Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge.

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

Broadly Protective Multivalent OspA Vaccine against Lyme Borreliosis, Developed Based on Surface Shaping of the C-Terminal Fragment.

The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, development might be restricted either to conserved antigens, which are often rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the United States and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions, based primarily on binding sites of monoclonal antibodies (MAbs). In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed, and, based on their immunogenicity, four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4) or with in vitro-grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro-grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.

Potential of Lipoprotein-Based Nanoparticulate Formulations for the Treatment of Eye Diseases.

Lipoproteins are naturally occurring nanoparticles and their main physiological function is the promotion of lipid metabolism. They can be prepared in vitro for use as drug carriers, and these reconstituted lipoproteins show similar biological activity to their natural counterparts. Some lipoproteins can cross the blood-retinal barrier and are involved in intraocular lipid metabolism. Drug-loaded lipoproteins can be delivered to the retina for the treatment of posterior eye diseases. In this review, we have discussed the therapeutic applications of lipoproteins for eye diseases and introduced the emerging animal models used for the evaluation of their therapeutic effects.

The Effect of the 10-Valent Pneumococcal Nontypeable Haemophilus influenzae Protein D Conjugate Vaccine on H. influenzae in Healthy Carriers and Middle Ear Infections in Iceland.

Vaccinations with the 10-valent pneumococcal conjugated vaccine (PHiD-CV) started in Iceland in 2011. Protein D (PD) from H. influenzae, which is coded for by the hpd gene, is used as a conjugate in the vaccine and may provide protection against PD-positive H. influenzae We aimed to evaluate the effect of PHiD-CV vaccination on H. influenzae in children, both in carriage and in acute otitis media (AOM). H. influenzae was isolated from nasopharyngeal swabs collected from healthy children attending 15 day care centers in 2009 and from 2012 to 2017 and from middle ear (ME) samples from children with AOM collected from 2012 to 2017. All isolates were identified using PCR for the hpd and fucK genes. Of the 3,600 samples collected from healthy children, 2,465 were culture positive for H. influenzae (68.5% carriage rate); of these, 151 (6.1%) contained hpd-negative isolates. Of the 2,847 ME samples collected, 889 (31.2%) were culture positive for H. influenzae; of these, 71 (8.0%) were hpd negative. Despite the same practice throughout the study, the annual number of ME samples reduced from 660 in 2012 to 330 in 2017. The proportions of hpd-negative isolates in unvaccinated versus vaccinated children were 5.6% and 7.0%, respectively, in healthy carriers, and 5.4% and 7.8%, respectively, in ME samples. The proportion of hpd-negative isolates increased with time in ME samples but not in healthy carriers. The number of ME samples from children with AOM decreased. The PHiD-CV had no effect on the proportion of the hpd gene in H. influenzae from carriage, but there was an increase in hpd-negative H. influenzae in otitis media. The proportions of hpd-negative isolates remained similar in vaccinated and unvaccinated children.

Lipoprotein Drug Delivery Vehicles for Cancer: Rationale and Reason.

Lipoproteins are a family of naturally occurring macromolecular complexes consisting amphiphilic apoproteins, phospholipids, and neutral lipids. The physiological role of mammalian plasma lipoproteins is to transport their apolar cargo (primarily cholesterol and triglyceride) to their respective destinations through a highly organized ligand-receptor recognition system. Current day synthetic nanoparticle delivery systems attempt to accomplish this task; however, many only manage to achieve limited results. In recent years, many research labs have employed the use of lipoprotein or lipoprotein-like carriers to transport imaging agents or drugs to tumors. The purpose of this review is to highlight the pharmacologic, clinical, and molecular evidence for utilizing lipoprotein-based formulations and discuss their scientific rationale. To accomplish this task, evidence of dynamic drug interactions with circulating plasma lipoproteins are presented. This is followed by epidemiologic and molecular data describing the association between cholesterol and cancer.

NanoSIMS imaging reveals unexpected heterogeneity in nutrient uptake by brown adipocytes.

