A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3’- region, named sopB4 and sopB5, were each fused to the gvpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.
Two chimeric recombinant fusion proteins (ch-rOspC and ch-rOspA) were created. They are composed of the immunodominant domains of OspC and OspA proteins described in the clinically most important strains of Bon-elia. The gene constructs for these chimeric proteins were inserted into plasmids pET28 allowing induced gene expressions in a bacterial system.The proteins were expressed in E. coli BL21 strains, purified and used for preparation of the vaccine. One dose of the tested vaccines contained 50 lug of each relevant protein (ch-rOspC, ch-rOspA, or ch-rOspC+chrOspA). PET GEL A (Seppic) or Aluminium hydroxide gel as the immune adjuvants were used.The dogs were vaccinated three times at 21 days intervals subcutaneously or intradermally and unvaccinated controls were also included.The vaccine-elicited serum action antibodies specific to OspA and OspC were determined using in-house ELISA sets.The immunisation induced specific antibody response in the vaccinated animals and OspC and OspA from representative genospecies B. garinii, B. afzelii, and B. burgdorferi sensu stricto were recognized The control dogs were without antibody response.ELISA examination enables determination of specific post-vaccination antibodies against OspA and OspC. Detection of these antibodies and their quantification may be used for evaluation of efficiency of vaccines. (C) 2015 Published by Elsevier B.V.
Analysis of human monoclonal antibodies (mAbs) developed from influenza infected donors have enormously contributed to the identification of neutralization sensitive epitopes of influenza virus. The HA protein is a crucial target of neutralizing antibodies and at monoclonal level only Abs binding to HA have been able to neutralize the virus. In this study, eight A (H1N1)pdm 2009 seropositive patients within the age range of 20-50 years (median=36 years) were recruited. Two anti-HA mAbs secreting stable clones, 2D8 and 2F12 were established under optimized conditions from the peripheral blood mononuclear cells (PBMCs) of the volunteers. These antibodies efficiently neutralized the homologous laboratory isolated strain of the pandemic virus as well as the reference strain. Our study suggests that the anti-HA antibodies derived from infected Indian patients display neutralization potential against the A(H1N1)pdm 2009 virus. This is the first ever study of generation of mAbs against the pandemic influenza virus involving the immune repertoire if Indian patients. Molecular characterization of the target regions will help in identifying potential immunogens in the Indian pandemic isolates and confer protective immunity against this virus.
Development of influenza vaccine processes requires virus quantification to optimize conditions in cell culture or in the associated downstream purification steps. Modern methods include qPCR, which utilizes TaqMan chemistry to detect and quantify viral RNA by comparison of a RNA standard of known concentration. Digital droplet PCR (ddPCR) is similar to qPCR in that it shares the same chemistry for nucleic acid detection. However, in ddPCR, the sample is diluted into partitions (‘droplets’) in order to separate and isolate single molecules. Upon PCR amplification, the droplet's fluorescent intensity depends on the presence or absence of the target; as such, positive and negative droplets are identified, which allows for absolute quantification of the viral genomes. The digital approach has enabled several key advantages. First, a standard is no longer required. Second, efficiency of the reverse transcription and the kinetics of the amplification, principles in qPCR, have no impact on the final digital PCR quantification. For this reason, the extracted RNA does not need to be purified from the reagents needed to lyse the virus. Also, viral associated RNA released by infected cells can be measured directly, further improving the quality of the data generated. Additional improvements to the approach include duplexing with a second assay that measures host cell DNA concentration. The method has been successfully implemented with automation in support of multiple upstream and downstream process development efforts for influenza vaccine manufacturing.
IL-15 cDNA of Tibetan pig was firstly cloned from its activated lymphocytes, and then was sub-cloned into VR1020 to construct recombinant VRTIL-15 plasmid to study the in vitro and in vivo biological effects on animal. The VRTIL-15 was entrapped with chitosan modified with PEG-PEI (CS-PEG-PEI) to transfect HEK293 cells for the preliminary study of its expression in eukaryotic cells. The total RNA of HEK293 cell was isolated in 48h, and the successful expression of IL-15 was detected by RT-PCR and the supernatant of HEK293 cells was found to stimulate significant proliferation of lymphoblasts of pig. Subsequently, VRTIL-15 packed with CS-PEG-PEI was utilized to intramuscularly inoculate Kunming female mice at the age of 21 days. Their bloods were collected before and after inoculation on 1, 2, 3, 4 and 5 weeks to detect the changes of innate and adaptive immunity of animals. The results were found that Th and Tc, specific antibody to FMD, IgG, IgG1, IgG2a content markedly increased in the blood of treated mice compared with the control group (P<0.05). The mRNA expression of TLR1, TLR4 TLR6, TLR9, TGF-β, IL-2, IL-4, IL-6 and IL-23 were significantly higher in the treated group than those of the control (P<0.05). These results indicate that the VRTIL-15 wrapped with CS-PEG-PEI can significantly improve the innate, humoral and cellular adaptive immunity of animal, which could inspire the development of effective immune adjuvant to improve the comprehensive immune protection of animals against FMD.
