Antibody-dependent Mechanisms Research Articles

Pan-Influenza A Protection by Prime-Boost Vaccination with Cold-Adapted Live-Attenuated Influenza Vaccine in a Mouse Model.

Influenza virus infections continually pose a major public health threat with seasonal epidemics and sporadic pandemics worldwide. While currently licensed influenza vaccines provide only strain-specific protection, antigenic drift and shift occasionally render the viruses resistant to the host immune responses, which highlight the need for a vaccine that provides broad protection against multiple subtypes. In this study, we suggest a vaccination strategy using cold-adapted, live attenuated influenza vaccines (CAIVs) to provide a broad, potent, and safe cross-protection covering antigenically distinct hemagglutinin (HA) groups 1 and 2 influenza viruses. Using a mouse model, we tested different prime–boost combinations of CAIVs for their ability to induce humoral and T-cell responses, and protective efficacy against H1 and H5 (HA group 1) as well as H3 and H7 (HA group 2) influenza viruses. Notably, even in the absence of antibody-mediated neutralizing activity or HA inhibitory activity in vitro, CAIVs provided a potent protection against heterologous and heterosubtypic lethal challenges in vivo. Heterologous combination of prime (H1)–boost (H5) vaccine strains showed the most potent cross-protection efficacy. In vivo depletion experiments demonstrated not only that T cells and natural killer cells contributed to the cross-protection, but also the involvement of antibody-dependent mechanisms for the cross-protection. Vaccination-induced antibodies did not enhance the infectivity of heterologous viruses, and prime vaccination did not interfere with neutralizing antibody generation by the boost vaccination, allaying vaccine safety concerns associated with heterogeneity between the vaccines and challenge strains. Our data show that CAIV-based strategy can serve as a simple but powerful option for developing a “truly” universal influenza vaccine providing pan-influenza A protection, which has not been achieved yet by other vaccine strategies. The promising results of potency, breadth, and safety demonstrated in the mouse model support further studies in higher animal models for clinical relevance.

A Case of Type 2 Hypersensitivity to Rasburicase Diagnosed with a Natural Killer Cell Activation Assay

Drug hypersensitivity reactions can lead to different clinical pictures depending on the underlying immunological mechanism. Diagnosis tests are already available to assess the most frequent drugs hypersensitivity reactions, which are mediated by specific IgE or T cells. However, it remains challenging to diagnose type 2 hypersensitivity reactions (T2HR), which can lead to severe cytopenia and liver failure. Here, we describe a case of T2HR to rasburicase, an uricolytic agent used to prevent tumor lysis syndrome. In this patient, sensitization was associated with the production of specific IgG able to bind to leukocytes. We found that patient NK cells were specifically activated in the presence of rasburicase and autologous serum, which led to exocytosis of lytic granules. This antibody-dependent cell cytotoxicity mechanism may lead to cytopenia observed in the patient. Moreover, this NK cell activation assay could be used to improve the diagnosis of a T2HR to rasburicase and, by extent, to other drugs. These data also suggest that NK cells could play an important role in the pathophysiological mechanism of T2HR.

In vitro inhibition of HIV-1 replication in autologous CD4+ T cells indicates viral containment by multifactorial mechanisms.

HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies (NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells, primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro, which was mediated by natural killer cells (NKs) and dependent on an Fc-Fc receptor interaction. Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.

Enhanced antiproliferative activity of antibody-functionalized polymeric nanoparticles for targeted delivery of anti-miR-21 to HER2 positive gastric cancer.

MiR-21 is an oncogenic miR frequently elevated in gastric cancer. Overexpression of miR-21 decreases the sensitivity of gastric cancer cells to trastuzumab, which is a humanized monoclonal antibody targeting human epidermal growth factor receptor 2. However, optimization of miRNA or its anti-miRNA oligonucleotides (AMOs) for delivery is a challenge. Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including AMOs. This study is a continuation of our earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target gastric cancer cells with HER2 receptor over-expression using anti-miRNA-21 antisense oligonucleotides (AMO-21). The antibody conjugates (HER-PEG-PCL NPs) act against target cells via antibody-dependent mechanisms and also based on encapsutalated AMO-21. X-ray photoelectron spectroscopy validated the presence of trastuzumab on NP surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a stable antibody expression. The cell line specificity, cellular uptake, AMO-21 delivery, and cytotoxicity of the HER-PEG-PCL NPs were investigated. We found that the antibody conjugates significantly enhanced the cellular uptake of NPs. The HER-PEG-PCL NPs effectively suppressed the target miRNA expression in gastric cancer cells, which further up-regulated phosphatase and tensin homolog (PTEN). As a result, the sensitivity of HER2-expressing gastric cancer cells to trastuzumab was enhanced. The approach enhances the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity of immune effector cells. The antitumor effects of AMO-21-HER-PEG-PCL NPs were compared with trastuzumab in xenograft gastric cancer mice. The results provide insight into the biological and clinical potential of targeted AMO-21 delivery using modified trastuzumab for gastric cancer treatment.

