Effective Cell-mediated Immunity Research Articles

A VCG-Based Multiepitope Chlamydia Vaccine Incorporating the Cholera Toxin A1 Subunit (MECA) Confers Protective Immunity Against Transcervical Challenge

Background/Objectives: We generated a novel recombinant Vibrio cholerae ghost (rVCG)-based subunit vaccine incorporating the A1 subunit of cholera toxin (CTA1) and a multiepitope Chlamydia trachomatis (CT) antigen (MECA) derived from five chlamydial outer membrane proteins (rVCG-MECA). The ability of this vaccine to protect against a CT transcervical challenge was evaluated. Methods: Female C57BL/6J mice were immunized thrice at two-week intervals with rVCG-MECA or rVCG-gD2 (antigen control) via the intramuscular (IM) or intranasal (IN) route. PBS-immunized mice or mice immunized with live CT served as negative and positive controls, respectively. Results: Vaccine delivery stimulated robust humoral and cell-mediated immune effectors, characterized by local mucosal and systemic CT-specific IgG, IgG2c, and IgA antibody and IFN-γ (Th1 cytokine) responses. The elicited mucosal and systemic IgG2c and IgA antibody responses persisted for 16 weeks post-immunization. Immunization with rVCG-MECA afforded protection comparable to that provided by IN immunization with live CT EBs without any side effects, irrespective of route of vaccine delivery. Conclusions: The results underline the potential of a multiepitope vaccine as a promising resource for protecting against CT genital infection and the potential of CTA1 on the VCG platform as a mucosal and systemic adjuvant for developing CT vaccines.

Brief Report: Detection of Urine Lipoarabinomannan Is Associated With Proinflammatory Innate Immune Activation, Impaired Host Defense, and Organ Dysfunction in Adults With Severe HIV-Associated Tuberculosis in Uganda.

The immunopathology of disseminated HIV-associated tuberculosis (HIV/TB), a leading cause of critical illness and death among persons living with HIV in sub-Saharan Africa, is incompletely understood. Reflective of hematogenously disseminated TB, detection of lipoarabinomannan (LAM) in urine is associated with greater bacillary burden and poor outcomes in adults with HIV/TB. We determined the relationship between detection of urine TB-LAM, organ dysfunction, and host immune responses in a prospective cohort of adults hospitalized with severe HIV/TB in Uganda. Generalized additive models were used to analyze the association between urine TB-LAM grade and concentrations of 14 soluble immune mediators. Whole-blood RNA-sequencing data were used to compare transcriptional profiles between patients with high- vs. low-grade TB-LAM results. Among 157 hospitalized persons living with HIV, 40 (25.5%) had positive urine TB-LAM testing. Higher TB-LAM grade was associated with more severe physiologic derangement, organ dysfunction, and shock. Adjusted generalized additive models showed that higher TB-LAM grade was significantly associated with higher concentrations of mediators reflecting proinflammatory innate and T-cell activation and chemotaxis (IL-8, MIF, MIP-1β/CCL4, and sIL-2Ra/sCD25). Transcriptionally, patients with higher TB-LAM grades demonstrated multifaceted impairment of antibacterial defense including reduced expression of genes encoding cytotoxic and autophagy-related proteins and impaired cross-talk between innate and cell-mediated immune effectors. Our findings add to emerging data suggesting pathobiological relationships between LAM, TB dissemination, innate cell activation, and evasion of host immunity in severe HIV/TB. Further translational studies are needed to elucidate the role for immunomodulatory therapies, in addition to optimized anti-TB treatment, in this often critically ill population.

Breakthrough infections due to SARS-CoV-2 Delta variant: relation to humoral and cellular vaccine responses.

