Cellular Immunity In Mice Research Articles

Research brief

Vaccines based on human adenovirus 5 (Ad5) induce protective immune responses against several pathogens in animal models, but the neutralising antibodies to Ad5 that most people make are likely to impair the immunological potency of such vaccines. To circumvent this potential problem, researchers have isolated more than 1000 chimpanzee adenovirus strains. Vaccine vectors derived from some of these viruses induce potent cellular immunity in mice. Moreover, in a phase 1 clinical trial, a chimpanzee adenovirus-based vector expressing non-structural hepatitis C virus (HCV) proteins induced broad, sustained T-cell responses to HCV.

Effects of magnetic induction hyperthermia and radiotherapy alone or combined on a murine 4T1 metastatic breast cancer model

Purpose: The purpose of this study was to explore the effects of MIH and radiotherapy alone or combined on metastatic breast cancer and the underlying mechanisms.Materials and methods: A murine 4T1 metastatic breast cancer model was established and randomly assigned into four treatment groups: C (control), R (radiotherapy), MIH, and MIH + R. Tumour volume, lung metastasis, the expression of Bax and MMP-9, T cell subsets, serum cytokine levels, and mouse survival were evaluated.Results: Group MIH + R showed significantly reduced tumour volume, lung metastasis, improved survival and increased Bax expression compared to group R or MIH (P < 0.05). MMP-9 expression in the primary tumour tissue was significantly increased in group R compared to the other groups (P < 0.05), which could be brought down by combined MIH treatment. Group MIH + R showed significantly higher CD4+ T cell percentage as well as CD4+/CD8+ cell ratio than group R (P < 0.05). Group MIH + R showed significantly higher serum levels of TNF-α, IFN-γ and IL-2 than group R (P < 0.05).Conclusions: MIH not only promotes the tumour-cell killing effect of radiotherapy through Bax-mediated cell death, but also improves cellular immunity in mice under radiotherapy and decreases the potential of radiotherapy to enhance MMP-9 expression, which leads to significant improvement in lung metastasis and overall survival of mice under combined treatment of MIH and R. This study is the first to have explored the effect of combined hyperthermia and radiotherapy on tumour metastasis and the underlying mechanisms. It provides insights into the application of MIH as an adjuvant to radiotherapy for metastatic breast cancer.

Next-generation dendritic cell vaccines

American researchers have devised two methods to maximize the uptake and presentation of antigens by dendritic cells and have demonstrated potent cellular immunity in mice and monkeys vaccinated using the techniques. Celldex already is using a related version of one of the methods in a Phase I/II trial of a cancer immunotherapy.

The effect of Nasal Healing Spraying Agent on red cell immune function in mice

Objective To study the immunological regulation of Nasal Healing Spraying Agent (NHSA). Methods Test models were setup by irradiation. The models were administrated with NHSA by lavage and immune adherence of red cells was tested by chaplet experiment with blank and positive control. Results NHSA could increase Red Blood Cell-C3b Receptor Rosette (RBC-C3bRR) and reduce Red Blood Cell-Immune Complex Rosette (RBC-ICR) in mice. Compared with blank control group, the results in the high dose group and middle dose group were statistically significant (P<0.01). The result also showed that NHSA could elevate the quantity and activity of Red Blood Cell-C3b Receptor (RBC-C3bR) and reduce the Immune Complex (IC) to enhance the red cell immune function in mice effectively. Conclusion NHSA could elevate the RBC immunity function in mice and increase immunoregulatory function. Key words: Nasal Healing Spraying Agent (NHSA) ; Test for red blood cellular immunity in mice; Immunoregulatory function

Evaluation of a new oil adjuvant for use in peptide‐based cancer vaccination

Vaccine therapies are increasingly being used for the treatment of various diseases, and the antigen molecules themselves are being expanded from whole microorganisms to fine molecules such as peptides. Accordingly, there is a need for new adjuvants to support these new applications. In this paper, we used pharmaceutical grade mineral oil and sorbitan monooleate to develop a new oil adjuvant formula, NH(2) , and investigated its effects on peptide vaccination at both the pre-clinical and clinical levels. The adjuvant effect of NH(2) on peptide-induced cellular immunity in mice was superior to that of Montanide ISA51VG, a commercially available incomplete Freund's adjuvant for clinical use, although no significant difference was observed between the two adjuvants on peptide-induced humoral immunity. The adjuvant effects of NH(2) were also confirmed in a Phase-I clinical trial of peptide vaccines for patients with advanced cancers. These results suggest that NH(2) is a suitable adjuvant for peptide vaccination, particularly for cancer vaccines (Phase-I clinical trial of pan-HLA type personalized peptide vaccine for advanced cancer patients, UMIN clinical trial registry number: UMIN 000000619).

