Procedia in vaccinology最新文献

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.

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.

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 Borrelia. 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 μg of each relevant protein (ch-rOspC, ch-rOspA, or ch-rOspC + ch-rOspA). 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.

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.

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.

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.

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.

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.

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 48 h, 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.