IFN-γ Production In Splenocytes Research Articles

Revealing novel and conservative CD8+T-cell epitopes with MHC B2 restriction on ALV-J

MHC B2 haplotype chickens have been reported to induce strong immune response against various avian pathogens. However, little is known about the CD8+T-cell epitope with MHC B2-restricted on subgroup J avian leukosis virus (ALV-J). In this study, we explored the ALV-J-induced cellular immune response in B2 haplotype chickens in vivo. We found that ALV-J infection significantly increased the proportion of CD8+T cells in chickens and up-regulated the expression of cytotoxic genes like Granzyme A and antiviral genes like IFIT5 at 14 days post-infection (dpi). We selected 32 candidate peptides based on the peptide-binding motif and further identified three MHC B2-restricted CD8+T epitopes on ALV-J, including Pol652−660, Gag374−382, and Gag403−411 which induced significant levels of chicken IFN-γ production in splenocytes from ALV-J infected chickens using the ELISpot assay. In addition, we also verified that the three identified epitopes stimulated memory splenocytes elevating TNF-α and IL-2 protein expression. Importantly, we found that the three positive peptides were highly conserved among ALV-A, ALV-B, ALV-E, ALV-J, and ALV-K. Taken together, we identified three MHC B2-restricted CD8+T cell epitopes on ALV-J, providing a foundation for developing effective T cell epitope vaccines targeting conserved internal viral proteins.

Immune Responses and Protection Profiles in Mice Induced by Subunit Vaccine Candidates Based on the Extracellular Domain Antigen of Respiratory Syncytial Virus G Protein Combined with Different Adjuvants.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of infants and older people. There is an urgent need for safe and effective vaccines against RSV infection. In this study, we analyzed the effects of the immune response and protection with the RSV recombinant G protein extracellular domain (Gecto) combined with various adjuvants as novel subunit vaccines in mice. All groups receiving RSV Gecto combined with adjuvants exhibited robust humoral and cellular immunity compared to those receiving an adjuvant alone or inactivated RSV vaccine. The greatest effect was observed in mice receiving Gecto combined with a CpG ODN + Alum salt adjuvant, resulting in the highest production of neutralizing antibodies against both RSV A and B subtypes, G-specific IgG and IFN-γ production in splenocytes, and interleukin-2 and interferon-γ expression in CD4+ T cells. Significant humoral and cellular immune responses were observed in mice immunized with Gecto combined with AddaS03™ or cyclosporin A adjuvants. The vaccine containing the AddaS03™ adjuvant showed significantly high expression of interleukin-4 in CD4+ T cells. Cross-protection against a challenge with either RSV A or B subtypes was observed in the Gecto plus adjuvant groups, resulting in a significant decrease in viral load and reduced pathological damage in the mouse lungs. These findings offer valuable insights into the development and application of recombinant RSV G-subunit vaccines with adjuvants.

