Lymphocyte Proliferative Responses Research Articles

Abstract IA07: Chimeric antigen receptor T cells for cancer immunotherapy

Abstract Background: T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR structure consists of two fundamental domains – the antigen binding portion (most commonly single chain variable fragment (scFv) derived from a monoclonal antibody) joined to one or more intracellular T cell signaling domains. To be effective CAR T cells must expand, persist, exhibit enduring anti-tumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape following infusion. T cells targeting the CD19 antigen is a novel therapeutic approach for patients with hematologic malignancies. To this end, we have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We now extend this novel treatment approach by testing of our CD19-specific CAR in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. Patients and Methods: Pediatric and young-adult patients with very high risk (VHR) or R/R CD19+ B-ALL were eligible for enrollment and leukapheresis. Patients with R/R B-ALL were eligible for infusion if they demonstrated residual disease following salvage chemotherapy. Once eligibility was determined isolation and transduction of T cells was performed using a retroviral vector encoding a CD19-specific CAR comprised of a CD19-specific scFv and the CD28 and CD3ζ signaling domains (termed 19-28z). All patients received conditioning chemotherapy followed 2 days later by 1x106 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment response was assessed at day 14-28 bone marrow aspirate including multiparameter flow cytometry for minimal residual disease (MRD). Results: To date, 28 patients with have been enrolled on protocol with a median age of 11.5 years (range 9 months to 20 years) at time of T cell collection. We have treated 11 patients with 19-28z CAR T cells with a median age 14.9 years (range 3-22 years). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 7/11 (64%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p<0.004) and fold expansion following CD3/28 bead activation during the generation of CAR T cells (p<0.03). Specifically, BM cellularity ≤50% and fold expansion (average >135 vs <40 fold expansion) was seen in responders compared to non-responders. Pre-collection peripheral blood absolute lymphocyte count, lymphocyte proliferative response (phytohemagglutinin proliferation assay), and CD3 absolute count did not correlate with response in this small sample size. Development of fever and cytokine release syndrome (CRS) occurred in responders including grade I-II (n=3) and grade III-IV (n=4). Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of CRS. Elevated serum cytokines (notably IFN-γ and IL-6) was noted in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. Conclusions: These early results demonstrate the feasibility and significant clinical impact of CAR T cells in patients with R/R B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the exportability of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Citation Format: Kevin J. Curran, Lewis B. Silverman, Rachel Kobos, Nancy A. Kernan, Steven P. Margossian, Jae H. Park, Craig S. Sauter, Victoria Szenes, Xiuyan Wang, Richard J. O'Reilly, Michel Sadelain, Isabelle Riviere, Reiner J. Brentjens. Chimeric antigen receptor T cells for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA07.

Enhanced humoral and cellular immune responses to PRRS virus GP5 glycoprotein by DNA prime-adenovirus boost vaccination in mice.

In order to investigate the induction of humoral and cellular immune responses against porcine reproductive and respiratory syndrome virus (PRRSV), BALB/c mice were immunized in a pcDNA3-GP5 prime-rAd-GP5 boost regimen. After humoral and cellular immune response detection, levels of PRRSV-specific antibodies, neutralizing antibodies, lymphocyte proliferation response, and cytotoxic T-lymphocyte response were significantly increased as compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. Significant difference was observed between heterogeneous and homologous vaccination. Induction of anti-GP5 antibody response was higher in all heterogeneous combinations than those of the homologous combinations. In the induction of lymphocyte proliferation response and CTL response, the homologous combination of pcDNA3-GP5/pcDNA3-GP5/pcDNA3-GP5was significantly stronger than that of rAd-GP5/rAd-GP5/rAd-GP5, but was relatively weaker than the heterogeneous combination of pcDNA3-GP5/pcDNA3-GP5/rAd-GP5 and pcDNA3-GP5/rAd-GP5/rAd-GP5. This heterogeneous combination was a most efficient immunization regimen in induction of PRRSV-specific cellular immune response just as the antibody response. These results suggested that DNA immunization followed by recombinant adenovirus boosting could be used as a potential PRRSV vaccine.