Acute myeloid leukemia targets for bispecific antibodies.

Despite substantial gains in our understanding of the genomics of acute myelogenous leukemia (AML), patient survival remains unsatisfactory especially among the older age group. T cell-based therapy of lymphoblastic leukemia is rapidly advancing; however, its application in AML is still lagging behind. Bispecific antibodies can redirect polyclonal effector cells to engage chosen targets on leukemia blasts. When the effector cells are natural-killer cells, both antibody-dependent and antibody-independent mechanisms could be exploited. When the effectors are T cells, direct tumor cytotoxicity can be engaged followed by a potential vaccination effect. In this review, we summarize the AML-associated tumor targets and the bispecific antibodies that have been studied. The potentials and limitations of each of these systems will be discussed.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Efficacy and safety of rituximab in neuromyelitis optica: Review of evidence.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system with preferential involvement in the optic nerve and spinal cord with a widespread spectrum of clinical features; multiple therapeutic agents have been used with different results. Recent evidence points to B-cell-mediated humoral immunity in the pathogenesis of NMO. Rituximab targets the CD20 antigen on B-cells. Treatment leads to profound B-cell depletion, principally over an antibody-dependent cell cytotoxicity mechanism. The aim of our study was to review clinical trials to elucidate the impact of rituximab on the relapse rate, Expanded Disability Status Scale (EDSS), and progression of disability in NMO. We performed a comprehensive review of all studies that evaluated clinical and paraclinical effects of rituximab on NMO. MEDLINE-PubMed, Web of Sciences, EMBASE, and Cochrane databases up to June 2016 included in our searches. In addition, reference lists from articles identified by search as well as a key review article to identify additional articles included in the study. Rituximab targets the CD20 antigen on B-cells and decreases attack frequency and severity in patients with NMO; however, it does not remove attacks, even when modifying treatment to achieve B-cell depletion. Most of the investigations revealed that EDSS significantly in all patients with rituximab treatment will be decreased after treatment with rituximab. No new or enlarged lesions or pathological gadolinium enhancement was observed in serial brain and spinal cord magnetic resonance imaging, except for those observed concomitantly with clinical relapses and the median length of spinal cord lesions was significantly reduced after therapy. Rituximab targets the CD20 antigen and decreases attack frequency and severity in patients with NMO.

Dengue Virus Isolation in Mosquito Aedes albopictus Captured During an Outbreak in Tokyo, 2014, by a Method Relying on Antibody-Dependent Enhancement Mechanism Using FcγR-Expressing BHK Cells.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.

Expression Profile of Human Fc Receptors in Mucosal Tissue: Implications for Antibody-Dependent Cellular Effector Functions Targeting HIV-1 Transmission

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in mucosal transmission and dissemination warrants further mechanistic studies.

Targets and Mechanisms Associated with Protection from Severe Plasmodium falciparum Malaria in Kenyan Children.

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum. Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n = 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P = 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82; P = 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12; P = 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Neuroinflammation: Ways in Which the Immune System Affects the Brain.

Neuroinflammation is the response of the central nervous system (CNS) to disturbed homeostasis and typifies all neurological diseases. The main reactive components of the CNS include microglial cells and infiltrating myeloid cells, astrocytes, oligodendrocytes, and the blood-brain barrier, cytokines, and cytokine signaling. Neuroinflammatory responses may be helpful or harmful, as mechanisms associated with neuroinflammation are involved in normal brain development, as well as in neuropathological processes. This review examines the roles of various cell types that contribute to the immune dysregulation associated with neuroinflammation. Microglia enter the CNS very early in embryonic development and, as such, play an essential role in both the healthy and diseased brain. B-cell diversity contributes to CNS disease through both antibody-dependent and antibody-independent mechanisms. The influences of these B-cell mechanisms on other cell types, including myeloid cells and T cells, are reviewed in relationship to antibody-mediated CNS disorders, paraneoplastic neurological diseases, and multiple sclerosis. New insights into neuroinflammation offer exciting opportunities to investigate potential therapeutic targets for debilitating CNS diseases.