Heterogeneity in the metabolic properties of adipocytes in white adipose tissue has been well documented. We sought to investigate metabolic heterogeneity in adipocytes of brown adipose tissue (BAT), focusing on heterogeneity in nutrient uptake. To explore the possibility of metabolic heterogeneity in brown adipocytes, we used nanoscale secondary ion mass spectrometry (NanoSIMS) to quantify uptake of lipids in adipocytes interscapular BAT and perivascular adipose tissue (PVAT) after an intravenous injection of triglyceride-rich lipoproteins (TRLs) containing [2H]triglycerides (2H-TRLs). The uptake of deuterated lipids into brown adipocytes was quantified by NanoSIMS. We also examined 13C enrichment in brown adipocytes after administering [13C]glucose or 13C-labeled mixed fatty acids by gastric gavage. The uptake of 2H-TRLs-derived lipids into brown adipocytes was heterogeneous, with 2H enrichment in adjacent adipocytes varying by more than fourfold. We also observed substantial heterogeneity in 13C enrichment in adjacent brown adipocytes after administering [13C]glucose or [13C]fatty acids by gastric gavage. The uptake of nutrients by adjacent brown adipocytes within a single depot is variable, suggesting that there is heterogeneity in the metabolic properties of brown adipocytes.

Characterization of recombinant OspA in two different Borrelia vaccines with respect to immunological response and its relationship to functional parameters.

BACKGROUND: Prevention of Lyme disease in dogs in North America depends on effective vaccination against infection by the tick vector-born spirochete Borrelia burgdorferi. Most vaccines effectively prevent spirochete transmission to dogs during tick feeding based on immunization with the outer-surface lipoprotein A (OspA) of B. burgdorferi. More recently, vaccines containing additional OspC protein moieties have been introduced. These are designed to enhance protection by forming a second line of defense within the vertebrate host, where OspC expression replaces OspA as the dominant surface antigen. However, supportive data for demonstration of OspC mediated protection is still lacking. Since OspA immunogenicity is of paramount importance to protection against spirochete transmission; this study was designed to compare the immunogenicity of two commercially available vaccines against the Borrelia burgdorferi OspA. We further characterized OspA antigen fractions of these vaccines with respect to their biochemical and biophysical properties. RESULTS: Two groups of beagle dogs (n = 9) were administered either: (1) a nonadjuvanted/monovalent, recombinant OspA vaccine (Recombitek® Lyme) or (2) an adjuvanted, recombinant OspA /OspC chimeric fusion vaccine (Vanguard® crLyme). The onset of the anti-OspA antibody response elicited by the nonadjuvanted/monovalent OspA vaccine was significantly earlier than that for the bivalent OspA /OspC vaccine and serum borreliacidal activity was significantly greater at all post-vaccination time points. As expected, only dogs inoculated with the bivalent OspA/OspC vaccine mounted a humoral anti-OspC response. However, only three out of nine dogs in that group had a positive response. Comparison of the OspA vaccine structures revealed that the OspA in the nonadjuvanted/monovalent vaccine was primarily in the lipidated form, eluting (SEC-HPLC) at a high molecular weight, suggestive of micelle formation. Conversely, the OspA moiety of the OspA/OspC vaccine was found to be nonlipidated and eluted as the monomeric protein. CONCLUSIONS: We hypothesize that these structural differences may account for the superior immunogenicity of the nonadjuvanted monovalent recombinant OspA vaccine in dogs over the adjuvanted OspA fraction of the OspA/OspC vaccine.

Characterizing blood-brain barrier perturbations after exposure to human triglyceride-rich lipoprotein lipolysis products using MRI in a rat model.

