Enhanced protection against Streptococcus pyogenes infection by intranasal vaccination with a dual antigen component M protein/SfbI lipid core peptide vaccine formulation

We investigated the efficacy of a synthetic Streptococcus pyogenes vaccine targeting two virulence factors using the Lipid Core Peptide (LCP) delivery system. BALB/c mice were immunised intranasally with LCPs containing peptides encompassing T-cell and B-cell epitopes of the conserved C-repeat region of the M protein (J8) or the fibronectin-binding repeats region (FNBR) of SfbI, or a combination formulation containing peptides representing both antigens. LCPs were co-administered with the TLR2/6 agonist MALP-2 as mucosal adjuvant. Humoral and cellular immune responses stimulated at systemic and mucosal levels were strongest in mice immunised with the dual antigen formulation. Mice were completely protected following a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain, whereas there was 70% and 90% survival in mice immunised with LCP-J8 and LCP-FNBR, respectively. This is the first report demonstrating the elicitation of better protective immunity by a dual antigen component S. pyogenes vaccine.     

Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums

A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis. Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis. Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state. Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes. Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M. bovis compared with animals vaccinated with non-formulated BCG. In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination. Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.     

Effect of bile salts, lipid, and humic acids on absorption of benzo[a]pyrene by isolated channel catfish (Ictalurus punctatus) intestine segments

Dietary absorption of lipophilic contaminants may be a significant route of exposure in aquatic organisms. Bile salts, lipids, and humic acids are important factors that may influence the intestinal absorption of a contaminant such as benzo[a]pyrene (BaP). We hypothesized that bile salts, monoglycerides, and free fatty acids would increase BaP intestinal absorption, while triglycerides, humic acids, and sediment would decrease BaP intestinal absorption. We have established and validated an in vitro model to examine modification of 3H-BaP absorption in everted intestinal segments from channel catfish (Ictalurus punctatus). Uptake of BaP into the everted intestinal segments continued to increase over the times examined in this study (60 min) and apparently occurs passively; thus, fugacity-based models of uptake are supported. Absorption of BaP into intestinal cells was significantly decreased by the addition of monoglycerides and free fatty acids to bile salts in the incubation media. Addition of triglycerides decreased BaP absorption even further. Humic acids may have decreased BaP intestinal absorption, while natural sediment may have increased BaP absorption. The results of this study suggest that all lipids may decrease intestinal uptake of lipophilic contaminants if they remain in unabsorbable excess in the intestinal lumen by retaining BaP in lipid/bile micelles. In contrast, if triglycerides are hydrolyzed into monoglycerides/free fatty acids prior to absorption, lipophilic contaminant uptake will likely be facilitated. Thus, it may be the hydrolytic state of lipids that determines its effects on BaP absorption. Humic acids alone may decrease dietary uptake of BaP, but our results suggest that other components in natural sediment may counteract this effect to cause a slight enhancement of BaP uptake. Further studies are needed to determine the dietary conditions necessary for bioaccumulation to contribute significantly to lipophilic contaminant body burdens in benthivorous fish. Finally, the everted intestinal segment technique has the potential to be used in other species and with different contaminants.     

Hepatic transport of bile salt and bile composition following total parenteral nutrition with and without lipid emulsion in the rat

The effect of total parenteral nutrition (TPN) on bile flow and composition and on hepatic bile acid transport maximum (Tm) and bile salt-independent bile flow (BSIF) was studied in the rat following seven days TPN containing 33% calories from Intralipid (IL) or Liposyn (LP) or 0% calories from lipid. All TPN regimes markedly reduced bile flow. In no case did TPN cause an increase in bile cholesterol concentration or saturation relative to bile acid and phospholipid. Bile acid Tm was reduced in rats receiving either 0% lipid or 33% IL; BSIF was reduced only in the 0% lipid group. Rats receiving 33% LP had a higher bile flow than the other TPN regimes while bile acid Tm and BSIF were similar to controls. It is proposed that in established TPN, bile flow is reduced largely as a result of decreased hepatic bile acid excretion. In the rat, TPN has no deleterious effect on the molar concentration of cholesterol, phospholipid or bile acid in bile secreted by the hepatocyte. The significant differences between the effect of the two lipid emulsions on hepatobiliary function require further study.     

Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters

Effective therapy for prion diseases is currently unavailable. Recently, vaccination was shown to be effective in mouse models of a particular neurodegenerative conditions: Alzheimer's disease (AD). Here, we report that vaccination with synthetic oligopeptides homologous to the hamster (Mesocricetus auratus) prion protein augments survival time in animals infected intraperitoneally with 263K scrapie agent. For each hamster included in the study, prion-specific serum antibodies as well as deposition of pathological prion protein (PrP(res)), glial fibrillary acidic protein (GFAP), and mRNA expression for cytokines (TNF alpha, IL-1beta, IL-10) in brain tissues were evaluated. In immunized animals, increased survival after challenge was associated with a reduction of cerebral lesion, PrP deposition and GFAP expression; in these animals, anti-prion protein peptide antibody levels were increased, and the expression of pro-inflammatory cytokines (TNF alpha and IL-1beta) was reduced. Vaccination could be an effective therapeutic approach to postpone disease onset.     

Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine. The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope.     

Enhanced surveillance of primary measles mumps and rubella (MMR) immunisation in Wales

In England and Wales routinely available data measure uptake of the measles mumps and rubella (MMR) vaccine at 2 years. This results in a delay in detecting change in uptake of the vaccine, which is scheduled at 12 months of age. The predictive value of uptake at 15–17 months is limited by the greater variability in uptake between quarters at the younger age. This can be overcome by presenting the data as a four-quarter annual rolling average. Uptake of the MMR vaccine at 2 years of age in Wales is predicted to stabilise at around 84% in the first three quarters of 2002.     

Silkworm pupae (Bombyx mori) are new sources of high quality protein and lipid

This study was performed to evaluate the nutritional value of silkworm pupae (Bombyx mori) and the content of α-glucosidase inhibitor. The percentages of total protein and lipid contents by dry weight were 55.6 and 32.2%, respectively. Silkworm pupae protein had high levels of essential amino acids such as valine, methionine and phenylalanine. The contents of essential amino acids in silkworm pupae protein satisfied the FAO/WHO/UNU suggested requirements (2007). In addition, they also possessed n-3 fatty acids, especially α-linolenic acid (36.3%), as a major component. The 50% ethanol extract of silkworm pupae contained 1-deoxynojirimycin (DNJ), which is a potent α-glucosidase inhibitor. These results suggest that silkworm pupae are a new source of high quality protein, lipid, and α-glucosidase inhibitor.     

Contraception by Ushercell (cellulose sulfate) in formulation: duration of effect and dose effectiveness

This study evaluated contraception by formulated Ushercell, a uniquely high-molecular-weight form of cellulose sulfate, in the rabbit. Variables included (1) dose effectiveness, (2) duration of effectiveness, and (3) formulation excipients. Vaginally applied carboxymethyl-cellulose-based Ushercell gel is contraceptive. A 6% gel is active for at least 18 h; partial activity is observed for at least 24 h. With an application-insemination interval of 0.5 h, Ushercell as low as 0.1% is contraceptive. Contraception is incomplete with 2% Ushercell and an application-insemination interval of 24 h. Ushercell formulations containing a relatively high concentration of Carbopol are ineffective contraceptives, whether the gel is applied before insemination or is premixed with spermatozoa before insemination. Contraceptive activity is restored in Ushercell formulations with lower Carbopol content. This study shows that formulated Ushercell is an effective, long-lasting contraceptive and, hence, is bioavailable when vaginally applied. Activity is dependent on the type and relative concentration of formulation excipients. These data support a projected successful outcome of further clinical trials.     

