Peptide functionalized biomimetic gene complexes enhance specificity for vascular endothelial regeneration.

Gene delivery presents great potential in endothelium regeneration and prevention of vascular diseases, but its outcome is inevitably limited by high shear stress and instable microenvironment. Highly efficient nanosystems may alleviate the problem with strong dual-specificity for diseased site and targeted cells. Hence, biomimetic coatings incorporating EC-targeting peptides were constructed by platelets and endothelial cells (ECs) for surface modification. A series of biomimetic gene complexes were fabricated by the biomimetic coatings to deliver pcDNA3.1-VEGF165 plasmid (pVEGF) for rapid recovery of endothelium. The gene complexes possessed good biocompatibility with macrophages, stability with serum and showed no evident cytotoxicity for ECs even at very high concentrations. Furthermore, the peptide modified gene complexes achieved selective internalization in ECs and significant accumulation in endothelium-injured site, especially the REDV-modified and EC-derived gene complexes. They substantially enhanced VEGF expression at mRNA and protein levels, thereby enabling a wound to heal completely within 24 h according to wound healing assay. In an artery endothelium-injured mouse model, the REDV-modified and EC-derived gene complexes presented efficient re-endothelialization with the help of enhanced specificity. The biomimetic gene complexes offer an efficient dual-targeting strategy for rapid recovery of endothelium, and hold potential in vascular tissue regeneration.

Bovine serum albumin-based biomimetic gene complexes with specificity facilitate rapid re-endothelialization for anti-restenosis.

Re-endothelialization is a critical problem to inhibit postoperative restenosis, and gene delivery exhibits great potential in rapid endothelialization. Unfortunately, the therapeutic effect is enormously limited by inefficient specificity, poor biocompatibility and in vivo stability owing largely to the complicated in vivo environment. Herein, we developed a series of platelet membrane (PM) cloaked gene complexes based on natural bovine serum albumin (BSA) and polyethyleneimine (PEI). The gene complexes aimed to accelerate re-endothelialization for anti-restenosis via pcDNA3.1-VEGF165 (VEGF) plasmid delivery. Based on BSA and PM coating, these gene complexes exhibited good biocompatibility, stability with serum and robust homing to endothelium-injured site inherited from platelets. Besides, they enhanced the expression of VEGF protein by their high internalization and nucleus accumulation efficiency, and also substantially promoted migration and proliferation of vascular endothelial cells. The biological properties were further optimized via altering PEI and PM content. Finally, rapid recovery of endothelium in a carotid artery injured mouse model (79% re-endothelialization compared with model group) was achieved through two weeks' treatment by the PM cloaked gene complexes. High level of expressed VEGF in vivo was also realized by the gene complexes. Moreover, neointimal hyperplasia (IH) was significantly inhibited by the gene complexes according to in vivo study. The results verified the great potential of the PM cloaked gene complexes in re-endothelialization for anti-restenosis. STATEMENT OF SIGNIFICANCE: Rapid re-endothelialization is a major challenge to inhibit postoperative restenosis. Herein, a series of biodegradable and biocompatible platelet membrane (PM) cloaked gene complexes were designed to accelerate re-endothelialization for anti-restenosis via pcDNA3.1-VEGF165 (VEGF) plasmid delivery. The PM cloaked gene complexes provided high VEGF expression in vascular endothelial cells (VECs), rapid migration and proliferation of VECs and robust homing to endothelium-injured site. In a carotid artery injured mouse model, PM cloaked gene complexes significantly promoted VEGF expression in vivo, accelerated re-endothelialization and inhibited neointimal hyperplasia due to their good biocompatibility and superior specificity. Overall, the optimized PM cloaked gene complexes overcomes multiple obstacles in gene delivery for re-endothelialization and can be a promising candidate for gene delivery and therapy of postoperative restenosis.

5-Boronopicolinic acid-functionalized polymeric nanoparticles for targeting drug delivery and enhanced tumor therapy.