Combined Costimulation Blockade Inhibits Accelerated Rejection Mediated by Alloantigen-primed Memory T Cells in Mice

Donor-reactive memory T cells threaten the survival of transplanted organs via multiple pathways. This study was undertaken to induce tolerance of cardiac allografts in mice, in which alloreactive memory T cells were adoptively transferred, by combined costimulatory blockade of both effector and memory T cells. We found that the median survival time (MST) of the grafts was 5.17 days in the untreated group, 10.33 days in the CTLA4Ig- and anti-CD40L-treated (2-combined) group, and more than 100 days in the CTLA4Ig-, anti-CD40L-, anti-LFA-1-, and anti-OX40L-treated (4-combined) group. Histological analysis revealed that the mean rejection level was Grade 4 in the untreated group, Grade 3 in the 2-combined treatment group, and Grade 0 in the 4-combined treatment group. CD44high T cells were detected only in the untreated group. The in vitro proliferation of lymphocytes of both untreated and 2-combined group was higher than that of the 4-combined treatment group (p < 0.01). Compared with the untreated group, the expression levels of IL-2, IFN-γ, and Foxp3 were lower in the 2-combined treatment group; the expression levels of these genes were the lowest in the 4-combined treatment group. IL-10 expression was significantly higher in the 4-combined treatment group than in the other groups. These results demonstrate the inhibition efficacy of combined costimulation blockade in accelerated-rejection models and the possible mechanisms underlying the suppression of cellular immunity in mice receiving grafts as well as in inducing the activation of IL-10-producing Tr1 cells in grafts.

Enhancement of humoral and cellular immunity in mice against Japanese encephalitis virus using a DNA prime–protein boost vaccine strategy

A synthetic multi-epitope gene containing critical epitopes of the Japanese encephalitis virus (JEV) envelope gene was cloned into both prokaryotic and eukaryotic expression vectors. The recombinant plasmid and purified recombinant protein (heterologously expressed in Escherichia coli) were used as immunogens in a mouse model. The results indicate that both the recombinant protein and the DNA vaccine induce humoral and cellular immune responses. Neutralising antibody titres in mice in the pcDNA-TEP plus rEP group increased considerably relative to mice immunised using either pcDNA-TEP or rEP alone (P<0.05). Furthermore, the highest levels of interleukin (IL)-2, interferon-gamma and IL-4 were induced following priming with the DNA vaccine and boosting with the recombinant protein. Together these findings demonstrate that a DNA-recombinant protein prime-boost vaccination strategy can produce high levels of antibody and trigger significant T cell responses in mice, highlighting the potential value of such an approach in the prevention of JEV infection.

Effect of Radix Isatidis polysaccharides on immunological function and expression of immune related cytokines in mice

To investigate the effect of Radix Isatidis polysaccharides (RIP) on the immunological function and expression of immune related cytokines in mice. Sixty mice were randomly divided into six groups, the normal group, cyclophosphamide (Cy) model group, Levamisole (positive control) group, RIP low, medium and high dose groups (0.08 g/kg, 0.16 g/kg, 0.32 g/kg, respectively), ten in each group. By detecting the value of abdominal macrophage phagocytic index, serum hemolysin (HC50), proliferation of lymphocytes and expression of related cytokines, interleukin (IL-2) and interferon gamma (INF-gamma), the effect of RIP on immunological function and its mechanism were studied. RIP could improve the level of hemolysin in immunological function depressed mice. The results showed that the value of macrophage phagocytic index in the high dose RIP group increased from 1.11+/-0.13 to 1.42+/-0.26. The level of IL-2 and INF-gamma could be decreased by Cy to 38.12+/-6.88 ng/L and 139.23+/-29.87 ng/L, respectively, while RIP in high dose could increase the secretion of IL-2 and INF-gamma to 53.54+/-14.43 ng/L and 189.91+/-32.63 ng/L, respectively. RIP could enhance non-specific immunological function, humoral immunity and cellular immunity in mice.

Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice.

The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells. We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice. Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells. The purpose of this study was to determine the phenotype of the cells involved in this phenomenon. This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment. Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells. It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers'. These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells.

Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine

It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1–422); N2 (residues: 1–109); N3 (residues: 110–422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1–1269 nt), N2 (1–327 nt) and N3 (328–1269 nt)—expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4+:CD8+ assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4+:CD8+ assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.

Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice

Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected. Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice. Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node-residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node-residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.

Expresión de marcadores en células dendríticas de pacientes chagásicos crónicos estimuladas con la proteína KMP-11 y el péptido K1 de Trypanosoma cruzi

The kinetoplastid membrane protein 11, KMP-11, from Trypanosoma cruzi elicits humoral and cellular immunity in mice that protects them from infection against further parasite challenge. To characterize the expression of surface markers on dendritic cells from chronic chagasic patients and healthy individuals, in response to the KMP-11 protein from Trypanosoma cruzi and its N-terminal peptide K1. Monocyte-derived dendritic cells from seven chronic chagasic patients and seven healthy individuals were stimulated with the KMP-11 protein and the K1 peptide. Seven days after culturing, the CD83, CD86, and HLA-DR membrane expression as well as the production of cytokines were evaluated by flow cytometry. Neither KMP-11 protein nor K1 peptide elicited the expression of the maturation marker CD83 on dendritic cells of patients or healthy control individuals. Dendritic cells from chronic chagasic patients exposed to K1 and LPS at the same time presented a significant reduction in CD86 and CD83 membrane expression in contrast to the cells exposed to LPS alone, whereas dendritic cells from healthy individuals did not show this behavior. The secretion of interleukin-12 was decreased in the cultures of dendritic cells from chronic chagasic patients but not from healthy controls. KMP-1 1 protein does not affect the maturation of dendritic cells, but in the presence of LPS the K1 peptide leads to a decreased expression of CD86 and CD83 as well as interleukin-12 production, This phenomenon may be associated with an impaired T cell stimulation.

Immunomodulative effect of glucan and/or glucan supplemented with zinc in albendazole therapy for murine alveolar echinococcosis

The effect of glucan immunomodulator (GI) and glucan supplemented with zinc (GIZn) administered separately or with albendazole (ABZ) on cellular immunity of mice with alveolar echinococcosis was observed. The stimulative effect of GI and GI + ABZ therapy on proliferative response of T lymphocytes was prolonged by GIZn or GIZn + ABZ from week 6 to 14 postinfection (p.i.). The increased proliferation of B lymphocytes was observed during combined therapies GI + ABZ and GIZn + ABZ from week 6 to 12 p.i. Number of splenic CD4 T cells in mice with GI or GI + ABZ therapy was increased only on weeks 6 and 8 p.i. GIZn and GIZn + ABZ therapy prolonged this stimulation from week 6 to 14 p.i. Serum concentration of interferon-gamma (IFN-gamma) was increased after GIZn therapy and reduced after GI therapy from week 8 to 12 p.i. GIZn + ABZ therapy had the highest effect on the IFN-gamma rise from week 8 to 22 p.i. Both GI and GIZn inhibited the serum concentration of interleukin-5 (IL-5) from week 6 p.i. The production of superoxide anion was increased after GI therapy from week 6 to 14 p.i. and after GI + ABZ or GIZn + ABZ therapies from week 12 to 18 p.i. The most effective antiparasitic therapy for alveolar echinococcosis was reached by GIZn + ABZ therapy.

Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A recombinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the downstream of the promoter (ATI-P7.5x20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous recombination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by genome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowlpox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were assayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4(+) T, CD8(+) T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cytokines (IFN-gamma and IL-2) of spleen lymphocytes stimulated by H-2(d)-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

Structure and immunogenicity of alternative forms of the simian immunodeficiency virus gag protein expressed using Venezuelan equine encephalitis virus replicon particles

Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gag myr+), full-length Gag lacking the myristylation signal (Gag myr−) or a truncated form of Gag myr− comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gag myr+) and distinctive cytoplasmic aggregates (VRP-Gag myr−) with each cell type. VRP were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-γ ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-γ-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gag myr+-VRP. Antibody responses to MA/CA-VRP and Gag myr+-VRP were not significantly different.