Abstract 2072: pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors

Abstract Background: IL-12 is a pro-inflammatory cytokine with antitumor potential that activates both innate and adaptive immunity. However, IL-12 shows poor pharmacokinetics and intolerable toxicities due to unspecific distribution. Herein, we present pH-sensitive polymeric micelles loading IL-12 (IL-12/m) that release fully active IL-12 after sensing intratumoral pH to safely potentiate immunotherapy. Methods: IL-12/m with 40 nm in diameter were prepared by self-assembly in phosphate buffer. The IL-12 release was tested at different pH. The stimulation of IFN-γ release from IL-12 and IL-12/m was studied in mouse splenocytes. Pharmacokinetic, pharmacodynamic, and antitumor activity were studied in immunosuppressed (B16F10 and 4T1) mouse tumor models (staged ≥ 200 mm3) and in a lung metastasis model (4T1). The safety of IL-12 was evaluated by tracing levels of cytokines and blood markers of toxicity. Results: IL-12/m selectively activated at intratumoral pH 6.5, releasing more than 80% of IL-12 in 24 h. While IL-12/m did not induce IFN-γ production in splenocytes, the released IL-12 showed similar activity to native IL-12. In mice, IL-12/m prolonged blood circulation with minimal leakage, yielding a 3-fold longer half-life than IL-12. Moreover, the accumulation in tumor for IL-12/m (7.5%ID/g of tumor) was 3-fold higher compared that of free IL-12, with more than 90% of IL-12/m being activated. The tumors treated with IL-12/m showed 2-3-fold higher levels of IFN-γ, IL-6 and TNF-α than those treated with IL-12, whereas anti-inflammatory IL-10 was 2-fold lower for IL-12/m. In blood and organs, IL-12/m lowered the cytokine levels compared to IL-12. Also, IL-12/m avoided the tachyphylaxis of IL-12 without peak effects of IFN-γ, and the blood markers of toxicity, i.e., BUN, ALT and AST, remained close to control levels, supporting the tumor selective activation of IL-12/m. IL-12/m was efficacious in the B16F10 tumor model (10 µg iv 3 injections every 4 days) as monotherapy, and in combination with anti-PD-1 (10 mg/kg IP 3 injections every 4 days), which led to 6 of 10 complete regressions (CR). IL-12/m was also effective at 1 µg (5 injections every 2 days) against 4T1 tumors. IL-12/m increase the number and activation of CD8+ T cells and NK cells in tumors. The combination of IL-12/m (10 µg iv 3 injections every 4 days) with anti-PD1 and anti-CTLA-4 (both 10 mg/kg IP 3 injections every 4 days) achieve complete CR in a spontaneous model of lung metastasis. Responders showed immunological memory after being rechallenged with fresh tumor cells. Conclusions: IL-12/m, a pH-activated polymeric micelle loading IL-12, masked the activity of IL-12 in healthy tissues, while it unleashed full potency of IL-12 in tumors, improving tolerability and efficacy. IL-12/m presented greater antitumor activity than IL-12 as monotherapy and in combination with immune checkpoint inhibitors. Citation Format: Horacio Cabral, Pengwen Chen, Kazuhiko Kakimi, Kazunori Kataoka, Takuya Miyazaki, Koji Nagaoka. pH-activatable IL-12-loaded polymeric micelles safely enhance antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2072.

A Comparison of Characterization and Its Actions on Immunocompetent Cells of Polysaccharides from Sijunzi Decoction

Sijunzi decoction (SJZD) is a classic recipe in Traditional Chinese Medicine (TCM) with strong immune-enhancement activity. To further understand the characterization and immunomodulatory effect of polysaccharides from SJZD, the monosaccharide compositions of crude polysaccharide (SJZDP), polysaccharide fraction (S-3), and homogeneous polysaccharide (S-3-AG) from SJZD were compared by GC analysis, as well as their immunomodulatory effects on Peyer's patch cells, splenocytes, and macrophages which are related to intestinal immunity, specific immunity, and nonspecific immunity. The results showed that S-3-AG mainly contained Ara with a proportion of 38.9%, while Glc accounted for the largest proportion in S-3 (55.6%) and SJZDP (87.6%). The SJZDP, S-3, and S-3-AG all showed strong capability to stimulate Peyer's patch cells to proliferate and produce IgA and promoted the proliferation and IFN-γ production of splenocytes and increased the NO production and TNF-α production of macrophages. However, S-3 and S-3-AG were able to stimulate splenocytes to secret IL-4, SJZDP had no effect on IL-4 production of splenocytes in the tested concentrations. In addition, S-3 could stimulate the phagocytic activity of macrophages, and S-3-AG restrained the proliferation of macrophages at the concentration of 50–200 µg/mL. These results suggested that SJZDP, S-3, and S-3-AG might have different immunomodulatory effects on intestinal immunity, specific immunity, and nonspecific immunity due to their different monosaccharide compositions. It will provide references for the material basis and mechanism of SJZD immunomodulation activity.

Induction of specific cell-mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate.

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n=12) by 2 intramuscular injections with a 2-week interval. Another group of 12 mice was used as non-vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B.melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18±0.57, and 1519.35±10.70pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02±0.02, and 210.01±17.58pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B.melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.