COVID-19 vaccines are expected to provide effective protection. However, emerging strains can cause breakthrough infection in vaccinated individuals. The immune response of vaccinated individuals who have experienced breakthrough infection is still poorly understood. Here, we studied the humoral and cellular immune responses of fully vaccinated individuals who subsequently experienced breakthrough infection due to the Delta variant of SARS-CoV-2 and correlated them with the severity of the disease. In this study, an effective humoral response alone was not sufficient to induce effective immune protection against severe breakthrough infection, which also required effective cell-mediated immunity to SARS-CoV-2. Patients who did not require oxygen had significantly higher specific (p=0.021) and nonspecific (p=0.004) cellular responses to SARS-CoV-2 at the onset of infection than those who progressed to a severe form. Knowing both humoral and cellular immune response could allow to adapt preventive strategy, by better selecting patients who would benefit from additional vaccine boosters. https://clinicaltrials.gov, identifier NCT04355351; https://clinicaltrials.gov, identifier NCT04429594.

Effect of iron deficiency on cell-mediated immunity in adults

Background Iron plays a vital role in immune system development, integrity, as well as proper function through its growth-promoting and differentiation-inducing properties in immune cells and its interference with cell-mediated immune effector pathways and cytokine activities. In the past few decades, several studies have found that iron deficiency markedly affects the immune system. However, data on the effect of iron deficiency on immune function are still confusing and contradictory. Aim To assess the effect of iron-deficiency anemia on cell-mediated immunity. Patients and methods The percentage and count of T-lymphocytes (CD3+) and their CD4+ and CD8+ subsets and CD4+/CD8+ ratio were evaluated in 25 iron-deficient and 15 healthy adult women. The effect of iron therapy on the same parameters was also assessed in patients after correction of anemia. Results In our study, there was no statistically significant difference between patient and control groups regarding CD3+, CD4+, and CD8+ cell counts and percentages and CD4+/CD8+ ratio, with no statistically significant difference after iron therapy. However, it was found that serum ferritin level was significantly positively correlated with CD4+/CD8+ ratio and CD4+ cell percentage and absolute count and significantly negatively correlated with CD8+ cell percentage. Conclusion Iron deficiency has no effect on cell-mediated immunity with respect to quantitative changes in T-lymphocyte subsets and CD4/CD8 ratio.

Development of alternative vaccination strategies to overcome Tc1 vaccine failure caused by chronic schistosomiasis

Abstract It is a global public health imperative to develop vaccine strategies that produce effective cell-mediated immunity in schistosome-endemic areas, such as sub-Saharan Africa, where HIV infection is common. Vaccine delivery techniques effective in immune-suppressed populations are critical to combating infectious diseases in these already compromised groups. It is well-known that immune suppression, such as by chronic schistosome infection, can cause Tc1/Th1 type vaccines to fail. Listeria, a strong inducer of cell-mediated immunity, can overcome the Th2 biasing and immune suppression caused by schistosome infection. However, safety concerns cause reluctance among the vaccine community and the public to incorporate this vector into immunization programs. Since neither DNA nor the Listeria vaccine vector are ideal for clinical translation, and Gag is likely not the ideal HIV vaccine antigen, we are using this system as a model for enumeration of Tc1 responses (IFNγ+ CD8+ cytotoxic T lymphocytes, CTL); CTLs are likely necessary components of functional responses for next-generation vaccines targeting diseases that currently lack effective vaccines (HIV, TB, malaria). Here, we investigate why DNA vaccine vectors fail during chronic schistosomiasis when Listeria vaccine vectors can generate robust CTL responses to HIV Gag. Specifically, we are examining live bacteria as adjuvants for an episomally expressed or concurrently administered DNA vaccine for HIV Gag. This project is one step toward our overall goal, which is to generate cell-mediated vaccine immunity in schistosome-infected, immune-suppressed patients.