DNA vaccines are simple to produce and can generate strong cellular and humoral immune response, making them attractive vaccine candidates. However, a major shortcoming of DNA vaccines is their poor immunogenicity when administered intramuscularly. Transcutaneous immunization (TCI) via microneedles is a promising alternative delivery route to enhance the vaccination efficacy. A novel dissolving microneedle array (DMA)-based TCI system loaded with cationic liposomes encapsulated with hepatitis B DNA vaccine and adjuvant CpG ODN was developed and characterized. The pGFP expression in mouse skin using DMA was imaged over time. In vivo immunity tests in mice were performed to observe the capability of DMA to induce immune response after delivery of DNA. The results showed that pGFP could be delivered into skin by DMA and expressed in skin. Further, the amount of expressed GFP was likely to peak at day 4. The immunity tests showed that the DMA-based DNA vaccination could induce effective immune response. CpG ODN significantly improved the immune response. The cationic liposomes could further improve the immunogenicity of DNA vaccine. In conclusion, the novel DMA-based TCI system can effectively deliver hepatitis B DNA vaccine into skin, and induce effective immune response.
The outer domain of gp120 is a relatively stable domain compared to the inner domain and bridging sheet at the CD4-binding site for the HIV-1 primary receptor. Therefore, the outer domain has been considered as an immunogen candidate for vaccine design. In this report, we focused on the VRC01 antibody binding epitope in the outer domain and evaluated the effects of introducing two disulfides to further stabilize the outer domain structure where the antibody binds for the purpose of generating a more effective immunogen. Our experimental data based on neutralization activities against HIV-1 of anti-sera produced from immunized guinea pigs demonstrated that this stabilized outer domain-based immunogen significantly enhances the specific immune response when compared to its wild-type outer domain counterpart. These findings strongly suggest that this structure-based designed epitope is effective in eliciting specific neutralizing antibodies against diverse HIV-1strains, including subtype C.
In order to develop cost-effective immunomodulator, the recombinant Pichia pastoris were firstly constructed to co-express porcine IL-2/4/6 genes, and then fermented to feed 45-days Tibetan piglets at different doses to evaluate its effects on immunity of piglets to PRRS vaccination, which simultaneously received intramuscular injection of inactivated PRRS vaccine. The results were found that the leukocytes, IgG and specific antibody to PRRSV, Th and Tc cells increased significantly in the blood of treated piglets in comparison with those of the control (P < 0.05); the mRNA expression of TLRs (TLR-2, 3, 4, 7, 9), IFN-γ, IL-2, IL-4, IL-6, IL-7, IL-12 and IL-15 genes were elevated significantly in the immune cells from the blood of treated piglets (P < 0.05). Moreover, the growth of the treated piglets also markedly improved whose average net weight gain was significantly higher than the control on 58 days post inoculation (P < 0.05). These results suggest that the recombinant yeast can effectively enhance the systematic innate and adaptive immunity of piglets as well as promote the growth of piglet, which could be further developed as cost-effective promising immunomodulator to improve the control of pig PRRS disease.
The prevention and treatment of infectious diseases is highly dependent on the availability of reliable diagnostic tests and protective or therapeutic vaccines. There also exists an urgent need to develop reliable biomarkers to monitor treatment success and to predict disease progression from asymptomatic to symptomatic disease in several disease scenarios. The elucidation of the disease-relevant antigens that elicit the protective immune responses is critical and required for the development of biomarkers, diagnostics, and vaccines. However; one of the main obstacles to the study of antigen specificity in human T cells is their low frequency in PBMC samples. To overcome this problem we have implemented strategies to generate memory T cell libraries and clones specific to the pathogen of interest. Due to the fact that memory T cells represent a repository of the human T cell response to infection, examination of their antigen specificity can efficiently reveal immunogenic and relevant antigens involved in the in vivo response to infection or vaccines. To examine the specificity of the memory T cells we use an unbiased collection of antigens together with an in silico analysis, namely positional scanning based biometrical analysis. Here we present a summary of our approach and ongoing work on the development of strategies for the culture of memory T cells from patients with Chagas disease. While most studies focus on the identification of vaccine candidates using preselected immunogenic proteins derived from animal models or by or bioinformatics prediction, here we present an innovative approach that directly examines the specificity of the memory response following infection or immunization in humans.