Mitogenic Activation and Proliferation of T Lymphocytes in TRPM7 Kinase-Dead Mutant Mice

Calcium and magnesium ions have been implicated in T lymphocyte proliferation in response to antigen recognition. Specifically, it is believed that calcium and magnesium elevations in the cytoplasm are necessary for efficient T cell proliferation. Research over the past two decades has focused on identifying the plasma membrane ion channels responsible for governing Ca2+ influx in lymphocytes and Orai-STIM, Kv1.3 and KCa3.1 channels were shown to be crucial for persistent calcium mobilization. Pharmacologic or genetic suppression of calcium influx gives rise to lymphoproliferative defects. In addition to Ca2+, several studies have demonstrated that Mg2+ influx positively regulates proliferation. TRPM7 is a channel-kinase highly expressed in T cells and reported to conduct both Ca2+ and Mg2+. TRPM7 channels give rise to outwardly rectifying currents inhibited by cytosolic Mg2+. TRPM7 protein appears to be necessary for murine thymocyte development, while small reductions in splenic T lymphocyte numbers were observed in TRPM7-deleted mice. Deletion of TRPM7 in a chicken B cell line resulted in severe proliferation defects. In this work we examine the role of TRPM7 kinase in T-cell blastogenesis and proliferation using a recently reported kinase-dead mutant mouse (Kaitsuka et al, Sci. Rep. 2014). Our goal was to compare the effects of extracellular Ca2+/Mg2+ between wildtype and kinase-dead mutant mouse splenic T cells stimulated with PMA/ionomycin. Increasing Ca2+ or Mg2+ stimulated proliferation in both wildtype and kinase-dead mouse lymphocytes but with significant differences. Using a Coulter counter we also measured the diameters of resting and activated T cells in response to changes in external Ca2+/Mg2+. The differences between WT and mutant T cell blastogenesis and proliferation parameters are discussed in the context of controversies regarding the role of TRPM7 channel and kinase function in cellular Mg2+ homeostasis.

A Rhesus Macaque Model of Pulmonary Nontuberculous Mycobacterial Disease.

In this study, we sought to develop a nonhuman primate model of pulmonary Mycobacterium avium complex (MAC) disease. Blood and bronchoalveolar lavage fluid were collected from three female rhesus macaques infected intrabronchially with escalating doses of M. avium subsp. hominissuis. Immunity was determined by measuring cytokine levels, lymphocyte proliferation, and antigen-specific responses. Disease progression was monitored clinically and microbiologically with serial thoracic radiographs, computed tomography scans, and quantitative mycobacterial cultures. The animal subjected to the highest inoculum showed evidence of chronic pulmonary MAC disease. Therefore, rhesus macaques could provide a robust model in which to investigate host-pathogen interactions during MAC infection.