Enhancing immune responses against HIV-1 DNA vaccine by coinoculating IL-6 expression vector

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. To analyze the immunogenicity of HIV-1 DNA vaccine which expressing the chimeric gene gag-gp120 of Chinese prevalent HIV-1 strain and the immunoregulatory activity of IL-6, DNA vaccine plasmid pVAX1-gag-gp120 and eukaryotic expression plasmid pVAX1-IL6 were constructed and the expression in vitro was detected by RT-PCR and Western blotting, the results showed that the gene of interest expressed in the transfected HeLa cells. To explore the immune response in mice coinoculated with HIV-1 DNA vaccine and IL-6 expression plasmid, BALB/c mice were injected i.m. with eukaryotic expression plasmid pVAX1-IL6 and DNA vaccine plasmid pVAX1-gag-gp120. The specific humoral and cellular immunity in mice could be induced by inoculating separately HIV-1 DNA vaccine plasmid or coinoculating with IL-6 expression plasmid, and the specific killing activities of spleen CTL and the level of serum antibodies in the coinoculation group were significantly higher than those in the separate inoculation group. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.

Identification of murine CD8 T cell epitopes in codon-optimized SARS-associated coronavirus spike protein

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus, SARS-associated coronavirus (SARS-CoV). CD8 T cells play an important role in controlling diseases caused by other coronaviruses and in mediating vaccine-induced protective immunity in corresponding animal models. The spike protein, a main surface antigen of SARS-CoV, is one of the most important antigen candidates for vaccine design. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding codon-optimized SARS-CoV spike protein. CD8 T-cell responses were mapped to two H-2 b-restricted epitopes (S436–443 and S525–532) and one H-2 d-restricted epitope (S366–374). The identification of these epitopes will facilitate the evaluation of vaccine strategies in murine models of SARS-CoV infection. Furthermore, codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in the context of replication-defective human and simian adenoviral vaccine carriers. The optimized recombinant adenoviral vaccine vectors encoding spike can generate robust antigen-specific cellular immunity in mice and may potentially be useful for control of SARS-CoV infection.

Expression and immunological evaluation of the Escherichia coli-derived hepatitis C virus envelope E1 protein.

Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia. The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1(340), amino acids 192-340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85%. We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1-encoding peptides. From this, 51.1% of human sera were found to react with E1(340). High-level antibodies against E1(340) were obtained in rabbits and mice when immunized. These antibodies had a similar peptide-recognition pattern to that described previously for human sera. The most reactive region was located at the N-terminus of the E1 protein. Cellular immunity in mice was evaluated by delayed-type hypersensitivity assay. It revealed the induction of a CD4+ T-cell-mediated response by this protein. This E1(340) protein and the animal-derived anti-E1 sera are immunological tools that could aid in the monitoring and development of anti-HCV therapies.

The potential role for urocanic acid and sunlight in the immune suppression associated with protein malnutrition

Irradiation of skin by sunlight or ultraviolet B (UVB, 290–320 nm) brings about a downregulation of cell-mediated immunity. An action spectrum for photoimmune suppression in mice indicates that trans-urocanic acid absorbs UV photons and is isomerized to the cis-isomer in the stratum corneum. Cis-urocanic acid is subsequently shown to suppress cellular immunity in mice. When histidine is elevated in a mouse diet, a higher level of urocanic acid is detected in mouse skin. These mice are more susceptible to photoimmune suppression. There is evidence that humans and animals experiencing protein malnutrition have very high levels of urocanic acid and/or histidine. Urocanic acid is formed by deamination of histidine in one enzymatic step. We discuss the protein malnutrition of kwashiorkor patients. They experience suppressed immunity and disturbed histidine metabolism. Here, we present a testable hypothesis: one cause of the immune deficiency observed in humans with protein malnutrition is the photoconversion by UVB of increased levels of trans-urocanic acid in skin to cis-urocanic acid, which suppresses the cellular immune system.