PURPOSE: Previous studies indicated hyperlipidemia may be a risk factor for Alzheimer's disease, but the contributions of postprandial triglyceride-rich lipoprotein (TGRL) are not known. In this study, changes in blood-brain barrier diffusional transport following exposure to human TGRL lipolysis products were studied using MRI in a rat model. METHODS: Male Sprague-Dawley rats (∼180-250 g) received an i.v. injection of lipoprotein lipase (LpL)-hydrolyzed TGRL (n = 8, plasma concentration ≈ 150 mg human TGRL/dL). Controls received i.v. injection of either saline (n = 6) or LpL only (n = 6). The (1) H longitudinal relaxation rate R1 = 1/T1 was measured over 18 min using a rapid-acquired refocus-echo (RARE) sequence after each of three injections of the contrast agent Gd-DTPA. Patlak plots were generated for each pixel yielding blood-to-brain transfer coefficients, Ki , chosen for best fit to impermeable, uni-directional influx or bi-directional flux models using the F-test. RESULTS: Analysis from a 2-mm slice, 2-mm rostral to the bregma showed a 275% increase of mean Ki during the first 20 min after infusion of human TGRL lipolysis product that differed significantly compared with saline and LpL controls. This difference disappeared by 40 min mark. CONCLUSION: These results suggest human TGRL lipolysis products can lead to a transient increase in rat BBB permeability. Magn Reson Med 76:1246-1251, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Mucosal immunization with PsaA protein, using chitosan as a delivery system, increases protection against acute otitis media and invasive infection by Streptococcus pneumoniae.

As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan-PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN-γ, IL-17A and IL-4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan-PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan-PsaA nanoparticles. Thus, intranasal immunization with chitosan-PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.

A randomised trial to evaluate the immunogenicity, reactogenicity, and safety of the 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) co-administered with routine childhood vaccines in Singapore and Malaysia.

BACKGROUND: The immunogenicity, reactogenicity, and safety of the 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) co-administered with routine childhood vaccines were evaluated among infants from Singapore and Malaysia, where PHiD-CV has been licensed. METHODS: In the primary vaccination phase, 298 infants from Singapore and 168 infants from Malaysia were randomised to receive the Phase III Clinical (Clin) or the Commercial (Com) lot of PHiD-CV at 2, 3, and 5 months of age. In the booster vaccination phase, 238 toddlers from Singapore received one dose of the PHiD-CV Commercial lot at 18-21 months of age. Immune responses to pneumococcal polysaccharides were measured using 22F-inhibition enzyme-linked immunosorbent assay (ELISA) and functional opsonophagocytic activity (OPA) assay and to protein D, using ELISA. RESULTS: Immune responses induced by primary vaccination with the PHiD-CV Commercial lot were non-inferior to the Phase III Clinical lot in terms of adjusted antibody geometric mean concentration (GMC) ratios for each vaccine pneumococcal serotype and protein D. For each vaccine pneumococcal serotype, ≥93.6% and ≥88.5% of infants from Malaysia and Singapore had post-primary vaccination antibody concentrations ≥0.2 µg/mL and OPA titres ≥8, in the Clin and Com groups, respectively. For each vaccine pneumococcal serotype, ≥60.8% and ≥98.2% of toddlers from Singapore had pre- and post-booster antibody concentrations ≥0.2 µg/mL, in the Clin and Com groups, respectively. All children, except one, had measurable anti-protein D antibodies and the primary and booster doses of the co-administered vaccines were immunogenic. The incidence of each grade 3 solicited symptom was ≤11.1% in both study phases. No serious adverse events considered causally related to vaccination were reported throughout the study. CONCLUSIONS: PHiD-CV given as three-dose primary vaccination to infants in Singapore and Malaysia and booster vaccination to toddlers in Singapore was shown to be immunogenic with a clinically acceptable-safety profile.This study has been registered at http://www.clinicaltrials.govNCT00808444 and NCT01119625.

[Recent development of natural and reconstituted lipoprotein based nano drug delivery vehicles].

Lipoproteins are biological lipids carriers. The natural and reconstituted lipoprotein based drug delivery systems have been extensively developed in recent years. This article reviews the development of natural and reconstituted low-density lipoprotein and high-density lipoprotein based vehicles in the antitumor area.

Optimisation of dose level and vaccination schedule for the VLA15 Lyme borreliosis vaccine candidate among healthy adults: two randomised, observer-blind, placebo-controlled, multicentre, phase 2 studies.