Strong specificity for cancer cells is still the main challenge to deliver drugs for the therapy of cancer. Herein, we developed a convenient strategy to prepare a series of 5-boronopicolinic acid (BA) modified tumor-targeting drug delivery systems (T-DDSs) with strong tumor targeting function. An anti-tumor drug of camptothecin (CPT) was encapsulated into poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-PEI) to form drug-loaded nanoparticles (NP/CPT). Then, the surface of NP/CPT was coated by BA with different polymer and BA molar ratios of 1:1, 1:5, 1:10 and 1:20 via electrostatic interaction to obtain T-DDSs with enhanced biocompatibility and specificity for tumor cells. The introduced BA can endow drug-loaded NPs with high targeting ability to tumor cells because of the overexpression of sialic acids (SA) in tumor cells, which possessed strong interaction with BA. Those T-DDSs exhibited good biocompatibility according to the results of MTT assay, hemolysis test and cellular uptake. Moreover, they were capable of decreasing the viability of breast cancer cell line 4T1 and MCF-7 cells with no obvious cytotoxicity for endothelial cells. Especially, T-DDS with 1:20 molar ratio displayed much higher cellular uptake than other groups, and also exhibited highly efficient in vivo anti-tumor effect. The significantly high targeting function and biocompatibility of T-DDSs improved their drug delivery efficiency and achieved good anti-tumor effect. The BA decorated T-DDSs provides a simple and robust strategy for the design and preparation of DDSs with good biocompatibility and strong tumor-specificity to promote drug delivery efficiency.

Delivery of benzoylaconitine using biodegradable nanoparticles to suppress inflammation via regulating NF-κB signaling.

Rheumatoid arthritis (RA) is a kind of systemic autoimmune disease, and patients with RA usually suffer serious pain, resulting in low quality of life. The development of drug delivery systems (DDSs) provides a valid approach for RA therapy via inhibiting the secretion of inflammatory cytokines from macrophages. As a prevailing drug nanocarrier with distinctive superiority, polymeric nanoparticles (NPs) have attracted much attention in recent years. However, low biocompatibility and limited exploitation of drug with high efficiency are still the main challenges in RA treatment. To overcome the limitations, we prepared a biocompatible copolymer methoxy-poly(ethylene glycol)-poly(lactide-co-glycolide) (mPEG-PLGA). Moreover, benzoylaconitine (BAC) with superior anti-inflammatory effect was selected as model drug. It was isolated from Aconitum kusnezoffii Reichb and encapsulated into mPEG-PLGA NPs (NP/BAC) to increase the bioavailablity of BAC. The NPs exhibited high cytocompatibility for activated macrophages and well compatibility with red blood cells. Furthermore, the anti-inflammatory property of NP/BAC was testified by substantially inhibiting secretion of pro-inflammatory cytokines. The TNF-α and IL-1ß cytokines of NP/BAC group reduced 70 % and 66 % compared with that of activated macrophages. Especially, NP/BAC reduced the overexpression of NF-κB p65 to inhibit NF-κB signaling pathway, which was a critical regulator of inflammatory responses. NP/BAC also showed efficient in vivo anti-inflammatory effect with high ear (69.8 %) and paw (87.1 %) swelling suppressing rate. These results revealed the anti-inflammatory mechanism of NP/BAC and proved it was a suitable DDS to suppress inflammation, providing a promising strategy for RA therapy and research of Aconitum kusnezoffii Reichb.

Immunogenicity Assessment of Rift Valley Fever Virus Virus-Like Particles in BALB/c Mice.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and is spread by arthropod vectors. Virus-like particle (VLP) vaccines, which have the advantages of strong immunogenicity and safety, play an important role in the prevention of this disease. VLPs for RVFV were successfully prepared by our research group using a baculovirus-insect cell expression system. To study the immunogenicity of these RVFV VLPs, a correct 3rd or 4th generation recombinant baculovirus, rBac-N-G, was identified and used to infect Sf9 cells, which were cultured in suspension at a large scale. Subsequently, cell debris was removed by centrifugation, and the VLPs were concentrated by ultracentrifugation and purified using a sucrose gradient, after which they were used to immunize BALB/c mice by intramuscular injection. The results showed that the RVFV VLPs prepared by our research group could effectively induce mice to produce RVFV neutralizing antibodies and that the prepared VLPs could stimulate mouse spleen cells to produce high levels of the cytokines IL-4 and IFN-γ. Moreover, the proportion of lymphocytes producing IL-4 and IFN-γ in the spleen of mice immunized with RVFV VLPs was significantly increased. Therefore, the RVFV VLPs prepared in this study had strong immunogenicity and could effectively activate humoral and cellular immunity in mice. This study lays a solid foundation for the development of RVFV VLP vaccine candidates and promotes the healthy development of animal husbandry and human public health.

