Glycosphingolipids promote pro-atherogenic pathways in the pathogenesis of hyperglycemia-induced accelerated atherosclerosis.

INTRODUCTION: Three out of four people with diabetes will die of cardiovascular disease. However, the molecular mechanisms by which hyperglycemia promotes atherosclerosis, the major underlying cause of cardiovascular disease, are not clear. OBJECTIVES: Three distinct models of hyperglycemia-associated accelerated atherosclerosis were used to identify commonly altered metabolites and pathways associated with the disease. METHODS: Normoglycemic apolipoprotein-E-deficient mice served as atherosclerotic control. Hyperglycemia was induced by multiple low-dose streptozotocin injections, or by introducing a point-mutation in one copy of insulin-2 gene. Glucosamine-supplemented mice, which experience accelerated atherosclerosis to a similar extent as hyperglycemia-induced models without alterations in glucose or insulin levels, were also included in the analysis. Untargeted plasma metabolomics were used to investigate hyperglycemia-associated accelerated atherosclerosis in three disease models. The effect of specific significantly altered metabolites on pro-atherogenic processes was investigated in cultured human vascular cells. RESULTS: Hyperglycemic and glucosamine-supplemented mice showed distinct metabolomic profiles compared to controls. Meta-analysis of three disease models revealed 62 similarly altered metabolite features (FDR-adjusted p < 0.05). Identification of shared metabolites revealed alterations in glycerophospholipid and sphingolipid metabolism, and pro-atherogenic processes including inflammation and oxidative stress. Post-multivariate and pathway analyses indicated that the glycosphingolipid pathway is strongly associated with hyperglycemia-induced accelerated atherosclerosis in these atherogenic mouse models. Glycosphingolipids induced oxidative stress and inflammation in cultured human vascular cells. CONCLUSION: Glycosphingolipids are strongly associated with hyperglycemia-induced accelerated atherosclerosis in three distinct models. They also promote pro-atherogenic processes in cultured human cells. These results suggest glycosphingolipid pathway may be a potential therapeutic target to block or slow atherogenesis in diabetic patients.

Glucosamine induces ER stress by disrupting lipid-linked oligosaccharide biosynthesis and N-linked protein glycosylation.

Glucosamine is an essential substrate for N-linked protein glycosylation. However, elevated levels of glucosamine can induce endoplasmic reticulum (ER) stress. Glucosamine-induced ER stress has been implicated in the development of diabetic complications, including atherosclerosis and hepatic steatosis. In this study, we investigate the potential relationship between the effects of glucosamine on lipid-linked oligosaccharide (LLO) biosynthesis, N-linked glycosylation, and ER homeostasis. Mouse embryonic fibroblasts (MEFs) were cultured in the presence of 0-5 mM glucosamine for up to 18 h, and LLO biosynthesis was monitored by fluorescence-assisted carbohydrate electrophoresis. ER stress was determined by quantification of unfolded protein response (UPR) gene expression. We found that exposure of MEFs to ≥1 mM glucosamine significantly impaired the biosynthesis of mature (Glc3Man9GlcNAc2) LLOs before the activation of the UPR, which resulted in the accumulation of an LLO intermediate (Man3GlcNAc2). The addition of 4-phenylbutyric acid (4-PBA), a chemical chaperone, was able to alleviate ER stress but did not rescue LLO biosynthesis. Other ER stress-inducing agents, including dithiothreitol and thapsigargin, had no effect on LLO levels. Together, these data suggest that elevated concentrations of glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. We hypothesize that this pathway represents a causative link between hyperglycemia and the development of diabetic complications.

Rotavirus NSP6 localizes to mitochondria via a predicted N-terminal a-helix.

Specific roles have been ascribed to each of the 12 known rotavirus proteins apart from the non-structural protein 6 (NSP6). However, NSP6 may be present at sites of viral replication within the cytoplasm. Here we report that NSP6 from diverse species of rotavirus A localizes to mitochondria via conserved sequences in a predicted N-terminal a-helix. This suggests that NSP6 may affect mitochondrial functions during rotavirus infection.

Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2ß1 integrin in mediating rotavirus infection.