BACKGROUND: Rising Lyme borreliosis incidence rates, potential for severe outcomes, and limitations in accurate and timely diagnosis for treatment initiation suggest the need for a preventive vaccine; however, no vaccine is currently available for human use. We performed two studies in adults to optimise the dose level and vaccination schedule for VLA15, an investigational Lyme borreliosis vaccine targeting outer surface protein A (OspA) serotypes 1-6, which are associated with the most common pathogenic Borrelia species in Europe and North America. METHODS: Both randomised, observer-blind, placebo-controlled, multicentre phase 2 studies included participants aged 18-65 years without recent history of Lyme borreliosis or tick bites. Study one was conducted at nine clinical research and study centre sites in the USA (n=6), Germany (n=2), and Belgium (n=1); study two was conducted at five of the study one US sites. Based on a randomisation list created by an unmasked statistician for each study, participants were randomly assigned via an electronic case report form randomisation module to receive 90 µg (study one only), 135 µg, or 180 µg VLA15 or placebo by intramuscular injection at months 0, 1, and 2 (study one) or 0, 2, and 6 (study two). Study one began with a run-in phase to confirm safety, after which the Data Safety Monitoring Board recommended the removal of the 90 µg group and continuation of the study. In the study one run-in phase, randomisation was stratified by study site, whereas in the study one main phase and in study two, randomisation was stratified by study site, age group, and baseline B burgdorferi (sensu lato) serostatus. All individuals were masked, other than staff involved in randomisation, vaccine preparation or administration, or safety data monitoring. The primary endpoint for both studies was OspA-specific IgG geometric mean titres (GMTs) at 1 month after the third vaccination and was evaluated in the per-protocol population. Safety endpoints were evaluated in the safety population: all participants who received at least one vaccination. Both studies are registered at ClinicalTrials.gov (study one NCT03769194 and study two NCT03970733) and are completed. FINDINGS: For study one, 573 participants were screened and randomly assigned to treatment groups between Dec 21, 2018, and Sept, 26, 2019. For study two, 248 participants were screened and randomly assigned between June 26 and Sept 3, 2019. In study one, 29 participants were assigned to receive 90 µg VLA15, 215 to 135 µg, 205 to 180 µg, and 124 to placebo. In study two, 97 participants were assigned to receive 135 µg VLA15, 100 to 180 µg, and 51 to placebo. At 1 month after the third vaccination (ie, month 3), OspA-specific IgG GMTs in study one ranged from 74·3 (serotype 1; 95% CI 46·4-119·0) to 267·4 units per mL (serotype 3; 194·8-367·1) for 90 µg VLA15, 101·9 (serotype 1; 87·1-119·4) to 283·2 units per mL (serotype 3; 248·2-323·1) for 135 µg, and 115·8 (serotype 1; 98·8-135·7) to 308·6 units per mL (serotype 3; 266·8-356·8) for 180 µg. In study two, ranges at 1 month after the third vaccination (ie, month 7) were 278·5 (serotype 1; 214·9-361·0) to 545·2 units per mL (serotype 2; 431·8-688·4) for 135 µg VLA15 and 274·7 (serotype 1; 209·4-360·4) to 596·8 units per mL (serotype 3; 471·9-754·8) for 180 µg. Relative to placebo, the VLA15 groups had more frequent reports of solicited local adverse events (study one: 94%, 95% CI 91-96 vs 26%, 19-34; study two: 96%, 93-98 vs 35%, 24-49 after any vaccination) and solicited systemic adverse events (study one: 69%, 65-73 vs 43%, 34-52; study two: 74%, 67-80 vs 51%, 38-64); most were mild or moderate. In study one, unsolicited adverse events were reported by 52% (48-57) of participants in the VLA15 groups and 52% (43-60) of those in the placebo groups; for study two these were 65% (58-71) and 69% (55-80), respectively. Percentages of participants reporting serious unsolicited adverse events (study one: 2%, 1-4; study two: 4%, 2-7) and adverse events of special interest (study one: 1%, 0-2; study two: 1%, 0-3) were low across all groups. A single severe, possibly related unsolicited adverse event was reported (worsening of pre-existing ventricular extrasystoles, which resolved after change of relevant concomitant medication); no related serious adverse events or deaths were reported. INTERPRETATION: VLA15 was safe, well tolerated, and elicited robust antibody responses to all six OspA serotypes. These findings support further clinical development of VLA15 using the 180 µg dose and 0-2-6-month schedule, which was associated with the greatest immune responses. FUNDING: Valneva.