Potentiation of Taishan Pinus massoniana pollen polysaccharide on the immune response and protection elicited by a highly pathogenic porcine reproductive and respiratory syndrome virus glycoprotein 5 subunit in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) heavily affects the global pork industry. Current available vaccine strategies have inherent drawbacks. In this work, the immune enhancement from Taishan Pinus massoniana pollen polysaccharide (TPPPS) and Freund's adjuvant on the efficacy of a PRRSV subunit vaccine were examined. Titers of specific anti-highly pathogenic PRRSV (HP-PRRSV) ELISA antibody and neutralizing antibody were significantly higher in pigs from the groups inoculated with medium- and high-dose TPPPS (mTPPPS, hTPPPS) adjuvant co-administered with a recombinant HP-PRRSV glycoprotein 5 subunit (GP5) than those from other groups (P < 0.05). Pigs inoculated with GP5 + Freund's adjuvant developed severely delayed humoral immune responses specific to GP5 within 28 days post-inoculation (dpi). The groups treated with mTPPPS and hTPPPS adjuvant exhibited the most potent immune enhancement effects on GP5 inoculation with cellular immunity developing, as shown by the level of T lymphocyte proliferation and the percentage of the CD3+ T lymphocyte subpopulation. Although complete Freund's adjuvant elicited cell-mediated immune responses, the level of T lymphocyte proliferation in this group decreased quickly and no significant differences were observed compared with other adjuvant-alone groups at 56 dpi (P > 0.05). The ratio between CD3+CD4+ and CD3+CD8+ T lymphocyte subpopulations indicated the inoculums of GP5 + mTPPPS and GP5 + hTPPPS induced consistently higher CD3+CD4+ T lymphocyte subpopulations than other inoculums (P < 0.05). The immune responses caused by complete Freund's adjuvant were mainly mediated by CD3+CD8+ T lymphocyte subpopulation (cytotoxic T lymphocytes) in the early stage of inoculation and had no significant difference compared with other adjuvant-alone groups after 28 dpi (P > 0.05). The low-dose TPPPS (lTPPPS) adjuvant also exhibited enhancement effects on humoral immune and T lymphocyte proliferation responses but these were significantly lower than the mTPPPS and hTPPPS doses (P < 0.05). Pigs challenged with HP-PRRSV from the GP5 + mTPPPS, GP5 + hTPPPS, and GP5 + Freund's adjuvant groups showed lower viremia, fewer clinical signs, and fewer pathological lung lesions compared with the groups of GP5-alone and GP5 + lTPPPS (P < 0.05). There were significant differences between the GP5-alone and GP5 + lTPPPS groups in detection indexes after viral challenge (P < 0.05). In conclusion, moderate doses of TPPPS as an adjuvant with GP5 show promise as a candidate for a HP-PRRSV subunit vaccine to efficiently prevent and control HP-PRRSV.

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.

Measuring Lymphocyte Proliferation in Response to Specific Antigen and Mitogen Stimuli Using Flow Cytometry.

We present a multivariate test system using flow cytometry after in-vitro lymphocyte stimulation using 5 mitogens and 7 antigens to describe in-vitro immunofunction. The present work is a crucial step towards establishing a simple, CFSE-based, multivariate test system that can describe the dynamics of stimulus-induced lymphocyte proliferation with considerably more precision than is possible with the radionucleotide method using 3H-thymidine. Using multicolour flow cytometry, our method allows additional phenotyping of the proliferating cells and quantifies the proliferation behaviour by precisely resolving daughter generations and determining the precursor frequency. </i> Taking the calculated apoptosis parameters into account not only provides additional information about the stimulus-specific response behaviour but also improves the validity of the commonly used proliferation indices. Not only can we confirm previous findings that healthy people have marked differences in a multivariate test system in terms of the individual in-vitro reactivity to various stimuli but also substantiate that the response pattern of an individual is remarkably constant. In follow-up studies we can show for the first time that the results of immunofunctional testing do not change over a period of at least 6 months and appear to be an inherent characteristic of the individual and thus possibly have a genetic basis.

DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.

Recombinant Toxoplasma gondii phosphoglycerate mutase 2 confers protective immunity against toxoplasmosis in BALB/c mice.