UNLABELLED: N-acetyl- and N-glycolylneuraminic acids (Sia) and α2ß1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2ß1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2ß1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE: Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

Rotavirus inhibits IFN-induced STAT nuclear translocation by a mechanism that acts after STAT binding to importin-α.

The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin α5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin α5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.

Has the role of veno-arterial extracorporeal membrane oxygenation in patients with cardiogenic shock following acute myocardial infarction been fully determined? A case report.

Background: The persistent challenge of high mortality rates in acute myocardial infarction-induced cardiogenic shock endures notwithstanding advancements in the diagnosis and treatment of this disease over the past two decades. While recent studies present conflicting evidence on the efficacy of veno-arterial extracorporeal membrane oxygenation (VA ECMO), observational research supports the benefits of early VA ECMO initiation. However, the current lack of robust support from randomized clinical trials for VA ECMO use in this context highlights the ongoing uncertainty surrounding its effectiveness. Case summary: A 52-year-old male with sudden, intense chest pain was diagnosed with cardiogenic shock due to non-ST-elevation acute myocardial infarction at a local hospital. Initial treatment included aspirin, clopidogrel, and noradrenaline. Upon transfer to our hospital, the patient's condition deteriorated, leading to acute respiratory distress and severe hypotension. Prior to emergent percutaneous coronary intervention, peripheral VA ECMO was initiated. Coronary angiography revealed left main coronary artery occlusion, and a successful intervention was performed. Post-intervention, the patient's haemodynamic parameters significantly improved, and after 7 days, ECMO was successfully discontinued. The patient was discharged in stable condition after 25 days, with favourable outcomes persisting at the 30-day mark. Continuous monitoring is planned during outpatient follow-up. Discussion: The clinical case illustrates a successful treatment outcome achieved through teamwork by the heart team, supporting the efficacy of the VA ECMO pre-percutaneous coronary intervention approach. The careful selection of appropriate candidates and strategic initiation of VA ECMO may play a role in enhancing outcomes for individuals experiencing acute myocardial infarction complicated by challenging cardiogenic shock.

Structural basis of rotavirus strain preference toward N-acetyl- or N-glycolylneuraminic acid-containing receptors.

The rotavirus spike protein domain VP8* is essential for recognition of cell surface carbohydrate receptors, notably those incorporating N-acylneuraminic acids (members of the sialic acid family). N-Acetylneuraminic acids occur naturally in both animals and humans, whereas N-glycolylneuraminic acids are acquired only through dietary uptake in normal human tissues. The preference of animal rotaviruses for these natural N-acylneuraminic acids has not been comprehensively established, and detailed structural information regarding the interactions of different rotaviruses with N-glycolylneuraminic acids is lacking. In this study, distinct specificities of VP8* for N-acetyl- and N-glycolylneuraminic acids were revealed using biophysical techniques. VP8* protein from the porcine rotavirus CRW-8 and the bovine rotavirus Nebraska calf diarrhea virus (NCDV) showed a preference for N-glycolyl- over N-acetylneuraminic acids, in contrast to results obtained with rhesus rotavirus (RRV). Crystallographic structures of VP8* from CRW-8 and RRV with bound methyl-N-glycolylneuraminide revealed the atomic details of their interactions. We examined the influence of amino acid type at position 157, which is proximal to the ligand's N-acetyl or N-glycolyl moiety and can mutate upon cell culture adaptation. A structure-based hypothesis derived from these results could account for rotavirus discrimination between the N-acylneuraminic acid forms. Infectivity blockade experiments demonstrated that the determined carbohydrate specificities of these VP8* domains directly correlate with those of the corresponding infectious virus. This includes an association between CRW-8 adaption to cell culture, decreased competition by N-glycolylneuraminic acid for CRW-8 infectivity, and a Pro157-to-Ser157 mutation in VP8* that reduces binding affinity for N-glycolylneuraminic acid.

Characterization of Retinal Microvascular Complications and the Effects of Endoplasmic Reticulum Stress in Mouse Models of Diabetic Atherosclerosis.