In vivo protein expression and immune responses generated by DNA vaccines expressing mycobacterial antigens fused with a reporter protein

We cloned six mycobacterial antigens into a mammalian expression vector as fusion proteins with the enhanced green fluorescent protein (EGFP). Plasmid DNA was injected intramuscularly, and the injection sites were examined 1 week later. Expression of each antigen-EGFP fusion protein was visualized as green fluorescence in muscle tissue sections. A plasmid expressing EGFP alone and a plasmid with a frameshift mutation served as positive and negative controls. Visualization of fluorescent protein in vivo was 100% specific when compared to in vitro results. In vivo sensitivity was only 37% based on individual injection sites, but increased to 100% when results from multiple injection sites were combined for each plasmid. EGFP alone was expressed in a higher proportion of myocytes than the antigen-EGFP fusion proteins (P < 0.001). There was a trend toward an inverse correlation between protein size and the proportion of myocytes with visible fluorescence (r = -0.68; P = 0.09). We compared antibody subtypes generated to Mycobacterium bovis antigen 85A, when it was expressed alone or as a fusion protein. Inclusion of EGFP modified the immune response toward a Th1 response, as indicated by the ratio of antigen 85A-specific IgG2a to IgG1 generated by each plasmid (antigen 85A alone 0.73 +/- 0.18 versus antigen 85A-EGFP 1.82 +/- 0.57, mean +/- S.D.; P < 0.01), though the magnitude of the antibody isotype shift was modest. Direct visualization of antigen-EGFP fusion proteins provided a simple and rapid method to confirm in vivo antigen expression.     

Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid)

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus na?ve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to na?ve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.     

Antibody responses after intravaginal immunisation with trimeric HIV-1 CN54 clade C gp140 in Carbopol gel are augmented by systemic priming or boosting with an adjuvanted formulation

Optimum strategies to elicit and maintain antibodies at mucosal portals of virus entry are critical for the development of vaccines against human immunodeficiency virus (HIV). Here we show in non-human primates that a novel regimen of repeated intravaginal delivery of a non-adjuvanted, soluble recombinant trimeric HIV-1_CN54 clade C envelope glycoprotein (gp140) administered in Carbopol gel can prime for B-cell responses even in the absence of seroconversion. Following 3 cycles of repeated intravaginal administration, throughout each intermenses interval, 3 of 4 macaques produced or boosted systemic and mucosally-detected antibodies upon intramuscular immunisation with gp140 formulated in AS01 adjuvant. Reciprocally, a single intramuscular immunisation primed 3 of 4 macaques for antibody boosting after a single cycle of intravaginal immunisation. Virus neutralising activity was detected against clade C and clade B HIV-1 envelopes but was restricted to highly neutralisation sensitive pseudoviruses.     

Comparison of prophylactic and therapeutic immunisation with an ErbB-2 (HER2) fusion protein and immunoglobulin V-gene repertoire analysis in a transgenic mouse model of spontaneous breast cancer

ErbB-2 is associated with several solid tumours of which breast cancer is the commonest cancer in women worldwide. Though anti-ErbB-2 antibody appears to play a significant role in prevention and therapy, naturally occurring anti-ErbB-2 antibody associated with the cleaved ectodomain of overexpressed ErbB-2 self antigen is detectable in patients. It is therefore essential to understand the course of antibody mediated protection during disease progression. 100% of FVB/N^neu mice expressing mutated, constitutively active ErbB-2 develop mammary carcinoma. It has been shown that vaccination with ErbB-2 associated with a T helper cell epitope P30 can offer protection against transplantable tumour but it is unclear whether the same vaccine protects against naturally developing tumour. We have analysed the course of the disease following prophylactic, and therapeutic vaccination in this spontaneous, eutopic mammary carcinoma model that more closely resembles the human disease. 100% protection against tumour development was observed subsequent to prophylactic immunisation but disease progression was unaffected by therapeutic vaccination. The antibody response exhibited restricted expansion of the Immunoglobulin (Ig) variable (V)-gene repertoire by ErbB-2 specific B cells compared with the non-antigen specific B cell pool and control mice. The serum antibody profile was similar in therapeutically injected mice without any effect on tumour burden.     