STRICT VEGAN DIET LEADING TO MALNUTRITION AND ACTIVE TB

SESSION TITLE: Monday Fellow Case Report Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: Cell mediated immunity is the basis of the body’s defense mechanisms against mycobacterium tuberculosis. Effective cell mediated immunity requires effective adequate phagocytosis and T-cell function. Vitamin B12 and Vitamin D deficiency has been linked to impaired oxygen burst reaction, which is needed for effective phagocytosis. Experimental data has shown protein energy malnutrition to impair T-cell mediated immune pathways. Strict vegan and vegetarian diets are known to be lacking in cobalamin, protein and vitamin D. CASE PRESENTATION: A 40 yr old HIV negative Caucasian female presented to pulmonary clinic for evaluation of chronic cough. Social history revealed adherence to a strict vegan diet for many years. CT scan of the chest revealed consolidation in the superior and anterobasal segments of the right lower lobe. Bronchoscopy revealed mucoid secretions in the superior segment of the right upper lobe with associated edema. As well as thick whitish mucus in the superior segment of the right lower lobe. BAL was performed and was positive for AFB in two smears in keeping with the diagnosis of acute MTB infection. The patient was admitted for initiation of RIPE therapy. Albumin levels were noted to be 2.8 g/dl during her admission and Vitamin D was noted to be 29ng/ml. Hain testing showed pryrazinamide and isoniazid resistance. Sputum conversion by smear and culture occurred in week 20 of treatment. DISCUSSION: Vit B12 deficiency, was hypothesized to contribute to increased susceptibility of vegetarian Asiatic Indians to active MTB. The prevalence of MTB infection was noted to be higher in strict vegetarians when compared to lactovegetarians and omnivores. Exclusion of diary products in the vegan diet further predisposes patients to Vit B 12 deficiency. Vitamin D deficient patients have been shown to have greater susceptibility to MTB and worse disease course prognosis. There is evidence to suggest that a fall in serum Vitamin D levels can decrease cell mediated immunity. Some have suggested a dual role of Vit D supplementation in treating MTB infection. In the setting of extended drug resistant and multidrug resistant TB low BMI and hypoalbuminemia have been associated with poorer treatment outcomes. Hypoalbuminemia independently is associated with increased in hospital mortality in patients with TB. Our patient had both low BMI and hypoalbuminemia at the time of presentation. Hind testing revealed poly-resistant TB (PRTB). It is unclear in this case how much her dietary restrictions may have contributed to her vitamin deficiencies and predisposition to conversion to active TB CONCLUSIONS: Further research is needed into the effects of the vegan diet on the immune system and how these changes may alter the course of TB infection Reference #1: Vegetarian diet as a risk factor for tuberculosis in immigrant south London Asians. - PubMed - NCBI [Internet]. [cited 2018 Jul 26]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/7701458 Reference #2: Kearns MD, Tangpricha V. The role of vitamin D in tuberculosis. J Clin Transl Endocrinol [Internet]. 2014 Aug 23 [cited 2018 Dec 12];1(4):167–9. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684962/ Reference #3: Vegetarian diet as a risk factor for tuberculosis in immigrant south London Asians. - PubMed - NCBI [Internet]. [cited 2018 Jul 26]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/7701458 DISCLOSURES: No relevant relationships by Armando Cabrera Alonso, source=Web Response No relevant relationships by Zeeshan Chauhan, source=Web Response No relevant relationships by Usha Deonarine, source=Web Response No relevant relationships by Gustavo Ferrer, source=Web Response No relevant relationships by Hector Vazquez Saad, source=Web Response

Quantification of Nitric Oxide and Reactive Oxygen Species in Leishmania-infected J774A.1 Macrophages as a Response to the in vitro Treatment with a Natural Product Compound

Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various mechanisms to evade the immune system and multiply inside the host. Among the main factors of the immunity that are recruited to confront the Leishmania infection are the macrophages (MΦs) that produce effector molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS). Therefore, efficient drug agents should combine the antileishmanial effect of these gaseous transmitters along with the enhancement of the host's adaptive immunity. In the quest of therapeutic alternatives, natural products have been extensively studied and are considered as candidate antileishmanial agents since they exhibit specific properties in modulating the host's immune response towards an effective anti-leishmanial cell-mediated immunity capable to eliminate parasitic dissemination. In the current protocol, Leishmania-infected MΦs (J774A.1 cell line) that have been treated with various increasing concentrations of a natural compound, are tested for the production of the aforementioned molecules. In order to detect NO production, we employ the Griess colorimetric nitrite assay and quantification relies on the construction of an accurate standard curve using appropriate standards of known concentration. ROS detection and quantification is achieved by flow cytometry and relies on the use of carboxy-H2DCFDA, an indicator that converts to a fluorescent form when interacts with ROS molecules.