Transitioning from Idiopathic to Explainable Autoimmune Hepatitis.

Autoimmune hepatitis lacks an identifiable cause, and its diagnosis requires the exclusion of etiologically defined diseases that resemble it. Insights into its pathogenesis are moving autoimmune hepatitis from an idiopathic to explainable disease, and the goal of this review is to describe the insights that are hastening this transition. Two types of autoimmune hepatitis are justified by serological markers, but they also have distinctive genetic associations (DRB1 and DQB1 genes) and autoantigens. DRB1 alleles are the principal susceptibility factors in white adults, and a six amino acid sequence encoded in the antigen-binding groove of class II molecules of the major histocompatibility complex can influence the selection of autoantigens. Polymorphisms, including variants of SH2B3 and CARD10 genes, may affect immune reactivity and disease severity. The cytochrome mono-oxygenase, CYP2D6, is the autoantigen associated with type 2 autoimmune hepatitis, and it shares homologies with multiple viruses that might promote self-intolerance by molecular mimicry. Chemokines, especially CXCL9 and CXCL10, orchestrate the migration of effector cells to sites of injury and are associated with disease severity. Cells of the innate and adaptive immune responses promote tissue damage, and possible deficiencies in the number and function of regulatory T cells may facilitate the injurious process. Receptor-mediated apoptosis is the principal mechanism of hepatocyte loss, and cell-mediated and antibody-dependent mechanisms of cytotoxicity also contribute. Insights that explain autoimmune hepatitis will allow triggering exogenous antigens to be characterized, risk management to be improved, prognostic indices to be refined, and site-specific therapeutic interventions to emerge.

Structural biology of antibody recognition of carbohydrate epitopes and potential uses for targeted cancer immunotherapies.

Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very diverse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy.

Diisocyanates, occupational asthma and IgE antibody: implications for hazard characterization.

Sensitization of the respiratory tract by chemicals resulting in rhinitis and asthma is an important occupational health issue. Occupational asthma is associated with significant morbidity and can be fatal. Tests for the identification and characterization of chemicals with the potential to cause sensitization of the respiratory tract are lacking. In spite of sustained interest there are no validated or widely accepted methods available, and this presents toxicologists with a considerable challenge. One important constraint on the development of appropriate testing strategies has been uncertainty and controversy about the immunological mechanisms through which chemicals may induce sensitization of the respiratory tract. By analogy with protein respiratory allergy it is legitimate to consider that IgE antibody-dependent mechanisms may play a pivotal role. However, although many aspects of chemical respiratory allergy are consistent with reactions caused by IgE antibody, uncertainty remains because among patients with occupational asthma caused by chemical respiratory allergens there are commonly a proportion, and sometimes a significant proportion, of subjects that lack detectable IgE antibody. Here we consider the relevance of IgE antibody responses for the development of a chemical respiratory allergy to diisocyanates. A case is made that IgE antibody responses are, either directly or indirectly, closely associated with occupational asthma to the diisocyanates (and to other chemical respiratory allergens). As such the argument is advanced here that IgE antibody represents an appropriate readout for the characterization of chemical respiratory allergens, and that uncertainty about mode of action should no longer represent a hurdle in the development of suitable test methods.

The complement receptors CD46, CD55 and CD59 are regulated by the tumour microenvironment of head and neck cancer to facilitate escape of complement attack

BackgroundMembrane-bound complement restriction proteins (mCRPs) CD46, CD55 and CD59 enable tumour cells to evade complement dependent cytotoxicity and antibody-dependent killing mechanisms. But less is known about the role of these mCRPs in head and neck cancer. MethodsIn this study we determined the expression of the mCRPs on head and neck squamous cell carcinoma (HNSCC) cell lines, on tumour tissue and TDLNs (tumour-draining lymph nodes) as well as on lymphocytes from HNSCC patients. The influence of the HNSCC microenvironment on the mCRP regulation was analysed using Flow Cytometry, Western blotting and small interfering RNAs (siRNA) transfection studies. ResultsWe examined the effects of the HNSCC tumour milieu on the expression levels of CD46, CD55 and CD59. We investigated the susceptibility of HNSCC cells to CDC (complement-dependent cytotoxicity) while silencing the mCRPs. Our results demonstrate a huge influence of the HNSCC tumour microenvironment on the regulation of mCRP expression and show a reciprocal regulation between the different mCRPs themselves. ConclusionsIn summary, our data indicate that HNSCC has evolved different strategies to evade complement attacks and that the tumour microenvironment leads to the enhancement of complement resistance of the surrounding tissue.