IL-10 Has Differential Effects on the Innate and Adaptive Immune Systems of Septic Patients.

Sepsis, a disease of divergent pro- and anti-inflammatory-mediated pathways, has a high prevalence of morbidity and mortality, yet an understanding of potential unifying mediators between these pathways that may improve clinical outcomes is largely unclear. IL-10 has classically been designated an immunosuppressive cytokine, although recent data suggest that under certain conditions IL-10 can be immune stimulatory. We sought to further investigate the effect of IL-10 on innate and adaptive immunity in an in vitro human observational cohort study in patients with sepsis via modulation of IL-10 on IFN-γ production by T cells and TNF-α production and HLA-DR expression by monocytes. These results were compared with critically ill nonseptic patients and healthy volunteers. ELISpot analysis was performed using PBMC fraction from patient whole-blood samples. Finally, to provide additional potential clinical relevance, we examined the effect of IL-10 on T cell IFN-γ production in an in vivo cecal ligation and puncture model of sepsis using C57 black/J6 female mice. We found that inhibition of IL-10 significantly increased both production of T cell IFN-γ and monocyte TNF-α, whereas addition of IL-10 increased T cell IFN-γ production but decreased monocyte production of TNF-α and HLA-DR expression. There was no significant effect of IL-10 on control cohorts. IL-10-treated septic mice demonstrated increased IFN-γ production in splenocytes. Thus, IL-10 demonstrates both pro- and anti-inflammatory effects in the septic microenvironment, which is likely cell and context dependent. Further elucidation of relevant signaling pathways may direct future therapeutic targets.

Anti-PD-1 Antibody Administration following Hip Fracture Surgery Reverses Immune Dysfunction and Decreases Susceptibility to Infection

The aim of this investigation was to assess expression of programmed death-1 (PD-1) and inflammatory status after hip fracture surgery in aged mice and to evaluate the effect of anti-PD-1 antibody intervention. Male C57BL/6 mice aged 22-28 months underwent hip fracture and femoral intramedullary pinning or a sham procedure. Expression of PD-1 was measured on CD4+ and CD8+ T cells. Additionally, the effects of anti-PD-1 antibody on lymphocyte apoptosis, cytokine production, bacterial clearance, and survival were determined. Expression of PD-1 on T cells was upregulated in mice after hip fracture and surgery compared to sham controls. Administration of anti-PD-1 antibody prevented T lymphocyte apoptosis, increased IFN-γ production in splenocytes, and decreased systemic inflammation. Antibody blockade of PD-1 significantly decreased susceptibility to bacteria and improved survival rates of aged mice after hip fracture and surgery followed by the induction of Pseudomonas aeruginosa pneumonia. This study showed that hip fracture and surgical trauma cause significant increases in PD-1 expression in aged mice. Antibody blockade of PD-1 partially reverses T cell apoptosis, decreases the systemic inflammatory response and susceptibility to bacteria, and reduces mortality.