Assembly of pigeon circovirus-like particles using baculovirus expression system.

Pigeon circovirus (PiCV) is able to infect racing and meat pigeons of all ages and is a key factor that triggers young pigeon disease syndrome (YPDS). PiCV vaccine research has been impeded because PiCV cannot be grown or propagated in cell cultures. Virus-like particles (VLPs), which can be generated by a wide range of expression systems, have been shown to have outstanding immunogenicity and constitute promising vaccines against a wide range of pathogens. Cap protein, which contains neutralizing antibody epitopes, is the only capsid protein of PiCV. In this study, the baculovirus expression system was utilized to express the PiCV Cap protein, which was self-assembled into VLPs with a spherical morphology and diameters of 15-18 nm. Specific antibodies against the Cap protein were induced after BALB/c mice immunized intramuscularly (i.m.) with VLPs combined with adjuvant. Based on these findings, PiCV VLPs may be a promising candidate vaccine against PiCV.

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2.

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

Equine-Origin Immunoglobulin Fragments Protect Nonhuman Primates from Ebola Virus Disease.

Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

Equine immunoglobulin F(ab')2 fragments protect mice from Rift Valley fever virus infection.

BACKGROUND: Rift Valley fever virus (RVFV) is an emerging arbovirus in Africa and the Arabian Peninsula, in which infection with RVFV poses a serious threat to humans and livestock globally. Approved treatments for RVFV infection, especially for use in humans, have not yet been developed. There is an urgent need for effective drugs to prevent RVFV disease. METHODS: In previous study, we developed RVFV virus like particles (VLPs) expressing the surface glycoproteins Gn and Gc. The morphology was shown to be similar to live RVFV under electron microscopy. In this study, we immunized horses with RVFV VLPs, prepared the immunoglobulin F(ab')2 fragments, and characterized its in vitro neutralization and in vivo efficacy in mice. RESULTS: F(ab')2 was found to potently neutralize RVFV in VeroE6 cells, and passive transfer of immunoglobulin F(ab')2 fragments resulting in reduced mortality in RVFV infected mice. CONCLUSION: Our results show that passive immunotherapy with equine immunoglobulin F(ab')2 fragments is a promising strategy to treat RVFV infections.

Packaging of Rift Valley fever virus pseudoviruses and establishment of a neutralization assay method.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV). RVF is mainly prevalent on the Arabian Peninsula, the African continent, and several islands in the Indian Ocean near southeast Africa. RVFV has been classified by the World Organisation for Animal Health (OIE) as a category A pathogen. To avoid biological safety concerns associated with use of the pathogen in RVFV neutralization assays, the present study investigated and established an RVFV pseudovirus-based neutralization assay. This study used the human immunodeficiency virus (HIV) lentiviral packaging system and RVFV structural proteins to successfully construct RVFV pseudoviruses. Electron microscopy observation and western blotting indicated that the size, structure, and shape of the packaged pseudoviruses were notably similar to those of HIV lentiviral vectors. Infection inhibition assay results showed that an antibody against RVFV inhibited the infective ability of the RVFV pseudoviruses, and an antibody neutralization assay for RVFV detection was then established. This study has successfully established a neutralization assay based on RVFV pseudoviruses and demonstrated that this method can be used to effectively evaluate antibody neutralization.

Immunization with recombinant rabies virus expressing Interleukin-18 exhibits enhanced immunogenicity and protection in mice.

