Journal of Immune Based Therapies and Vaccines - Most viewed articles

Immunostimulatory effects of three classes of CpG oligodeoxynucleotides on PBMC from HCV chronic carriers

2008-06-09

Background: Chronic hepatitis C virus (HCV) infection results from weak or absent T cell responses. Pegylated-interferon-alpha (IFN-α) and ribavirin, the standard of care for chronic HCV, have numerous immune effects but are not potent T cell activators. A potent immune activator such as TLR9 agonist CpG oligodeoxynucleotide (CpG) may complement current treatment approaches. Methods: Peripheral blood mononuclear cells (PBMC) obtained from HCV chronic carriers who failed previous treatment and from healthy donors were incubated in vitro with the three main CpG classes (A, B or C), recombinant IFN-α-2b (IntronA) and/or ribavirin. Proliferation and cytokine secretion (IFN-α, IL-10 and IP-10) were evaluated. Results: CpG induced proliferation and cytokine secretion in patterns expected for each CpG class with similar group means for HCV and healthy donors. IntronA and ribavirin, alone or together, had no detectable effects. IntronA and C-Class CpG together induced more IFN-α than CpG alone in most subjects. IFN-α secretion was proportional to the number of plasmacytoid dendritic cells in PBMC from healthy donors but not HCV donors in whom responses were highly heterogeneous. Conclusion: The strong immune stimulatory effect of CpG on PBMC isolated from treatment-failed HCV patients suggests possible utility alone or in combination with current HCV antiviral treatment.

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

2008-04-28

Background: As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described. Methods: Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity. Results: By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells. Conclusion: Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.

A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology

2008-03-06

Background: Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor cell lysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies and viral infection with great promise. One of the factors that has limited more widespread use of these vaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivation of mDC in static culture vessels are labor- and time- intensive, and also require many culture vessels. Here, we describe a new method for the large-scale generation of human mDC from human PBMC from leukopheresis or buffy coat products using roller bottles, never attempted before for mDC generation. We have tested this technology using 850 cm2 roller bottles compared to conventional T-175 flat-bottom static culture flasks. Methods: DC were generated from adherent human PBMC from buffy coats or leukopherisis products using GM-CSF and IL-4 in T-175 static flasks or 850 cm2 roller bottles. The cells were matured over two days, harvested and analyzed for cell yield and mature DC phenotype by flow cytometry, and then functionally analyzed for their ability to activate allogeneic T-cell or recall antigen peptide-specific T-cell responses. Results: Monocytes were found to adhere inside roller bottles to the same extent as in static culture flasks. The phenotype and function of the mDC harvested after maturation from both type of culture systems were similar. The yield of mDC from input PBMC in the roller bottle system was similar as in the static flask system. However, each 850 cm2 roller bottle could be seeded with 4–5 times more input PBMC and could yield 4–5 times as many mDC per culture vessel than the static flasks as a result. Conclusion: Our results indicate that the roller bottle technology can generate similar numbers of mDC from adherent PBMC as traditional static flask methods, but with having to use fewer culture vessels. Thus, this may be a more practical method to generate mDC in large-scale cutting down on the amount of laboratory manipulations, and can save both time and labor costs.

An HIV/AIDS Prophylactic vaccine is possible

Qiu Zhong and Ronald B Luftig — 2007-12-19

One needs to think outside of the box, as one of us (Ronald B Luftig) learned from many years as a mathematician, and a biophysicist.In this short Review, the need to focus on producing high levels of neutralizing antibodies (NAbs) to incoming and conformationally altered virus after it has bound to CD4+ cells is essential.Increasing the number of gp120 molecules on the surface of L-2 particles, could allow for an enhanced number of NAbs.The attempt at increasing CD8+ T cell responses in recent vaccine trials has not worked perhaps because it may have allowed HIV to enter into remote sanctuaries. Our approach focuses on increasing NAbs, before high levels of CD8+ T cells are produced.

Rapid generation of an anthrax immunotherapeutic from goats using a novel non-toxic muramyl dipeptide adjuvant

2007-10-22

Background: There is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections. Methods: Using recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication. Results: Anti-PA83 IgG conferred 100% protection at 7.5 μg in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo. Conclusion: The protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations.

Specific antibody response of mice after immunization with COS-7 cell derived avian influenza virus (H5N1) recombinant proteins

2007-10-03

To develop avian influenza H5N1 recombinant protein, the hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural (NS1) of avian influenza H5N1 isolates from Thailand were engineered to be expressed in prokaryotic (E. coli) and mammalian cell (COS-7) system. The plasmid pBAD-His and pSec-His were used as vectors for these inserted genes. Mice immunized with purified recombinant proteins at concentration 50–250 μg intramuscularly with Alum adjuvant at week 0, week 2, and week 3 showed a good immunogenicity measured by ELISA and neutralization assay. The HA and NS recombinant proteins produced in COS-7 cells can induce specific antibody titer detected by neutralization assay significantly higher than corresponding recombinant proteins produced in E. coli system. The antibody produced in immunized mice could neutralize heterologous avian influenza virus determined by micro-neutralization assay. This study shows that avian influenza virus H5N1 recombinant proteins produced in mammalian cell system were able to induce neutralizing antibody response.

Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors

2007-05-03

Background: Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors. Methods: Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFα+IL-1β+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells. Results: Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro. Conclusion: Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

Dendritic cells: In the forefront of immunopathogenesis and vaccine development – A review

Mansour Mohamadzadeh and Ronald Luftig — 2004-01-13

Dendritic cellls (DCs) comprise an essential component of the immune system. These cells, as antigen presenting cells (APCs) to naïve T cells, are crucial in the initiation of antigen specific immune responses. In the past years, several DC subsets have been identified in different organs which exert different effects in order to elicit adaptive immune responses. Thus, identification of such DC subsets has led to a better understanding of their distribution and function in the body. In this review, several key properties of the immunobiology, immunopathogenesis and vaccine strategies using DCs will be discussed.

BCG vaccination at three different age groups: response and effectiveness

George Briassoulis, Irene Karabatsou, Vasilis Gogoglou and Athina Tsorva — 2005-04-01

Background: The protection, which some BCG vaccines could confer against the development of tuberculosis (TB) in childhood, might be indirectly reflected by the subsequent development of BCG immune response. The objectives of the study were to examine effectiveness and possible differences of post-vaccination reaction to a lyophilized BCG at different age groups and to evaluate its protection against TB in a decade's period. Methods: We studied the post-vaccination PPD-skin reaction and scar formation at three different school levels, corresponding to ages of 6, 12 and 15 years old, vaccinated by a lyophilized BCG vaccine (Pasteur Institute), currently used in our country. During a 10-year follow up the reported TB cases in vaccinated and non-vaccinated adolescences up to 24-years old were analyzed and compared to the number of cumulative cases observed in the adult population of two neighboring territories (vaccinated and non-vaccinated).Results and DiscussionThere was a significant correlation (r2 = 0.87, p < 0.0001) between tuberculin induration and scar formation. There was no statistically significant difference between the three age groups (6, 12, and 15 year-old, respectively) in regard to the diameter of tuberculin induration or scar formation. Although 34% of 10-year later indurations were unpredictably related to the initial ones (increased or decreased), they were significantly correlated (r2 = 0.45, p = 0.009). The relative percentage of TB for the 14–24 years-age group to the adult studied population was significantly lower among the immunized children compared to the non-immunized population of the same age group (17/77, 22% vs. 71/101, 70%, p < .0001). Conclusion: Our data suggest that the lyophilized BCG vaccine used for BCG programs at different age groups is equally effective and may confer satisfactory protection against tuberculosis in puberty.

Age-related waning of in vitro Interferon-γ levels against r32kDaBCG in BCG vaccinated children

2007-06-07

Background: Mycobacterium bovis BCG vaccine has displayed inconsistent efficacy in different trials conducted in various geographical regions. Nevertheless, it significantly reduces the risk of severe childhood tuberculosis and continues to be used to prevent tuberculosis in many countries. Many studies revealed that efficacy of vaccine wanes with age. Most of the studies were based on in vivo and in vitro responses to tuberculin. With the advent of newer tests such as in vitro interferon-γ assays and identification of potent immunogenic mycobacterial proteins there is a need to corroborate the observations. This study aims at ascertaining the need for a booster at a later age as indicated by in vitro release of IFN-γ while evaluating Ag85A as an antigen. Methods: Ninety healthy children who were without any clinical evidence of the disease, 45 with a BCG-scar and the remaining 45 without scar and 25 with tuberculosis were included in the study. The incidence of TB was analyzed in 216 children attending a DOTS clinic during 1996–2005. CD3+, CD4+ and CD8+ cell counts were measured by Flow cytometry. r32kDaBCG (Ag85A-BCG) protein was used to stimulate T cells in in vitro T cell responses and interferon-γ (IFN-γ) cytokine levels in the supernatants were measured by ELISA. Results: High incidence of TB was observed in age group 13–14 years followed by children in the age group 10–12 years (Chi-square 242.22; p < 0.000). T cell subsets were within the normal range in all subjects. 79% of vaccinated children showed positive proliferative responses with a mean SI value of 4.98 ± 1.99 while only 39% of the unvaccinated and 58% of the tuberculosis children showed positive responses with mean values of 2.9 ± 1.6 (p < 0.001) and 2.9 ± 1.7(p < 0.057), respectively. The stimulation indices in vaccinated children decreased in the older children concurring with an increase in the incidence of TB. Conclusion: Significantly high levels of in vitro IFN-γ demonstrated in BCG vaccinated children in our study substantiate the observation that BCG is effective in children, but the effect may wane with age. The immunity could be boosted using modified r32kDa (Ag85A) of BCG.