Abstract A021: Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) expressed during development in the nervous system. Aberrant expression of ALK drives several tumor malignancies. About 3-7% of non-small cell lung cancers (NSCLC) harbor ALK rearrangements. Treatment with the Tyrosine Kinase inhibitor (TKI) crizotinib results in a high objective rate response (ORR) of ∼60%, but median progression-free survival (mPFS) is only 8-10 months, owing to the rapid emergence of acquired drug resistance. Alectinib and ceritinib, a second generation of TKIs developed to overcome crizotinib resistance, induce a transient response followed by tumor relapses. Currently, there are at least 7 additional ALK TKIs in clinical development. Emergence of mechanisms of resistance to the new TKI is expected. Thus, innovative therapeutic approaches are needed to complement the partial success obtained by TKIs in ALK-positive NSCLC patients. Three properties make of ALK a rather unique shared antigen: 1) ALK is a driver oncogene that is expressed in all the tumoral cells; 2) ALK is not expressed by normal cells with only marginal expression in few cells in the nervous system of adults, an immuneprivileged tissue, anticipating limited if any autoimmune side effects by an ALK-specific immunotherapy; 3) ALK is immunogenic in humans because ALK-specific antibodies and T cell responses are detected in ALK-positive lymphoma and lung cancer patients. As an additional advantage, the availability of ALK TKI to reduce tumor might facilitate immunotherapeutic approaches. Therefore, we aim to develop a strategy of vaccination in human patients to treat ALK-positive tumors in combination with TKIs and/or immunocheckpoints blockade. We recently developed an ALK vaccine based on a DNA plasmid that generates an efficacious anti-ALK immune response in pre-clinical mouse models. This vaccine potentiates the effects of ALK TKI and can be combined with immune checkpoint blockade therapy. However, a consistent theme in human DNA vaccine trials has been their suboptimal immunogenicity when compared with protein-based vaccine approaches. To move towards a peptide-based vaccine, we sought to identify antigenic peptides responsible for these immune responses by in vitro screening with ALK overlapping peptides in splenocytes from ALK vaccinated mice and by MHC-I binding predictive algorithms. Four peptides induced IFN-γ production in splenocytes and one of these peptides contained a predicted CD8+ T-cell epitope. To validate these results in vivo, mice were vaccinated with these peptides. The peptide containing the predicted CD8+ 9mer was confirmed to induce CD8+ T-cell responses while the others induced CD4+ T-cell responses. To increase immunogenicity, peptides were modified with an amphiphilic tail. As we recently showed, this conjugation promotes binding to endogenous albumin, increasing lymph node accumulation and T-cell priming. In prophylactic experiments, mice vaccinated with the CD8+ T-cell epitope, but not the CD4+ T-cell epitopes, almost completely rejected ALK-expressing lung orthotropic tumors. To validate the efficacy of the vaccine in a therapeutic protocol, EML4-ALK transgenic mice that spontaneously develop ALK-positive lung tumors were vaccinated with a combination of these peptides. The vaccine significantly delayed the progression of primary lung tumors. We propose that a peptide-based ALK vaccine, combined with ALK TKI treatment and/or immune checkpoint blockade therapy, could represent a novel therapeutic option for ALK-positive lung cancer patients. A Phase I clinical trial with a peptide-based ALK vaccine in NSCLC patients is currently under preparation. Citation Format: Rafael B. Blasco. Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A021.

A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1.

BackgroundPersistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB) for the treatment of HPV58 (+) cancer.MethodsPVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI)-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor.ResultsPVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the recombinant antigen HPV58 E6E7-GST. Furthermore, the vaccine also induced antitumor responses in the HPV58 (+) B16-HPV58 E6E7 tumor challenge model as evidenced by delayed tumor development.ConclusionThe recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB efficiently generates cellular immunity and antitumor efficacy in immunized mice. These data provide a basis for the further study of this recombinant vaccine as a potential candidate vaccine.

Identification of CD8 T cell epitopes in VP2 and NS1 proteins of African horse sickness virus in IFNAR(-/-) mice.

African horse sickness virus (AHSV) is an Orbivirus of the family Reoviridae that causes severe pathology in equids. Previous work in our laboratory showed the presence of AHSV-specific CD8+ T cells in mice immunized with recombinant Modified Vaccinia Ankara (rMVA) expressing VP2 and NS1 proteins. In the present work, we selected potential CD8 T cell epitopes (MHC-class I binding peptides) for the 129 mouse strain from the VP2 and NS1 proteins of AHSV-4, using a combination of four epitope prediction algorithms (SYFPEITHI, BYMAS, NetMHC I and NetMHCpan). ELISPOT and Intracellular Cytokine Staining (ICS) analysis showed that the VP2-720 (MSLLNFGAV), VP2-1044 (YTFGNKFLL), and NS1-83 (CVIKNADYV) peptides elicited IFN-γ production in splenocytes of MVA-VP2 and MVA-NS1 immunized mice and were identified as CD8+ T cell epitopes. In addition, these three MHC-class I-binding peptides induced the expression of CD107a in CD8+ T cells, an indirect marker of cytotoxic activity. Importantly, VP2-1044 and NS1-83 epitopes are conserved among all nine AHSV serotypes. These data demonstrate the activation of AHSV specific T-cell epitopes during vaccination with rMVAs expressing VP2 and NS1. Furthermore, the characterization of these CD8+ T-cell epitopes provides information useful for the design of novel marker multiserotype vaccines against AHSV.