Toxoplasmosis is one of the most widespread zoonoses worldwide. It has a high incidence and can result in severe disease in humans and livestock. Effective vaccines are needed to limit and prevent infection with Toxoplasma gondii. In this study, we evaluated the immuno-protective efficacy of a recombinant Toxoplasma gondii phosphoglycerate mutase 2 (rTgPGAM 2) against T. gondii infection in BALB/c mice. We report that the mice nasally immunised with rTgPGAM 2 displayed significantly higher levels of special IgG antibodies against rTgPGAM 2 (including IgG1, IgG2a and IgAs) and cytokines (including IFN-γ, IL-2 and IL-4) in their blood sera and supernatant of cultured spleen cells compared to those of control animals. In addition, an increased number of spleen lymphocytes and enhanced lymphocyte proliferative responses were observed in the rTgPGAM 2-immunised mice. After chronic infection and lethal challenge with the highly virulent T. gondii RH strain by oral gavage, the survival time of the rTgPGAM 2-immunised mice was longer (P < 0.01) and the survival rate (70%) was higher compared with the control mice (P < 0.01). The reduction rate of brain and liver tachyzoites in rTgPGAM 2-vaccinated mice reached approximately 57% and 69% compared with those of the control mice (P < 0.01). These results suggest that rTgPGAM 2 can generate protective immunity against T. gondii infection in BALB/c mice and may be a promising antigen in the further development of an effective vaccine against T. gondii infection.

Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2.

Infradian rhythm in hematology, cell mediated immunity and serum micronutrient concentration around peripartum periods in high yielding crossbred cows before and after micronutrient supplementation

Alteration in hematology and cell-mediated immunity around peripartum periods before and after micronutrient supplementation was studied in this investigation. Twelve (12) high-yielding cross-bred cows in advanced pregnancy were selected for the experiment and divided into two equal groups viz. supplemented (n = 6) and unsupplemented (n = 6). Supplemented animals were provided with a micronutrient mixture (@ 25 g per cow) as per the recommendation of NRC over the normal feeding. Unsupplemented group of cows were kept as control with standard farm feeding management. Blood samples (15 ml/cow) were collected from all the animals during days 30th, 15th, and 7th before calving, on the day of calving, and during days 7th, 15th, and 30th after calving and analyzed for hematology viz. hemoglobin (Hb), total erythrocyte counts (TEC), total leukocyte counts, and differential leukocyte counts. In vitro phagocytic index (PI) of neutrophils and lymphocyte proliferation response (LPR) were also evaluated. Copper (Cu), zinc (Zn), manganese, and selenium (Se) concentrations in serum were estimated by atomic absorption spectrophotometer. The Hb concentration was found to be significantly (p < 0.01) higher in supplemented group compared to the unsupplemented group of cows. There were no significant differences in Hb concentration during different days of peripartum in both the group of cows. There was a significantly (p < 0.001) higher TEC in supplemented group compared to unsupplemented group of cows, though it did not vary significantly between different days of peripartum in both the group of cows. There was a significantly (p < 0.001) higher PI in supplemented group compared to the unsupplemented group of cows. In both the groups, the PI of blood neutrophils was significantly (p < 0.001) lower during calving. The LPR did not differ significantly between the groups, but it differed significantly (p < 0.01) between different days of peripartum and was lowest on the day of calving in both the groups. The Cu, Zn, and SE concentration was found to be significantly (p < 0.01) higher in supplemented group compared to the unsupplemented group of cows, but remained unaltered throughout the peripartum period. The study indicated that there was a marked immune depression around peripartum (15 days precalving to 15 days postcalving) in terms of in vitro phagocytic activity of blood neutrophils and in vitro LPR. Supplementation of micronutrients improves the hematological status as well as immune status, so it was recommended to supplement these micronutrients for better productivity and disease resistance in high-yielding cross-bred cows around peripartum.

Multi-Center Clinical Trial of CAR T Cells in Pediatric/Young Adult Patients with Relapsed B-Cell ALL