Purpose: Recent evidence suggests that there is a correlation between the micro- and macrovascular complications of diabetes mellitus. The aim of this study is to investigate the molecular mechanisms by which diabetes promotes the development of microvascular disease (diabetic retinopathy [DR]) through characterization of the effects of hyperglycemia in the retina of mouse models of diabetic atherosclerosis. Methods: Hyperglycemia was induced in apolipoprotein E-deficient (ApoE-/-) mice, a model of accelerated atherosclerosis, either through streptozotocin (STZ) injection or introduction of the Ins2Akita mutation (ApoE-/-Ins2+/Akita). Another subset of ApoE-/- mice was supplemented with glucosamine (GlcN). To attenuate atherosclerosis, subsets of mice from each experimental group were treated with the chemical chaperone, 4-phenylbutyric acid (4PBA). Eyes from 15-week-old mice were either trypsin digested and stained with periodic acid-Schiff (PAS) or frozen for cryostat sectioning and immunostained for endoplasmic reticulum (ER) stress markers, including C/EBP homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). PAS-stained retinal flatmounts were analyzed for microvessel density, acellular capillaries, and pericyte ghosts. Results: Features of DR, including pericyte ghosts and reduced microvessel density, were observed in hyperglycemic and GlcN-supplemented mice. Treatment with 4PBA reduced ER stress in the retinal periphery and attenuated DR in the experimental groups. Conclusions: Mouse models of diabetic atherosclerosis show characteristic pathologies of DR that correlate with atherosclerosis. The increased magnitude of these changes and responses to 4PBA in the peripheral retina suggest that future studies should be aimed at assessing regional differences in mechanisms of ER stress-related pathways in these mouse models.

Untargeted Metabolomics in the Discovery of Novel Biomarkers and Therapeutic Targets for Atherosclerotic Cardiovascular Diseases.

BACKGROUND: Cardiovascular Disease (CVD) is the leading cause of mortality and morbidity worldwide. Four out of five CVD deaths are due to myocardial infarction or stroke. Despite many initiatives that have been established for CVD prevention and risk management, and new therapies to treat existing CVD, patients continue to die from cardiac events. Clearly, we need to identify new therapeutic targets and strategies. Metabolomics offers a novel solution to this problem, as metabolomics-based biomarkers do not only indicate the presence or absence of a disease, but are also capable of assessing risks of developing the disease and detecting the disease prior to the appearance of overt clinical symptoms. METHOD: In this review, we describe the analytical techniques and workflow used in untargeted metabolomics. We also identify several case studies that highlight the use of untargeted metabolomics in cardiovascular research. RESULTS: Five case studies that employ untargeted metabolomics approaches to identify biomarkers for cardiovascular risk, myocardial ischemia, transient ischemic attack, incident coronary heart disease, and myocardial infarction risk prediction are described. The use of the untargeted metabolomics is still relatively new in cardiovascular research. As such, there remains a need for future advancement in metabolomic technologies. CONCLUSION: Early diagnosis of CVDs and identification of patients at high risk of developing adverse events would allow for timely intervention that prevents serious consequences or death. There is a need to establish sensitive and non-invasive CV biomarkers, and novel therapeutic targets for the prevention and treatment of CVDs.

4-phenylbutyrate and valproate treatment attenuates the progression of atherosclerosis and stabilizes existing plaques.

BACKGROUND AND AIMS: Recent evidence suggests that endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/ß is involved in the activation of pro-atherosclerotic processes. In this study, we examined the effects of small molecules that interfere with ER stress-GSK3α/ß signaling on the progression and regression of atherosclerosis in a mouse model. METHODS: To examine atherosclerotic progression, low-density lipoprotein receptor deficient (Ldlr-/-) mice were placed on a high-fat diet (HFD) and treated with the chemical chaperone, 4-phenylbutyrate (4PBA, 3.8  g/L drinking water), or the GSK3α/ß inhibitor, valproate (VPA, 625 mg VPA/kg diet), for 10 weeks. To examine potential effects on atherosclerotic regression, 4 week old Ldlr-/- mice were placed on a HFD for 16 weeks. Subsets of mice were harvested at this time or switched to a chow (low fat) diet, or a chow diet with 4PBA or VPA treatment for 4 weeks. RESULTS: In the progression model, the 4PBA- and VPA-treated mice had significantly reduced lesion and necrotic core size. Treatments had no effect on metabolic parameters, including plasma and hepatic lipid levels, or plaque composition. In the regression model, mice with 4PBA or VPA treatment showed no alterations in lesion size, but the lesions had significantly smaller necrotic cores, increased vascular smooth muscle cell content, and increased collagen content. These features are consistent with more stable plaques. CONCLUSIONS: The pharmacological attenuation of ER stress or inhibition of GSK3α/ß impedes the development of atherosclerosis in Ldlr-/- mice and appears to promote the stabilization of existing lesions.

Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.

Irritable bowel syndrome (IBS) is a common disorder characterized by altered gut function and often is accompanied by comorbid anxiety. Although changes in the gut microbiota have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role for commensal gut bacteria in IBS, we colonized germ-free mice with the fecal microbiota from healthy control individuals or IBS patients with diarrhea (IBS-D), with or without anxiety, and monitored gut function and behavior in the transplanted mice. Microbiota profiles in recipient mice clustered according to the microbiota profiles of the human donors. Mice receiving the IBS-D fecal microbiota showed a taxonomically similar microbial composition to that of mice receiving the healthy control fecal microbiota. However, IBS-D mice showed different serum metabolomic profiles. Mice receiving the IBS-D fecal microbiota, but not the healthy control fecal microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation, and anxiety-like behavior. These results indicate the potential of the gut microbiota to contribute to both intestinal and behavioral manifestations of IBS-D and suggest the potential value of microbiota-directed therapies in IBS patients.

Comprehensive Plasma Metabolomic Analyses of Atherosclerotic Progression Reveal Alterations in Glycerophospholipid and Sphingolipid Metabolism in Apolipoprotein E-deficient Mice.

Atherosclerosis is the major underlying cause of most cardiovascular diseases. Despite recent advances, the molecular mechanisms underlying the pathophysiology of atherogenesis are not clear. In this study, comprehensive plasma metabolomics were used to investigate early-stage atherosclerotic development and progression in chow-fed apolipoprotein E-deficient mice at 5, 10 and 15 weeks of age. Comprehensive plasma metabolomic profiles, based on 4365 detected metabolite features, differentiate atherosclerosis-prone from atherosclerosis-resistant models. Metabolites in the sphingomyelin pathway were significantly altered prior to detectable lesion formation and at all subsequent time-points. The cytidine diphosphate-diacylglycerol pathway was up-regulated during stage I of atherosclerosis, while metabolites in the phosphatidylethanolamine and glycosphingolipid pathways were augmented in mice with stage II lesions. These pathways, involving glycerophospholipid and sphingolipid metabolism, were also significantly affected during the course of atherosclerotic progression. Our findings suggest that distinct plasma metabolomic profiles can differentiate the different stages of atherosclerotic progression. This study reveals that alteration of specific, previously unreported pathways of glycerophospholipid and sphingolipid metabolism are associated with atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis.

Revisiting the role of histo-blood group antigens in rotavirus host-cell invasion.

Histo-blood group antigens (HBGAs) have been proposed as rotavirus receptors. H type-1 and Lewis(b) antigens have been reported to bind VP8* from major human rotavirus genotypes P[4], P[6] and P[8], while VP8* from a rarer P[14] rotavirus recognizes A-type HBGAs. However, the role and significance of HBGA receptors in rotavirus pathogenesis remains uncertain. Here we report that P[14] rotavirus HAL1166 and the related P[9] human rotavirus K8 bind to A-type HBGAs, although neither virus engages the HBGA-specific α1,2-linked fucose moiety. Notably, human rotaviruses DS-1 (P[4]) and RV-3 (P[6]) also use A-type HBGAs for infection, with fucose involvement. However, human P[8] rotavirus Wa does not recognize A-type HBGAs. Furthermore, the common human rotaviruses that we have investigated do not use Lewis(b) and H type-1 antigens. Our results indicate that A-type HBGAs are receptors for human rotaviruses, although rotavirus strains vary in their ability to recognize these antigens.

VP7 of Rhesus monkey rotavirus RRV contributes to diabetes acceleration in association with an elevated anti-rotavirus antibody response.