Shifts in global immunisation goals (1984-2004): unfinished agendas and mixed results

The turn of the millennium has been marked by a large-scale mobilisation of resources for immunisation programmes in developing countries. The resources have been generated by public and private sector parties collaborating in the Global Alliance for Vaccines and Immunization (GAVI). GAVI was formed in response to deteriorating immunisation coverage rates occurring in the late 1990s. GAVI is the latest in a line of vaccine initiatives, which have operated over the past 20 years. This article reviews the five most important global immunisation initiatives that have taken place over those past 20 years. It analyses their origins, shifts in global immunisation goals, identifies key actors, assesses the initiatives' capacity to mobilise resources and increase immunisation coverage, and points to possible unintended effects of the initiatives. The study argues that shifts in global immunisation goals lead to fragmentation in the implementation of vaccine programmes at the local level in developing countries. It also suggests that global actors involved in the formulation of these initiatives appear to miss opportunities to build on past experiences and fail to learn from previous mistakes. This raises questions about the initiatives' sustainability and relevance to the overall objective of preventing vaccine-preventable deaths.     

Cholesterol and bile acid synthesis during total parenteral nutrition with and without lipid emulsion in the rat

The origin of excess plasma free cholesterol known to accumulate in plasma of patients or animals given total parenteral nutrition (TPN) with lipid emulsion was investigated. Rats were infused for 8 days with a specially formulated TPN solution plus either lipid emulsion (lipid-TPN) or an equicaloric volume of 25% dextrose (dextrose-TPN). Laboratory diet-fed controls were sham operated. Lipid-TPN suppressed hepatic HMG CoA reductase (HMG CoAR) activity but elevated cholesterol 7 alpha-hydroxylase (7 alpha-OH) activity. HMG CoAR activity, however, was increased in adipose tissue and skeletal muscle by lipid-TPN when compared to dextrose-TPN. Plasma lecithin/cholesterol acyl transfer activity was similar among all groups. It is suggested that in lipid-TPN excess plasma free cholesterol does not arise from decreased hepatic clearance or plasma esterification but may originate from extrahepatic tissue, possibly through leaching of membrane cholesterol by mesophase phospholipid present in the lipid emulsion. The changes in hepatic HMG CoAR and 7 alpha-OH activity imply that during lipid-TPN plasma free cholesterol is cleared by the liver and catabolized to bile acid.     

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.     

高血压脑出血患者血浆氨基末端脑利钠肽前体和C反应蛋白变化的临床意义

目的探讨高血压脑出血(HICH)患者静脉血浆氨基末端脑利钠肽前体(NT－pro－BNP)和C反应蛋白(CRP)的变化及其临床意义。 方法选取2016年1月至2017年12月江苏大学附属金坛医院重症医学科(ICU)收治的HICH患者65例(观察组)和30例同期到院进行健康体检者(对照组)为研究对象进行回顾性病例对照分析。对HICH患者和对照组健康者血浆NT－pro－BNP和CRP进行比较。观察组病例依据入ICU后格拉斯哥昏迷评分(GCS),分为GCS评分≤8分组、GCS评分>8分组,依据转归分为生存组与死亡组。比较组间NT－pro－BNP和CRP的差异。 结果观察组患者静脉血NT－pro－BNP、CRP水平较对照组增高[NT－pro－BNP水平分别为：(823.67±3 095.51)pg/L、(43.60±51.06)pg/L,CRP水平分别为：(52.26±52.50)mg/L、(1.25±2.29)mg/L,P均<0.05]。死亡组HICH患者的静脉血NT－pro－BNP和CRP水平高于生存组[NT－pro－BNP：(2 172.50±5 272.32)pg/L、(179.92±143.75)pg/L,CRP：(98.50±59.16)mg/L、(30.19±30.53)mg/L,P均<0.05]。GCS评分≤8分HICH患者静脉血CRP水平高于GCS评分>8分的患者[(73.04±56.34)mg/L、(26.45±32.98)mg/L,P<0.05],但两者NT－pro－BNP水平差异无统计学意义(P>0.05)。 结论血浆NT－pro－BNP、CRP水平可用于指导HICH的临床诊断,CRP水平能反映病情严重程度。两者联合应用可作为诊断和判断HICH预后的实验室指标。