T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR T cells targeting the CD19 antigen is a novel therapeutic approach for patients with relapsed B cell acute lymphoblastic leukemia (B-ALL). We have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed B-ALL. The primary objective of this study (NCT01860937) is to extend the use and test the safety of CD19 specific CAR T cells in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. To date, 24 patients with very high risk (VHR) or relapsed B-ALL have been enrolled on protocol with a median age of 12 years (range 2-20 years) at time of T cell collection. We have treated 9 patients with relapsed B-ALL with a median age 15 years (range 3-22 years) using patient derived T cells expressing a CD19 specific CAR (19-28z). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 5/9 (55%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p<0.02) and fold expansion following CD3/28 bead activation during the generation of CAR T cells (p<0.02). Specifically, BM cellularity ≤50% and fold expansion (average >150 vs <40 fold expansion) was seen in responders compared to non-responders. Pre-collection peripheral blood absolute lymphocyte count, lymphocyte proliferative response (phytohemagglutinin proliferation assay), and CD3 absolute count did not correlate with response in this small sample size. Development of fever and cytokine release syndrome (CRS) occurred in responders including grade I-II (n=2) and grade III-IV (n=3). Systemic immunosuppressants (corticosteroids or anti-IL6 receptor antibody tocilizumab) abrogated clinical symptoms of CRS. Elevated serum cytokines of IFN-g (>20 fold), fractalkine (>20 fold), Flt-3L (>55 fold), IL-5 (>15 fold), IL-6 (>100 fold), and IL-10 (>15 fold) were demonstrated in patients with CRS. Monitoring of bone marrow demonstrated peak 19-28z CAR T cell detection within 1-2 weeks following infusion with gradual contracture over 1-2 months. These early results demonstrate the feasibility and significant clinical impact of this approach in patients with relapsed B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the "exportability" of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. DisclosuresCurran:Juno Therapeutics: Consultancy. Off Label Use: CAR T cells for B-ALL. Riviere:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Prockop:Atara Biotherapeutics: Other: I have no financial disclosures, but Atara Biotherapeutics has exercised a licensing agreement with Memorial Sloan Kettering Cancer Center and MSKCC and some investigators at MSKCC have a financial interest in Atara.. Park:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy. O'Reilly:Atara Biotherapeutics: Research Funding. Sadelain:Juno Therapeutics: Other: Co-founder, stockholder and consultant. Brentjens:Juno Therapeutics: Other: Co-founder, stockholder and consultant.

Evaluation of Salmonella Gallinarum ghost formulated with Montanide™ ISA 70 VG adjuvant as a vaccine against fowl typhoid.

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent adjuvant. Salmonella Gallinarum ghosts carrying LTB (S. Gallinarum-LTB ghosts) were genetically constructed using a plasmid, pJHL187-LTB, designed for the co-expression of the LTB and E lysis proteins. This study evaluates the immunopotentiating effects of Montanide™ ISA 70 VG on S. Gallinarum-LTB ghost vaccination against fowl typhoid. Five-week-old layer chickens were injected intramuscularly with sterile PBS (non-immunised control, Group A), S. Gallinarum-LTB ghost (Group B) or S. Gallinarum-LTB ghost emulsified with Montanide™ ISA 70 VG adjuvant (Group C). Chickens from both Groups B and C showed significant induction of antigen-specific systemic IgG response compared to controls; in addition, Group C showed enhanced induction of systemic IgG response compared to Group B. We observed significant induction of antigen-specific lymphocyte proliferative response and increased mRNA levels of Th1 cytokines (IFN-γ and IL2) in both Groups B and C. Furthermore, in the challenge experiment with a virulent strain of S. Gallinarum, Group C showed higher survival rates compared with other groups. These results indicate that vaccination with the S. Gallinarum-LTB ghost in combination with Montanide™ ISA 70 VG may enhance the protective immunity against fowl typhoid.