Antibody-mediated enhancement of parvovirus B19 uptake into endothelial cells mediated by a receptor for complement factor C1q.

Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors α5β1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection. Importance: Both efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we demonstrated that uptake of B19V into endothelial cells most probably does not rely on the classical receptor-mediated route via the primary B19V receptor P antigen and coreceptors, such as α5β1 integrins, but rather on antibody-dependent mechanisms. Since the strong antibody-dependent enhancement (ADE) of B19V entry requires the CD93 surface protein, it very likely involves bridging of the B19V-antibody complexes to this receptor by the complement factor C1q, leading to enhanced endocytosis of the virus.

Expansion of CD16 positive and negative human NK cells in response to tumor stimulation

NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.

Cytomegalovirus-based cancer vaccines expressing TRP2 induce rejection of melanoma in mice

Cytomegalovirus (CMV) induces strong and long-lasting immune responses, which make it an attractive candidate for a cancer vaccine vector. In this study, we tested whether a tumor antigen expressed in CMV can induce a strong anti-tumor effect. We expressed an unmodified melanoma antigen, mouse tyrosinase-related protein 2 (TRP2), in mouse cytomegalovirus (MCMV). Prophylactic vaccination of the mice with a single dose of MCMV-TRP2 induced rejection of B16 melanoma challenge; therapeutic vaccination with MCMV-TRP2 prolonged the survival of the mice challenged with B16 cells. Additionally, vaccination with MCMV-TRP2 five months before tumor challenge still induced tumor rejection, which indicated that the vaccine induced long-term protection. Furthermore, MCMV-TRP2 protected mice against B16 melanoma challenge regardless of the pre-existing CMV infection. We found that vaccination with MCMV-TRP2 induced long-lasting TRP2 specific antibodies but not CD8 T cells. In addition, depletion of CD4 and CD8 T cells did not compromise the antitumor effect by MCMV-TRP2; while in B cell deficient (μMT) mice, the vaccine lost its antitumor effect. These results indicate that antibodies, not T cells, are important in mediating the antitumor effect during the effector phase by the vaccine. We also made a spread deficient MCMV-TRP2 lacking the essential glycoprotein gL, which showed a similar antitumor effect. In conclusion, our study indicates that tumor antigen (TRP2) expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody-dependent mechanism. Our findings demonstrate that CMV might be a promising vector for the development of cancer vaccines.

Serological characterization of dengue virus infections observed among dengue hemorrhagic fever/dengue shock syndrome cases in upper Myanmar

In Myanmar, dengue fever (DF)/dengue hemorrhagic fever (DHF) is one of the leading causes of morbidity and mortality among children. From Pyinmana Hospital in 2004 and Mandalay Children Hospital in 2006, 160 patients diagnosed clinically to have DHF/dengue shock syndrome (DSS) were examined for immunoglobulin M (IgM) and IgG levels. A focus reduction neutralization test was also used to determine primary or secondary dengue virus (DENV) infection. By using IgM-capture ELISA, 139 cases were confirmed as DENV infections. Of these IgM-positives, 94 samples were collected 7-24 days from the onset of illness, to which 13 (14%) and 81 (86%) were determined to be primary and secondary DENV infections, respectively. The 13 primary DENV infection cases were spread among the various severity groups (DHF grade I-IV and DSS) and represented age groups ranging from <1 year of age to 9 years of age. The patients in these primary infection cases showed a remarkably high IgM with a low IgG titer response compared with the secondary infection cases. No significant differences were observed in IgG titers with clinical severity. The data obtained in this study suggest that primary DENV infection cases exist certainly among DHF/DSS cases in Myanmar, and that additional mechanism(s) aside from the antibody-dependent enhancement mechanism could have influenced the clinical severity in DHF/DSS cases.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.