Several studies have shown that interleukin-18 (IL-18) plays an important role in both innate and adaptive immune responses. In this study, we investigated the pathogenicity and immunogenicity of recombinant rabies virus expressing IL-18 (rHEP-IL18). Experimental results showed that Institute of Cancer Research (ICR) mice that received a single intramuscular immunization with rHEP-IL18 elicited the highest titers of serum neutralizing antibodies and the strongest cell-mediated immune responses to prevent the development of rabies disease, compared with immunization with the parent virus HEP-Flury. Mice inoculated with rHEP-IL18 developed significantly higher IFN-γ responses, increased percentages of CD4+ and CD8+ T-lymphocytes compared to HEP-Flury. Flow cytometry results show that rHEP-IL18 recruited more activated T- and B-cells in lymph nodes or peripheral blood, which is beneficial for virus clearance in the early stages of infection. A higher percentage of mice immunized with rHEP-IL18 survived wild-type rabies virus (RABV) challenge, compared to HEP-Flury mice. Our results show that rHEP-IL18 is promising as a novel vaccine for RABV prevention and control.

Marburg virus-like particles by co-expression of glycoprotein and matrix protein in insect cells induces immune responses in mice.

BACKGROUND: Marburg virus (MARV) causes severe haemorrhagic fever in humans and nonhuman primates and has a high mortality rate. However, effective drugs or licensed vaccines are not currently available to control the outbreak and spread of this disease. METHODS: In this study, we generated MARV virus-like particles (VLPs) by co-expressing the glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. MARV VLPs and three adjuvants, Poria cocos polysaccharide (PCP-II), poly(I:C) and aluminium hydroxide, were evaluated after intramuscular vaccination in mice. RESULTS: Murine studies demonstrated that vaccination with the MARV VLPs induce neutralizing antibodies and cellar immune responses. MARV VLPs and the PCP-II adjuvant group resulted in high titres of MARV-specific antibodies, activated relatively higher numbers of B cells and T cells in peripheral blood mononuclear cells (PBMCs), and induced greater cytokine secretion from splenocytes than the other adjuvants. CONCLUSION: MARV VLPs with the PCP-II adjuvant may constitute an effective vaccination and PCP-II should be further investigated as a novel adjuvant.

DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice.

The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 104) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4+ and CD8+ T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection.

Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation.

MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans with a case fatality rate of over 39%, and poses a considerable threat to public health. A lack of approved vaccine or drugs currently constitutes a roadblock in controlling disease outbreak and spread. In this study, we generated MERS-CoV VLPs using the baculovirus expression system. Electron microscopy and immunoelectron microscopy results demonstrate that MERS-CoV VLPs are structurally similar to the native virus. Rhesus macaques inoculated with MERS-CoV VLPs and Alum adjuvant induced virus-neutralizing antibodies titers up to 1:40 and induced specific IgG antibodies against the receptor binding domain (RBD), with endpoint titers reaching 1:1,280. MERS-CoV VLPs also elicited T-helper 1 cell (Th1)-mediated immunity, as measured by ELISpot. These data demonstrate that MERS-CoV VLPs have excellent immunogenicity in rhesus macaques, and represent a promising vaccine candidate.

Passive immunotherapy for Middle East Respiratory Syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model.

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab')2 from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab')2 efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab')2 for potential use in the prophylactic or therapeutic treatment of exposed or infected patients.

Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection.

Recent successes with monoclonal antibody cocktails ZMapp(TM) and MIL77 against Ebola virus (EBOV) infections have reignited interest in antibody-based therapeutics. Since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. We produced purified equine antisera from horses hyperimmunized with EBOV virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. BALB/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. To decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate F(ab')2 fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. Full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other Fc-dependent antibody mechanisms may also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab')2 at or starting at 1 or 2 dpi were also fully protected from EBOV infection. These results justify future efficacy studies for purified equine products in NHPs.

Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection.

Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.

Isatis indigotica root polysaccharides as adjuvants for an inactivated rabies virus vaccine.

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.