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses.

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.

Immune response and protective effect against chronic Toxoplasma gondii infection induced by vaccination with a DNA vaccine encoding profilin

BackgroundToxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals. T. gondii profilin (TgPF) plays a crucial role in parasite motility and host cell invasion, and has shown promise against toxoplasmosis. DNA vaccine was considered to elicit effective humoral and cell-mediated immunity against T. gondii infection. The objective of the present study was to evaluate the immunogenicity of TgPF in mice using a DNA vaccination strategy.MethodsA DNA vaccine (pVAX-PF) encoding TgPF gene was constructed and then was intramuscularly injected into mice with and without a plasmid encoding IL-15 (pVAX-IL-15). The immune responses in immunized Kunming mice including lymphocyte proliferation, levels of cytokines, antibody titers and T lymphocyte subclasses were analyzed. The protective efficacy against chronic T. gondii infection was observed at 4 weeks post-infection with the cyst-forming PRU strain of T. gondii (Genotype II).ResultsEitherpVAX-PF with or without pVAX-IL-15 could elicit higher level of IgG and IgG2a antibodies and produce strong cellular immune responses in the immunized mice. The brain cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 (1843 ± 215.7) and pVAX-PF (1897 ± 337.8) were reduced 40.82% and 39.08%, respectively, compared to that in mice received nothing (3114 ± 168.8), and the differences were statistically significant (P < 0.0001). However, the T. gondii cyst numbers in mice immunized with pVAX-PF + pVAX-IL-15 were not statistically significantly different compared to that in mice immunized with pVAX-PF alone [t(10) = 0.33, P > 0.05].ConclusionsThe present study indicated that TgPF could be a promising vaccine candidate against chronic toxoplasmosis, which can be further used to develop multi-epitope vaccine formulations in food-producing animals against T. gondii infection.

Bifidobacteria and Their Molecular Communication with the Immune System.

Bifidobacterium represents a genus within the phylum Actinobacteria which is one of the major phyla in the healthy intestinal tract of humans. Bifidobacterium is one of the most abundant genera in adults, but its predominance is even more pronounced in infants, especially during lactation, when they can constitute the majority of the total bacterial population. They are one of the pioneering colonizers of the early gut microbiota, and they are known to play important roles in the metabolism of dietary components, otherwise indigestible in the upper parts of the intestine, and in the maturation of the immune system. Bifidobacteria have been shown to interact with human immune cells and to modulate specific pathways, involving innate and adaptive immune processes. In this mini-review, we provide an overview of the current knowledge on the immunomodulatory properties of bifidobacteria and the mechanisms and molecular players underlying these processes, focusing on the corresponding implications for human health. We deal with in vitro models suitable for studying strain-specific immunomodulatory activities. These include peripheral blood mononuclear cells and T cell-mediated immune responses, both effector and regulatory cell responses, as well as the modulation of the phenotype of dendritic cells, among others. Furthermore, preclinical studies, mainly germ-free, gnotobiotic, and conventional murine models, and human clinical trials, are also discussed. Finally, we highlight evidence supporting the immunomodulatory effects of bifidobacterial molecules (proteins and peptides, exopolysaccharides, metabolites, and DNA), as well as the role of bifidobacterial metabolism in maintaining immune homeostasis through cross-feeding mechanisms.

Impact of heat shock protein 60KD in combination with outer membrane proteins on immune response against Brucella melitensis.