Effect of In Vitro Copper and Zinc Supplementation on Neutrophil Phagocytic Activity and Lymphocyte Proliferation Response of Transition Dairy Cows

Three experiments were conducted to assess the effect of in vitro copper (Cu) and zinc (Zn) supplementation on neutrophil phagocytic activity (NPA) and lymphocyte proliferation response (LPR) of transition dairy cows. In the first experiment, six multiparous crossbred (Karan-Fries: Tharparkar × Holstein-Friesian) cows were selected; the neutrophils and lymphocytes were isolated from the blood samples of different peripartum days of each cow. To determine the effect of in vitro Cu and Zn supplementation on the NPA and LPR, 2.360 μM Cu and 9.175 μM Zn were added separately into the culture media of neutrophils and lymphocytes. It was found that the addition of Zn in the culture media increased the NPA and B lymphocyte LPR significantly (P < 0.05) as compared to non-supplemented neutrophils and lymphocytes. In the second experiment, another six multiparous crossbred cows were selected and fed with 20 ppm Cu/cow/day individually from 45 days pre-calving to 45 days post-calving. Their blood samples were collected during different peripartum days and 2.360 μM Cu was incorporated separately into the culture media of NPA and LPR to know whether there is need for extra Cu after Cu supplementation. Addition of Cu in the culture media of lymphocyte of Cu fed animals increased B lymphocytes LPR significantly (P < 0.05). On the day of calving, the B and T lymphocyte LPR increased significantly (P < 0.05) and phagocytic activity increased by 15.38 %. In the third experiment, six more multiparous crossbred cows were selected and fed with 80 ppm Zn/cow/day individually from 45 days pre-calving to 45 days post-calving. Their blood samples were collected during different peripartum days and 9.175 μM Zn was added separately into the culture media of NPA and LPR to know whether there is need for extra Zn after Zn supplementation. A significantly (P < 0.05) higher LPR (T and B) was found on 3 days before parturition with in vitro addition of Zn in the culture media and it remained higher up to 7 days post-partum. Peripartum days had (P < 0.05) significant effect on both in vitro NPA and LPR in all three experiments. It is concluded that extra Cu and Zn supplementation around peripartum period has beneficial effects on in vitro NPA and LPR.

Phytochemical screening of the dichloromethane⿿ethanolic extract of Eriosema campestre var. macrophylum roots and its antiproliferative effect on human peripheral blood lymphocytes

Eriosema campestre var. macrophylum (Grear) Fortunato, Fabaceae, is a native plant of the Brazilian Cerrado and the decoction of its roots has been used by folk medicine for the therapy of inflammatory diseases. In this study we aimed to investigate the effect of the dichloromethane⿿ethanolic extract of E. campestre roots on the proliferative response of lymphocytes and to examine the profile of IL-2 production. The effect of dichloromethane⿿ethanolic extract of E. campestre on the proliferation of phytohemagglutinin-stimulated lymphocytes was evaluated by using flow cytometry and the cell supernatants were assayed for IL-2 concentrations by using an enzyme-linked immunosorbent assay. The phytochemical screening of E. campestre roots was performed to determine the main secondary metabolites through chromogenic and precipitation reactions and by using HPLC-PAD. In addition to the presence of subclasses of flavonoids (flavones and flavonols) in dichloromethane⿿ethanolic extract of E. campestre, we observed that the extract induced a concentration-dependent decrease in IL-2 levels on the supernatant of the cell cultures as well as an antiproliferative effect on T lymphocytes, including CD4+ and CD8+ cells. The anti-inflammatory effects attributed to E. campestre by folk medicine may partly be explained by its antiproliferative action on T lymphocytes.

Single immunization with a recombinant multiple-epitope protein induced protection against FMDV type Asia 1 in cattle

To develop recombinant epitope vaccines against the foot-and-mouth disease virus (FMDV) serotype Asia 1, genes encoding six recombinant proteins (A1–A6) consisting of different combinations of B-cell and T-cell epitopes from VP1 capsid protein (VP1) of FMDV were constructed. These proteins were expressed in Escherichia coli and used to immunize animals. Our results showed that A6 elicited higher titers of neutralizing antibodies after single inoculation in guinea pigs than did the other five recombinant proteins, as determined by micro-neutralization tests. In addition, a strong lymphocyte proliferation response and Th1 type immunity were observed in splenocytes from the mice immunized with A6. Further tests carried out in cattle demonstrated that a single inoculation with A6 generated comparable levels of neutralizing antibodies as inactivated vaccine and protected 4 of 5 cattle against challenge with FMDV type Asia 1. Our results suggest that A6 might be a promising recombinant vaccine against FMDV type Asia 1 in cattle.