T cell-receptor transgenic NOD8.3 mice provide a model for spontaneous type 1 diabetes development. Infection of 5 week-old NOD8.3 mice with Rhesus monkey rotavirus (RRV) accelerates the onset of their diabetes. This acceleration requires virus replication and relates to the presence and level of serum anti-rotavirus antibodies, but the role of individual RRV genes is unknown. Here we assessed the importance for diabetes acceleration of the RRV genes encoding VP4 and VP7, by infecting NOD8.3 mice with parental and reassortant rotaviruses. Diabetes was accelerated by reassortant rotaviruses containing RRV VP7 on a UK rotavirus genetic background, but not by parental UK or a UK reassortant containing RRV VP4 without VP7. Diabetes acceleration by reassortant rotaviruses containing RRV VP7 depended on the development of a high serum anti-rotavirus antibody titer. This study shows that VP7, together with an elevated anti-rotavirus antibody response, contributes to the acceleration of diabetes onset by RRV.

Efficacy and safety of five injectable anesthetic regimens for chronic blood collection from the anterior vena cava of Guinea pigs.

Despite several published methods of inducing surgical anesthesia in guinea pigs, viable methods of anesthesia for blood collection from the vena cava are inadequate. We compared 5 anesthesia regimens and their efficacy in inducing anesthesia for blood sampling in guinea pigs: ketamine-xylazine (30 and 2.5 mg/kg) administered subcutaneously, intramuscularly, or intraperitoneally; pentobarbital (37 mg/kg) administered intraperitoneally; and medetomidine (0.5 mg/kg) administered intramuscularly. Parameters measured included time to onset of anesthesia, time to recovery from anesthesia, and complete blood count (CBC) and serum chemistry values. CBC values did not differ among the 5 regimens, but serum glucose, BUN, phosphorous, and creatine phosphokinase levels varied among groups. Based on our data, intraperitoneal ketamine-xylazine appears to emerge as a preferable injectable anesthetic regimen in guinea pigs for blood collection from the anterior vena cava.

No evidence for consistent virus-specific immunity in simian immunodeficiency virus-exposed, uninfected rhesus monkeys.

Defining the immune correlates of the protection against human immunodeficiency virus type 1 (HIV-1) acquisition in individuals who are exposed to HIV-1 but do not become infected may provide important direction for the creation of an HIV-1 vaccine. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to determine whether monkeys can be repeatedly exposed to a primate lentivirus by a mucosal route and escape infection and whether virus-specific immune correlates of protection from infection can be identified in uninfected monkeys. Five of 18 rhesus monkeys exposed 18 times by intrarectal inoculation to SIVmac251 or SIVsmE660 were resistant to infection, indicating that the exposed/uninfected phenotype can be reproduced in a nonhuman primate AIDS model. However, routine peripheral blood lymphocyte gamma interferon enzyme-linked immunospot (ELISPOT), tetramer, and intracellular cytokine staining assays, as well as cytokine-augmented ELISPOT and peptide-stimulated tetramer assays, failed to define a systemic antigen-specific cellular immune correlate to this protection. Further, local cell-mediated immunity could not be demonstrated by tetramer assays of these protected monkeys, and local humoral immunity was not associated with protection against acquisition of virus in another cohort of mucosally exposed monkeys. Therefore, resistance to mucosal infection in these monkeys may not be mediated by adaptive virus-specific immune mechanisms. Rather, innate immune mechanisms or an intact epithelial barrier may be responsible for protection against mucosal infection in this population of monkeys.

Comparative evaluation of three different intramuscular delivery methods for DNA immunization in a nonhuman primate animal model.

Although plasmid DNA vaccines induce potent cell-mediated immune responses and prime for antibody responses in experimental laboratory animals, their immunogenicity in humans has been less remarkable. A number of strategies have been proposed to improve the immunogenicity of these vaccines, including using novel means of vaccine delivery. In the present study, the immunogenicity of three different methods of intramuscular plasmid DNA administration was compared in cynomolgus monkeys: needle and syringe, Biojector 2000, and Mini-Ject. The elicited cellular and humoral immune responses were comparable in monkeys immunized using these different delivery techniques, suggesting that the needle-free approaches to vaccine administration do not significantly improve the immunogenicity of the plasmid DNA vaccine used in the study.