Brucellosis caused by the bacterium Brucella affects various domestic and wild species. The outer membrane proteins 25 and 31 play key roles on stimulation of cell-mediated immune response against Brucella. GroEL as one of the major Brucella antigens stimulates the immune system and increases intracellular survival of bacteria. In the present study, we assumed injection of GroEL in combination with OMP25 and OMP31 would offer higher immunity levels. So, the impact of GroEL with different concentrations of recombinant outer membrane proteins emulsified in Chitosan Nanoparticles on immune responses was evaluated in mice model. Results showed both univalent (except rGroEL) and divalent immunized groups induced higher IFN-γ, TNF-α, and IL-4 titers in comparison to negative control groups. While GroEL showed negative effect on TNF-α titer, there were positive increase trends in IFN-γ in some treatments. Analysis of humoral antibody response revealed both univalent and divalent immunized groups induced higher IgG2a titer than IgG1 titer, indicating strong bent of Th1 immune response. Also, results showed GroEL can have positive impact on lymphocyte proliferation response. Overall, mice immunization using individual OMP25 or OMP31 demonstrated more effective cell-mediated immunity, although some combinations of rGroEL and rOMP31 vaccines were more efficient than other divalent ones.

Profiling the Targets of Protective CD8+ T Cell Responses to Infection

T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

Rabies Virus Antibodies from Oral Vaccination as a Correlate of Protection against Lethal Infection in Wildlife.

Both cell-mediated and humoral immune effectors are important in combating rabies infection, although the humoral response receives greater attention regarding rabies prevention. The principle of preventive vaccination has been adopted for strategies of oral rabies vaccination (ORV) of wildlife reservoir populations for decades to control circulation of rabies virus in free-ranging hosts. There remains much debate about the levels of rabies antibodies (and the assays to measure them) that confer resistance to rabies virus. In this paper, data from published literature and our own unpublished animal studies on the induction of rabies binding and neutralizing antibodies following oral immunization of animals with live attenuated or recombinant rabies vaccines, are examined as correlates of protection against lethal rabies infection in captive challenge settings. Analysis of our studies suggests that, though serum neutralization test results are expected to reflect in vivo protection, the blocking enzyme linked immunosorbent assay (ELISA) result at Day 28 was a better predictor of survival. ELISA kits may have an advantage of greater precision and ability to compare results among different studies and laboratories based on the inherent standardization of the kit format. This paper examines current knowledge and study findings to guide meaningful interpretation of serology results in oral baiting monitoring.

CD8+T cells with distinct cytokine‐producing features and low cytotoxic activity in eosinophilic and non‐eosinophilic chronic rhinosinusitis with nasal polyps

CD8(+) T cells are important effectors of cell-mediated immunity; however, their contribution to the pathogenesis of CRS is unclear. This study aimed to characterize the cytokine-producing features and cytotoxic activity of CD8(+) T cells, and their correlation with inflammation patterns in CRS with nasal polyps. The expression of IFN-γ, IL-4, IL-5, IL-17A, forkhead box P3 (FOXP3), perforin, and granzyme B in CD8(+) T cells was studied by means of flow cytometry, immunohistochemistry, and immunofluorescence. The expression of CD8(+) T-cell subset relevant chemokines and chemokine receptors was detected by means of real-time RT-PCR or ELISA. The cytotoxic activity of sorted CD8(+) T cells was defined by anti-CD3-redirected killing assay. Compared with controls, elevated percentages of total CD8(+) T cells and cytotoxic T lymphocyte (Tc) 1 (IFN-γ(+) ), Tc2 (IL-4(+) ), and Tc17 (IL-17A(+) ) cell subset, and decreased percentages of FOXP3(+) CD8(+) regulatory T cells, were found in both eosinophilic and non-eosinophilic polyps with a Tc2-skewed and Tc1/Tc17-dominated response in eosinophilic and non-eosinophilic polyps, respectively. Nasal CD8(+) T cells were found to produce similar or even higher levels of IFN-γ and IL-4 compared with CD4(+) T cells. Tc1 and Tc17, and Tc2 (IL-4(+) and IL-5(+) ) cell subset percentages positively correlated with neutrophil and eosinophil counts in sinonasal mucosa, respectively. Strikingly, the expression of perforin and granzyme B and cytotoxic activity were significantly reduced in nasal CD8(+) T cells compared with their counterparts in peripheral blood. The expression of CXCL16, CCL17, and CCL20 positively correlated with Tc1, Tc2, and Tc17 cell subset number in sinonasal mucosa, respectively. CD8(+) T cells have low cytotoxic activity; nevertheless, they are a significant and previously underappreciated source of inflammatory cytokine production in polyps. Different Tc cell subset domination may contribute to distinctly biased granulocyte inflammation in eosinophilic and non-eosinophilic polyps.