NKT Cells Determine Titer and Subtype Profile of Virus-Specific IgG Antibodies during Herpes Simplex Virus Infection

Invariant NKT cells (iNKT cells) are innate lymphocytes that recognize lipid-derived Ags presented by the MHC class I-related protein CD1d. In this study, we analyzed the role of iNKT cells in the generation of Abs against HSV type 1 (HSV-1). In sera from healthy hman donors, we found a correlation between HSV-1-specific IgG titers and proportions of CD4(+) iNKT cells. In HSV-1-infected iNKT cell-deficient mice, the amount of specific IgM and IgG Abs were significantly reduced compared with wild-type mice. Moreover, iNKT cell-deficient mice were unable to upregulate CD1d on B cells and failed to establish an IFN-γ-driven subtype profile of HSV-1-specific IgG Abs. In spleens of HSV-1-infected wild-type mice, the percentage of iNKT cells expressing CCR6, a marker for inflammatory iNKT cells secreting IFN-γ, was significantly decreased at 6 mo postinfection, suggesting that these cells were released from the spleen to other tissues. Finally, in vitro experiments showed that in the absence of CD1d-restricted cells, HSV-1 induced markedly lower IFN-γ production in splenocytes from naive mice. Taken together, our results indicate that iNKT cells shape the Ab response to HSV-1 infection and provide a basis for rational development of antiviral vaccines.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization

The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Vaccination with Recombinant Leishmania donovani Gamma-Glutamylcysteine Synthetase Fusion Protein Protects Against L. donovani Infection

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.

Heat shock protein 110 improves the anti-tumor effects of the cytotoxic T lymphocyte epitope E7 in mice

Several strategies have been used to enhance the vaccine-induced immunity of peptide vaccines and effective therapeutic benefits, including the utilization of heat shock proteins (HSP), especially the HSP70 family. HSP110 exhibits a higher binding affinity with protein and is capable of enhancing the immunogenicity of protein antigens; however, whether HSP110 can also increase the efficiency of peptide vaccine remains unclear. Here, we investigated mHSP110 as a chaperone immunoadjuvant to enhance the immune response to HPV16 oncoprotein E7-derived CTL epitopeE749-57 in a mouse model.We developed the HSP110- E749-57 complex and demonstrated that mHSP110 could form complexes with peptide E749-57 using FITC-labeled E749-57 as the tracer. Inoculation of the mHSP110-E749-57 complex was capable of priming strong epitope-specific immune response as determined by its ability to elicit an epitope-specific splenocytes proliferation and a cytotoxic T cell response, and IFN-γ production in splenocytes. Results also showed that immunization with the mHSP110- E749-57 complex completely protected mice against subsequent challenge with tumor cells. More importantly, immunization of this complex also significantly inhibited the growth of established tumors and prolonged the survival time of the tumor-bearing animals. Thus, mHSP110-E749-57 complex vaccine represents a potentially powerful approach for use in the immunotherapy of cervical cancer associated with HPV16 infection. More importantly, the multi-epitopes derived from E7 and other E proteins can be applied to the strategy described in this study to form a multi-antigenic vaccine to induce an improved anti-tumor immune response to cervical cancer in the future.

Opioid-Like Activity of Naltrexone on Natural Killer Cell Cytolytic Activity and Cytokine Production in Splenocytes: Effects of Alcohol