Impaired Immune Response in Old Mice Suffering from Obesity and Premature Immunosenescence in Adulthood.

Obesity and aging share an impaired immune system and oxidative and inflammatory stress. Therefore, the hypothesis of obesity as a possible model of premature immunosenescence has been proposed. In this study, we investigated whether adult obese mice, as a consequence of being fed with a fat-rich diet during their adolescence, showed premature immunosenescence and if this was aggravated with aging. Peritoneal cell suspensions were obtained when ICR/CD1 obese female mice were adults (28 weeks) and old (72 weeks), and several functions and antioxidant defenses were evaluated. The results showed that the chemotaxis of both macrophages and lymphocytes, phagocytosis of macrophages, activity of natural killer cells, proliferative response of lymphocytes, interleukin-1β, tumor necrosis factor-alpha, interleukin-6, interleukin-2, and interleukin-10 released in leukocyte cultures, as well as antioxidant and oxidant capacity were significantly impaired in adult obese mice with respect to adult nonobese mice, with values similar to those in chronologically old mice. When these obese animals grew older, although having been fed with a standard diet, they showed a higher deterioration of their immune functions in comparison with the old control group. In conclusion, these results demonstrate that a high fat intake during adolescence can produce an obesity state in adult age associated with a premature immunosenescence, which is aggravated through aging.

Dried fruit extract of Terminalia chebula modulates the immune response in mice

Terminalia chebula, traditionally regarded as universal panacea, is known for its pleiotropic effects, including the effects on oxidative stress, malignant tumor growth, wound healing, and insulin sensitivity in Type 2 diabetes. This study reports the effect of dry fruit extract of T. chebula (TCE) on Th1/Th2-mediated immune responses in mice. TCE was administered orally for 10 consecutive days, after which mice were immunized with goat RBC (gRBC) or ovalbumin. TCE enhanced the expression of Th1 cytokine, interferon γ, decreased interleukin 4, and increased the number of plaque-forming cells in gRBC-immunized mice. The percentage of CD4+ cells and delayed-type hypersensitivity response also increased in these mice. Treatment is reported to increase lymphocyte proliferation and macrophage phagocyte response, but decrease nitrite production. The bone marrow cellularity and WBC count also increased in the treated mice. None of the group showed any sign of toxicity. The data indicate that TCE elicits a significant dose-dependent Th1 response.

Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Immunostimulatory potential and proteome profiling of Leishmania donovani soluble exogenous antigens.

Isolation of the soluble exogenous antigens (SEAgs), its immune response study and proteome profiling is an essential prerequisite for understanding the molecular pathogenesis of Leishmania donovani. The immunostimulatory potential of L. donovani SEAgs, purified from culture of L. donovani clinical isolate, was evaluated for their ability to induce cellular responses in treated/cured hamsters. SEAgs induced significant proliferative responses in lymphocytes (SI 5.6 ± 2.3; P < 0.01) isolated from cured hamster. In addition, significant NO production in response to SEAgs was also noticed in macrophages of hamsters, mouse and human cell lines (J774A-1 and THP1). Western blot analyses with antibodies against proteophosphoglycan (PPG; surface-expressed and secreted molecule) of L. donovani revealed that PPG molecules are also present in L. donovani SEAgs. Mass spectrometry (MS)-based proteome analysis of 12 protein bands of SEAgs through MALDI-TOF/TOF endorsed the identification of some Th1-stimulatory immunogenic proteins. These immunogenic proteins may offer increased hope for the discovery of new promising vaccine candidates against visceral leishmaniasis (VL). The overall results suggest that immunostimulatory molecules are present in the SEAgs, which may be further exploited, for developing a subunit vaccine against VL a fatal human disease.