Impact of conjugation to different lipids on the lymphatic uptake and biodistribution of brush PEG polymers.

Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.

Metabolism of triglyceride-rich lipoproteins and transfer of lipids to high-density lipoproteins (HDL) in vegan and omnivore subjects.

BACKGROUND AND AIMS: Vegan diet excludes all foodstuffs of animal origin and leads to cholesterol lowering and possibly reduction of cardiovascular disease risk. The aim was to investigate whether vegan diet improves the metabolic pathway of triglyceride-rich lipoproteins, consisting in lipoprotein lipolysis and removal from circulation of the resulting remnants and to verify whether the diet alters HDL metabolism by changing lipid transfers to this lipoprotein. METHODS AND RESULTS: 21 vegan and 29 omnivores eutrophic and normolipidemic subjects were intravenously injected triglyceride-rich emulsions labeled with (14)C-cholesterol oleate and (3)H-triolein: fractional clearance rates (FCR, in min(-1)) were calculated from samples collected during 60 min for radioactive counting. Lipid transfer to HDL was assayed by incubating plasma samples with a donor nanoemulsion labeled with radioactive lipids; % lipids transferred to HDL were quantified in supernatant after chemical precipitation of non-HDL fractions and nanoemulsion. Serum LDL cholesterol was lower in vegans than in omnivores (2.1 ± 0.8, 2.7 ± 0.7 mmol/L, respectively, p < 0,05), but HDL cholesterol and triglycerides were equal. Cholesteryl ester FCR was greater in vegans than in omnivores (0.016 ± 0.012, 0.003 ± 0.003, p < 0.01), whereas triglyceride FCR was equal (0.024 ± 0.014, 0.030 ± 0.016, N.S.). Cholesteryl ester transfer to HDL was lower in vegans than in omnivores (2.7 ± 0.6, 3.5 ± 1.5%, p < 0,05). Free-cholesterol, triglyceride and phospholipid transfer were equal, as well as HDL size. CONCLUSION: Remnant removal from circulation, estimated by cholesteryl oleate FCR was faster in vegans, but the lipolysis process, estimated by triglyceride FCR was equal. Increased removal of atherogenic remnants and diminution of cholesteryl ester transfer may favor atherosclerosis prevention by vegan diet.

Harnessing a physiologic mechanism for siRNA delivery with mimetic lipoprotein particles.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip-mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

The rOmp22-HpaA fusion protein confers protective immunity against helicobacter pylori in mice.

Helicobacter pylori (H. pylori) plays an essential role in the development of various gastroduodenal diseases; however, no vaccines preventing H. pylori infection have been available now. This study was to evaluate the protective effect of rOmp22-HpaA fusion protein against H. pylori infection in mouse model and to screen the candidate to be used in the development of an oral vaccine against H. pylori. rOmp22, rHpaA, rOmp22+rHpaA, and rOmp22-HpaA groups were used to immunize mice with mLT63 as adjuvant by intragastric route, respectively, four times at 1-week intervals. Two weeks after last immunization, all of the animals were orally challenged with H. pylori NCTC11637 and then were killed after another 2 weeks. The mice gastric tissue of all groups was separated to detect the presence of infection by urease tests, to culture H. pylori, and to observe the histological characteristics. The protective effect against H. pylori challenge in mice immunized with rOmp22-HpaA fusion protein and mLT63 adjuvant was significantly higher than PBS and mLT63 control groups (P < 0.05), but no significant difference was detected among rOmp22, rHpaA, rOmp22+rHpaA, and rOmp22-HpaA groups (P > 0.05). rOmp22-HpaA fusion protein retained immunogenicity and could be used as an antigen candidate in the development of an oral vaccine against H. pylori infection.

The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis.

Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease.

Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2).

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.