The high incidence of Campylobacter jejuni-associated diarrhoea, the increase in the frequency of drug resistance, and the correlation between infection and Guillain-Barré syndrome, has heightened the need to develop effective anti-Campylobacter vaccines. Due to the risk of auto-immunity, vaccinations using whole-cells or attenuated cells are not clinical candidates. Subunit vaccines are viable alternatives and have been shown to be effective against other enteric pathogens. This research tested the efficacy of the catalase (KatA) from C. jejuni as a potential vaccine candidate against the pathogen. The protein was found to be immunogenic following an intramuscular murine immunization. A balanced IgG1/IgG2a response was generated when KatA was co-administered with monophosphoryl lipid A and alum. The antibodies elicited reduced the adhesion and invasion of C. jejuni to human epithelial cells (IECs) in vitro. The next phase of testing will be in combination with a proteoliposome (PL) derived from the outer membrane of V. cholerae. PL nano-vesicles were generated with an average size of ≈70nm and had a zeta potential of ≈-40mV.
Background: Guidelines for protecting young infants from pertussis now recommend maternal pertussis booster vaccination (dTpa) during pregnancy (3 rd trimester) over postpartum maternal vaccination.Methods: We undertook a cross-sectional survey of 723 postpartum women on the postnatal ward of a private hospital in Sydney, Australia. We excluded 47% of women who were 'up to date' with their pertussis booster prior to pregnancy. We surveyed the remaining 53% of women who were eligible for the pertussis booster vaccine during their pregnancy (no dTpa<5years).Results: In our study, pertussis booster vaccine uptake during pregnancy was only 8.7%. We confirmed that vaccination mostly occurred at the family doctor's practice (25/33; 75.8%), at an average of 34 weeks gestation ( 5wks). Women vaccinated during pregnancy were more likely to report that they received a pregnancy-specific pertussis vaccine recommendation (<0.001), and had no vaccine safety concerns (0.004) or transport difficulties in accessing an immunization provider (0.032). They also had twice the odds of receiving an influenza vaccine during pregnancy (0.002). Additionally, 70% of vaccinated women reported that their partner had also been recently vaccinated against pertussis. Amongst unvaccinated women we found that 80% intended to be vaccinated, yet 33% would only do so if the vaccine was free (publically funded). Additionally, 40% of unvaccinated women would have agreed to pertussis vaccination during pregnancy had their doctor recommended it.Conclusion: Providers should highlight pertussis vaccine safety and recommend vaccination during late pregnancy. (C) 2015 The Authors. Published by Elsevier B.V.
Vulvovaginal candidiasis (VVC) affects a significant number of women, especially in working age. In an estimated 75% of women an episode of acute vulvovaginal candidiasis occurs during lifetime and another 5 - 10% of women develop recurrent vulvovaginal candidiasis (RVVC). This is mainly characterized by intense burning, itching, pain, abnormal discharge, dyspareunia. Immune response to candidiasis is both cellular (CMI) (natural protection mechanisms) and humoral (antibody production). Understanding the principles of immunity in candidiasis is also important for development of candida vaccines. CANDIVAC contains lyophilized Candida lysate (C. albicans, C. krusei, C. glabrata) together with immunostimulatory bacterial strain of Propionibacterium acnes. The product is taken orally in capsules for 10 days followed by a 20-day pause. It is administered for 3 to 6 months. The product has been tested in a total of 75 women at the age of 18 - 45 years. In these women at least 4 episodes of vulvovaginal candidiasis have been microscopically or laboratory diagnosed during the last 12 months. Following CANDIVAC administration, statistically significant changes occurred in the evaluation of subjective and some objective criteria. The most important marker of product efficiency is a significant reduction in recurrence compared to the recent state. This criterion has a fundamental importance in patient satisfaction. Before medication the patients suffered from at least 4 attacks, while after medication an attack occurred in only 31% of women and more than 2 attacks in only 3% of treated women. Compromised balance of immune system plays a major role in recurrent vulvovaginal candidiasis. Specific oral product CANDIVAC, prepared from the most common strains of yeast infections, supports immune mechanisms, ensuring resistance of the human organism against yeasts. Its administration significantly prolongs remission, leads to a reduction in application of antimycotics and also changes properties of cellular and humoral immunity in medicated patients. (C) 2015 The Authors. Published by Elsevier B.V.
Staphylococcus aureus is the most important pathogen found in sea foods. Food poisoning in human may happen due to the consumption of aqua products contaminated with this bacteria and its enterotoxin. The procedures carried out to maintain and preserve the quality of these products, from the time they are fished and transported to stores until they are consumed, can play a major role in the generation and growth of pathogenic bacteria and toxins. A total of 300 samples were collected, including (fresh and frozen, farm and marine). Consistent with the Iran National Standards, a number of phenotypical and molecular assays were utilized for screening S. aureus in order to detect Staphylococcus aureus. They study was conducted from September 2013 to March 2014. Baird Parker agar containing egg-yolk and tellurite emulsion were used for isolation. Isolates were identified using the following criteria: production of coagulase, DNase, catalase, mannitol fermentation, hemolytic zone on 5% sheep blood agar, VP test and Gramstaining A total of 74 samples (24.6%), were contaminated with Staphylococcus aureus. Due to the presence of Staphylococcus aureus in shrimps, it is necessary to enforce quality control standards by the fisheries and carefully monitor fishing, farming, preparation, freezing, and transporting marine products, and ensure the health of workers. The results of this study also showed it is necessary to produce and develop a vaccine to prevent the disease and sea-food poisoning caused by Staphylococcus aureus. (C) 2015 Published by Elsevier B.V.
Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis.
For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised.
Cervical cancer is a major public health concern in Morocco where it represents the second most common and lethal cancer in women. Human papillomavirus (HPV) vaccines have been licensed in Morocco since 2008 but there is no available data on their acceptability. This study aimed to assess awareness of HPV and the vaccine, and to identify factors associated with acceptability of the vaccine among parents in Morocco. A questionnaire-based survey using face-to-face interviews was conducted in a sample of 852 parents (670 mothers and 182 fathers) with at least one unmarried daughter ≤26 years. The study took place within public and private health centres and clinics in four regions in Morocco between July and August 2012. The main outcome measure was willingness to consider vaccinating a daughter against HPV. Responses revealed very low awareness of HPV infection (5%) and the HPV vaccine (14%). None of the participants had vaccinated their daughters against HPV and only 35% (32% of mothers and 45% of fathers) were willing to consider doing so in the future. Higher education and income, previous awareness of the HPV vaccine and endorsement of the belief that a recommendation from the ministry of health or a doctor to have the vaccine would be encouraging, were associated with mothers’ HPV vaccine acceptance. Non-acceptance among mothers was associated with having more than two daughters, believing the vaccine was expensive and lack of information. The only factor associated with the fathers’ acceptance of the vaccine was the cost. Increasing HPV and HPV vaccine awareness through educational campaigns, along with active recommendation by physicians and a publically funded vaccination programme could increase parental acceptance of the vaccine in Morocco.
Nanoparticles (NPs) have great potential as advanced delivery systems for cancer immunotherapy. PEGylated-Poly- lactide-co-glycolic acid-based (PLGA-PEG) NPs were prepared by double-emulsion solvent evaporation technique, using ovalbumin (OVA) as a model antigen. Glycol Chitosan and block co-polymer Pluronic F127 were used in order to best attain the most efficient parameters for cancer immunotherapy. OVA-loaded PLGA-PEG NPs presented a narrow size distribution with an average size of 167nm witha polidisperity index (PdI)0.167 and zeta potential values close to neutrality (-1.66mV), which is desired for a particulate cancer vaccine to overcome their premature capture by macrophages. The encapsulation efficiency (EE) and loading capacity (LC) of these NPs were 57.5% and 29μg/mg, respectively. PLGA-PEG NPs modified with Pluronic F127 presented slightly higher Z- Average (180nm with a PdI 0.18), and ZP (ZP -1.78mV), but lower EE and LC (32% and 16μg/mg). The effect of NPs on dendritic cell viabilitywas evaluated using Alamar Blue® assays.
Visceral leishmaniasis is a chronicand lethal parasite disease against which no human vaccine is available. Hepatosplenomegaly and a progressive suppression of the cellular immune response are among its most important clinical signs. The characteristic cellular immunosupression was described as being mediated in part, through the spatial segregation of dendritic cells (DCs) and T cell lymphocytes due to altered frequencies and migration capabilities of DCs. In this investigation, we measured the spleen/body relative weight, the spleen parasite load and the total counts of spleen DCs of C57BL6 mice infected with Leishmania chagasi. All the variables achieved their maximum at 30 days after infection. We detected in infected animals a 5.08 fold increase of spleen relative weight, a 19.6 fold increase of parasite load and a 4.55 increase of total DCs counts, when compared to naive controls. We further analysed the efficacy of the NH36 and F3 vaccines formulated in saponin in prevention of visceral leishmaniasis. When compared to the infected controls, both vaccines determined strong protection. The F3 vaccine induced the highest efficacy showing 95% and 49% reduction the parasite load and splenomegaly, respectively. The NH36 vaccine, on the other hand, developed a slightly lower but still significant protection reducing by 87% the parasite load and by 39% the spleen relative weight. Both vaccines also prevented the increase in total counts of DCs with no significant difference between them (36% by the NH36 and 26% by the F3 vaccine). Our results suggest that vaccination against murine visceral leishmaniasis with the NH36 vaccine can prevent the development of the disease by preventing the DCs dysfunction-related immunosupression. Additionally, they disclose the potential use of the NH36 C-terminal moiety, the F3 peptide for optimization of the vaccine efficacy. (C) 2014 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/3.0/).