Chronic alcohol consumption has been shown to decrease the activity of natural killer (NK) cell cytolytic function and the production of various cytokines from the spleen. We have recently shown that naltrexone, an opiate receptor antagonist, when administered for a period of 2 weeks suppresses micro-opiate receptor binding but increases partial differential-opiate receptor activity in rat splenocytes. However, the effects of long-term naltrexone treatment on alcohol-induced alteration of NK cell cytolytic activity and cytokines production in splenocytes have not been determined. Male rats were pair-fed an isocaloric liquid diet or fed an ethanol-containing liquid diet for a period of 3 weeks. These rats were additionally treated after a week with a subcutaneous implant of either a naltrexone pellet or placebo pellet for 2 weeks. Splenocytes were isolated and used for determination of various cytokines interleukin (IL)-2, IL-4, and IL-6, and interferon-gamma (IFN-gamma) using enzyme-linked immunosorbent assay (ELISA), and the basal and IL-2-, IL-12-, or IL-18-induced NK cytolytic activity was measured using a standard 4-h (51)Cr release assay against YAC-1 lymphoma target cells. Ethanol consumption resulted in a reduction of the production of IL-2, IL-4, and IL-6 as well as the basal and cytokine-activated NK cell cytolytic activity and IFN-gamma production in splenocytes. Naltrexone administration increased the production of IL-2, IL-4, and IL-6 and the basal and cytokine-activated NK cell cytolytic activity and IFN-gamma production in the splenocytes of pair-fed and alcohol-fed rats. These results indicated that naltrexone treatment increases NK cell cytolytic activity and cytokine production in the spleen in vivo. Furthermore, these results identify the potential of the use of naltrexone in the treatment of immune deficiency in alcoholic and non-alcoholic patients.

Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses

Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-γ production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-β mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen.

Co-delivery of lymphopoietic cytokines in live Salmonella vaccines enhances the T cell response (47.17)

Abstract Live bacteria vaccine S. Typhi elicits strong mucosal and systemic immune responses. It is useful as a live vector to deliver heterologous antigens. Additionally, delivery of a eukaryotic expression vector by attenuated Salmonella leads to temporary expression of the coded gene in host cells after invasion. Because Salmonella invades preferentially macrophages and dendritic cells, the live bacteria could also be used to deliver immunomodulatory molecules to antigen-presenting cells. We hypothesize that co-delivery of lymphopoietic cytokine such as IL-2, IL-7 and IL-15, which has potential in promoting immune memory, enhances the efficacy of vaccination. Murine IL-2, IL-7 and IL-15 were cloned into eukaryotic expression vector pCMS-EGFP, and introduced into S. Typhi(F1), which carries Y. pestis protective antigen F1. Mice were immunized intranasally with above derivative strains, and boosted with purified antigen F1 in alum adjuvant Alhydrogel. This prime-boost strategy elicited strong mucosal and systemic antibody responses, as well as cell-mediated responses, e.g. antigen-specific proliferation and IFN-γ production of splenocytes. Inclusion of cytokine, however, further enhanced the cell-mediated responses. Antibody responses were not affected. Our data strongly support our hypothesis that production of lymphopoietic cytokine during priming vaccination promotes memory T cell responses.

DNA immunization with 2C FMDV non-structural protein reveals the presence of an immunodominant CD8 +, CTL epitope for Balb/c mice

Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-γ) induction in CD8 +, CD44 hi splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-γ production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses.

A novel intranasal Protollin ™-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin ™ ( Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG 1:IgG 2a ratios and MV-specific IFNγ production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced T H1/T H2-type response.

Construction of Recombinant BCGs Overexpressing Antigen 85 Complex and Their Protective Efficacy againstMycobacterium tuberculosisInfection in a Mouse Model

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate BCG vaccine is urgently needed. We constructed three recombinant Mycobacterium bovis BCG (rBCG) strains over-expressing antigen (Ag) 85A, Ag85B, or both of M. tuberculosis using their own promoter and secretory sequence, or hsp60 promoter. SDS-PAGE analysis of rBCG proteins showed overexpression of Ag85A and Ag85B proteins in higher level than of those in their parental strain of BCG. In addition, rBCG(rBCG/B.FA) over-expressing Ag85A and Ag85B induced strong IFN-γ production in splenocytes. However, there was no significant difference in protective efficacy between rBCG and their parental BCG strain. In this study, therefore, rBCG over-expressing Ag85A, Ag85B, or both failed to show enhanced protection against M. tuberculosis infection in a mouse model. (Tuberc Respir Dis 2004; 57:125-131)