Generation of interleukin-13 receptor alpha2 antigen expressing modified vaccinia ankara recombinant virus for potential cancer immunotherapy

Genetically modified recombinant poxviruses have shown promise in preclinical models of cancer immunotherapy due to their ability to induce effective cell-mediated immunity against target tumor-associated antigens (TAA). One such vector, recombinant Modified Vaccinia Ankara (MVA), is capable of expressing foreign genes in infected host cells. MVA is replication restricted in most mammalian cells exemplifying a unique safety profile. We have demonstrated that the interleukin-13 receptor α2 (IL-13Rα2) is selectively expressed in various solid tumors but not in normal tissues making it a promising TAA. Prophylactic and therapeutic vaccination with a plasmid vector expressing IL-13Rα2 caused only partial regression of established tumors [1], suggesting that host immune responses against IL-13Rα2 needed further enhancement. Thus, we constructed a recombinant MVA (rMVA-IL13Rα2) expressing both IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by immunostaining using anti-vaccinia antibody and anti-IL-13Rα2 antibody confirmed the identity and purity of the recombinant MVA. Western Blot analysis showed the presence of IL-13Rα2 protein (65 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells infected with rMVA-IL13Rα2 virus (T98G-IL13Rα2) demonstrated surface expression of IL-13Rα2, indicating the infectivity potential of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0-100 ng/ml) of IL13-PE (interleukin-13 fused to truncated Pseudomonas exotoxin [2] resulted in depletion of GFP+ T98G-IL13Rα2 cells in a concentration-dependent manner. Higher concentrations of IL13-PE (10-1000 ng/ml) also inhibited the protein synthesis in T98G-IL13Rα2 compared to cells infected with control pLW44-MVA. We further observed that IL13-PE treatment of rMVA-IL13Rα2 infected chicken fibroblast, DF-1 cells led to a reduction in virus titer compared to untreated cells. These results indicate that rMVA-IL13Rα2 virus can successfully infect mammalian cells and express IL-13Rα2 in a biologically active form on the cell surface. The immunization studies of rMVA-IL13Rα2 are ongoing in a syngeneic mouse model of metastatic breast carcinoma. Based on in vitro results, we expect the rMVA-IL13Rα2 to be a useful agent in tumor immunotherapy as a vaccine alone and in combination with other therapeutic agents to eradicate metastatic tumors.

PKCθ Regulates T Cell Motility via Ezrin-Radixin-Moesin Localization to the Uropod

Cell motility is a fundamental process crucial for function in many cell types, including T cells. T cell motility is critical for T cell-mediated immune responses, including initiation, activation, and effector function. While many extracellular receptors and cytoskeletal regulators have been shown to control T cell migration, relatively few signaling mediators have been identified that can modulate T cell motility. In this study, we find a previously unknown role for PKCθ in regulating T cell migration to lymph nodes. PKCθ localizes to the migrating T cell uropod and regulates localization of the MTOC, CD43 and ERM proteins to the uropod. Furthermore, PKCθ-deficient T cells are less responsive to chemokine induced migration and are defective in migration to lymph nodes. Our results reveal a novel role for PKCθ in regulating T cell migration and demonstrate that PKCθ signals downstream of CCR7 to regulate protein localization and uropod formation.

The prolactin-inducible-protein (PIP): A regulatory molecule in adaptive and innate immunity

The Prolactin-inducible-protein (PIP)/Gross Cystic Disease Fluid Protein-15 (GCDFP-15) gene is highly expressed in salivary, lacrimal and sweat glands and the protein abundantly found in the secretions that originate from these glands; saliva, tears and sweat. PIP is thus considered to be strategically located at sites viewed as the first port of entry for invading organisms. PIP is also found over-expressed under abnormal and pathological conditions of the breast and prostate. The function of PIP has yet to be defined but it has been implicated to play a role in immunity, with respect to bacterial and viral infection, cancer and fertility. Despite such predictive functions, there is still no clear demonstration of an immunoregulatory role for PIP. In this review we will focus on accumulating evidence that suggests a role for PIP in both innate and adaptive immunity. Moreover, we will discuss recent evidence that defines a modulatory role for PIP with regards to a CD4+ T cell immune response, identifying for the first time, a critical role for PIP in effective cell-mediated immunity against an intracellular pathogen.

