a flagella swarming assay and electron microscopy.To analyze the effects of chimeric flagellin,the cell-mediated immune response and cytotoxic T lymphocyte(CTL)effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat).The results showed that SL5928(fliC/esat)intranasal immunization can strongly elicit an ESAT-6-specific T helper(Th)1-type immune response in mucosal lymphoid tissues,such as nasopharynx-associated lymph nodes,lung and Peyer’s patches,and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes.Furthermore,intranasal immunization of SL5928(fliC/esat)produced efficient CTL effects,as demonstrated using a 5-and 6-carboxyfluorescein diacetate succinimidyl ester(CFSE)assay.Thus,our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization.Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.

A DNA-Binding Bromodomain-Containing Protein Interacts with and Reduces Rx1-Mediated Immune Response to Potato Virus X

Plant NLR proteins enable the immune systemto recognize and respond to pathogen attack.An early consequence of immune activation is transcriptional reprogramming.SomeNLRs have been shownto act in the nucleus and interact with transcription factors.The Rx1 NLR protein of potato binds and distorts doublestranded DNA.However,the components of the chromatin-localized Rx1 complex are largely unknown.Here,we report a physical and functional interaction between Rx1 and NbDBCP,a bromodomaincontaining chromatin-interacting protein.NbDBCP accumulates in the nucleoplasmand nucleolus,interacts with chromatin,and redistributes Rx1 tothe nucleolus in a subpopulation of imaged cells.Rx1 overexpression reduces the interaction between NbDBCP and chromatin.NbDBCP is a negative regulator of Rx1-mediated immune responses to potato virus X(PVX),and this activity requires an intact bromodomain.Previously,Rx1 has been shown to regulate the DNA-binding activity of a Golden2-like transcription factor,NbGlk1.Rx1 and NbDBCP act synergistically to reduce NbGlk1 DNA binding,suggesting a mode of action for NbDBCP’s inhibitory effect on immunity.This study provides new mechanistic insight into the mechanism by which a chromatin-localized NLR complex co-ordinates immune signaling after pathogen perception.