Prognostic Significance of Immunohistochemical Phenotypes in Patients Treated for High-Grade Cervical Intraepithelial Neoplasia

Strong evidence exists that the host's immune system plays a crucial role for the development of human papillomavirus-related cervical premalignant and malignant lesions. In particular, effective cell-mediated immunity (CMI) promotes spontaneous infection clearance and cancer precursors regression in healthy subjects, while immunosuppressed individuals are more likely to experience infection persistence, cervical intraepithelial neoplasia (CIN) lesions, and cervical cancer. In this study, the prognostic significance of immunohistochemical profiling of CD4+ T-cells, CD8+ T-cells, dendritic cells (CD11c+), T-bet+, and GATA-3+ transcription factors has been studied in surgical specimens of 34 consecutive women affected by high-grade cervical intraepithelial neoplasia (CIN2-3) submitted to cervical conization. Results have been correlated with the clinical outcomes at 24 months after treatment and statistically analyzed. Higher rates of CD4+ T-cells, CD11c+ dendritic cells, and T-bet+ transcription factor positivity showed a strong statistically significative correlation with favourable clinical outcomes (P ≤ 0.0001). These data reinforce the evidence of the relevance of the host's immune status in the natural history of HPV-related cervical disease and add a prognostic significance of the cervical immunological profile in terms of predicting significant lower recurrence rates.

Evaluation of Immunological Changes in Patients Treated With SBRT and Radiation Therapy -- A Prospective Cohort Study

Evaluation of Immunological Changes in Patients Treated With SBRT and Radiation Therapy -- A Prospective Cohort Study

HIV vaccines: progress to date.

The quest for an effective and safe HIV-1 vaccine has been and still is the aspiration of many scientists and clinicians worldwide. Until recently, the hopes for an effective vaccine were thwarted by the disappointing results and early termination in September 2007 of the STEP study, which saw a subgroup of male vaccine recipients at an increased risk of HIV-1 infection, and the failure of earlier trials of vaccines based on recombinant envelope proteins to provide any level of protection. The results of the STEP study raised important questions in the field of HIV vaccines, including the use of recombinant adenovirus vectors as immunogens, the rationale for the development of T-cell-based vaccines and the development pathway for these vaccines, in terms of assessment of immunogenicity and the challenge models used. The study of neutralizing antibodies has demonstrated that the induction of high-titre, broadly neutralizing antibodies in the majority of recipients is likely to be highly problematic. However, the results of the RV144 Thai trial released in September 2009 have brought new optimism to the field. This study employed envelope-based immunogens delivered as a priming vaccination with a recombinant poxvirus vector and boosting with recombinant proteins. This regimen provided modest protection to HIV-1 infection in a low-risk population. Although the correlates of protection are currently unknown, extensive studies are underway to try to determine these. Neutralizing antibodies were not induced in the RV144 study; however, considerable titres of binding antibodies to HIV-1 viral envelope (Env) were. It is speculated that these antibodies may have provided a means of protection by a mechanism such as antibody-dependent cell-mediated cytotoxicity. In addition, no CD8+ T-cell responses were induced, but robust CD4+ T-cell responses were, and correlates of protection are being sought by analysing the quality of this aspect of the vaccine-induced immune response. The current paradigm for an optimal HIV-1 vaccine is to design immunogens and vaccination protocols that allow the induction of both broadly neutralizing humoral and broadly reactive and effective cell-mediated immunity, to act at sites of possible infection and post-infection, respectively. However, this is challenged by the results of the RV144 trial as neither of these responses were induced but modest protection was observed. Understanding the biology and immunopathology of HIV-1 early following infection, its modes of transmission and the human immune system's response to the virus should aid in the rational design of vaccines of increased efficacy.