Use Of Vaccination Strategies Research Articles

The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis

The Tol–Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in E. coli, and subsequently been identified in many other bacterial genera. However, the function of the Tol–Pal system in Salmonella Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of S. Choleraesuis. We found that mutations in tolA, tolB, and tolR caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased S. Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition, tolA, tolB, and tolR mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from ΔtolB efficiently enhanced murine viability in contrast to OMVs from the wild-type S. Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the S. Choleraesuis Tol–Pal system.

In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes

The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as β-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated β(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1–150 μgml−1. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25–250 μgml−1 in comparison to control (sterile water), cellulose ether, MacroGard® and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 μgml−1. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1β), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.

The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2

ABSTRACTMuramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242–596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239–598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848–1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.

Impact of Preventive Responses to Epidemics in Rural Regions

Various epidemics have arisen in rural locations through human-animal interaction, such as the H1N1 outbreak of 2009. Through collaboration with local government officials, we have surveyed a rural county and its communities and collected a dataset characterizing the rural population. From the respondents’ answers, we build a social (face-to-face) contact network. With this network, we explore the potential spread of epidemics through a Susceptible-Latent-Infected-Recovered (SLIR) disease model. We simulate an exact model of a stochastic SLIR Poisson process with disease parameters representing a typical influenza-like illness. We test vaccine distribution strategies under limited resources. We examine global and location-based distribution strategies, as a way to reach critical individuals in the rural setting. We demonstrate that locations can be identified through contact metrics for use in vaccination strategies to control contagious diseases.

Adjuvants that stimulate TLR3 or NLPR3 pathways enhance the efficiency of influenza virus-like particle vaccines in aged mice

There is intense interest in the design and use of vaccine strategies against influenza to enhance protective immune responses in the elderly. To address the need for improved influenza vaccines for the aged, two inflammatory adjuvants, Imject(®) alum (a stimulator of the Nod-like receptor, Nalp3) and poly I:C (a toll-like receptor type 3 ligand), were used during vaccination with novel influenza virus-like particles (VLP). Adult (4 month old) or aged (24 month old) mice were vaccinated with VLPs alone or in combination with adjuvant. VLP-vaccinated adult mice were protected from a lethal influenza virus challenge without the use of either adjuvant. In contrast, only aged mice that were vaccinated with VLPs plus adjuvant survived challenge, whereas ∼33% of the mice vaccinated with VLP only survived challenge. Mice vaccinated with adjuvant only did not survive challenge despite similar levels of activation of CD11b(+)/CD11c(+) dendritic cells in the lungs. The protection was not associated with HAI titers or HA specific CD8(+) T cells, since both adjuvants boosted the VLP-induced serum HAI titers and CD8(+) responses in adult mice, but not aged mice. Influenza VLPs used in combination with two different inflammatory adjuvants during vaccination allow for the immune system to overcome the deficiency in the aged immune system to influenza virus infection.

DNA vaccines to target the cancer testis antigen PASD1 in human multiple myeloma

We previously described PASD1 as a new cancer testis antigen in multiple myeloma (MM) that is retained post-therapy, suggesting the use of vaccination strategies to induce anti-PASD1 immunity in a setting of minimal residual disease. We have focused on DNA fusion gene vaccines, coupling fragment C domain (DOM) of tetanus toxin with PASD1 sequence, and examined efficacy in Human Leukocyte Antigen (HLA)-A2 (HHD) transgenic mice using a human MM cell line expressing PASD1 protein and chimeric HLA-A2 class I molecules as target. DNA vaccines encoded two HLA-A2-restricted epitopes (p.DOM-PASD1(1), p.DOM-PASD1(2)) and full-length PASD1 (p.DOM-PASD1FL). p.DOM-PASD1(1) proved superior to p.DOM-PASD1(2) in generating T-cell responses in HHD mice, able to lyse the chimeric murine RMA-HHD cells. Boosting by electroporation significantly enhanced p.DOM-PASD1(1). Only p.DOM-PASD1(1) induced cytotoxic T-lymphocytes (CTLs) were able to lyse human MM target cells expressing endogenous antigen. The p.DOM-PASD1FL vaccine predominantly induced strong PASD1(1) over PASD1(2) T-cell immune responses, indicative of immunodominance. Importantly, p.DOM-PASD1FL generated immune-mediating killing of native chimeric MM cells, in the absence of exogenous added peptide, implicating PASD1(1) specific CTLs. These data demonstrate that PASD1-derived epitopes are both efficiently and selectively processed and presented by native human MM cells. Notably, they permit the use of PASD1-encoding DNA vaccine therapy in a clinical setting.

Vaccinia viruses with mutations in the E3L gene as potential replication-competent, attenuated vaccines: Intra-nasal vaccination

Vaccinia virus (VACV) has been used as the vaccine to protect against smallpox, and recombinant VACVs have been used to develop vaccine candidates against numerous cancers and infectious diseases. Although relatively safe for use in humans, the strains of VACV that were used as smallpox vaccines led to several complications including, progressive infection in immune compromised individuals, eczema vaccination in individuals with a history of atopic dermatitis, and encephalitis and perimyocarditis in apparently healthy individuals. The work described in this paper focuses on attenuated strains of VACV that may have the potential for use as vaccine vectors with reduced pathogenicity. We have generated several VACV mutants in a WR background with specific mutations in the E3L gene that were at least a 1000-fold less pathogenic compared to wtVACV upon intra-nasal infection of mice. Many of these mutant viruses replicated to high titers in the nasal mucosa of mice following intra-nasal administration. Despite replication to high titers in the nose, there was little spread to other organs in infected animals. Intra-nasal vaccination with doses as low as 100-1000 pfu (plaque forming units) of these replicating VACV constructs were sufficient to protect the host from challenge with large doses of wtVACV. Similar constructs in a Copenhagen and a NYCBH background were highly attenuated, yet effective as vaccines in the mouse model. These recombinant VACV constructs may be promising vector candidates for use in vaccination strategies against smallpox and other pathogens.

Evaluation of the HER2/neu‐derived peptide GP2 for use in a peptide‐based breast cancer vaccine trial

E75 and GP2 are human leukocyte antigen (HLA)-A2-restricted immunogenic peptides derived from the HER2/neu protein. In a E75 peptide-based vaccine trial, preexisting immunity and epitope spreading to GP2 was detected. The purpose of this study was to further investigate GP2 for potential use in vaccination strategies. Importantly, a naturally occurring polymorphism (I-->V at position 2, 2VGP2) associated with increased breast cancer risk was addressed. Prevaccination peripheral blood samples (PBMC) from HLA-A2 breast cancer patients and CD8+ T cells from HLA-A2 healthy donors were stimulated with autologous dendritic cells (DC) pulsed with GP2 and tested in standard cytotoxicity assays with HER2/neu+ tumor cells or GP2- or 2VGP2-loaded T2 targets. Additional cytotoxicity experiments used effectors stimulated with DC pulsed with E75, GP2, or the combination of E75+GP2. GP2-stimulated prevaccination PBMC from 28 patients demonstrated killing of MCF-7, SKOV3-A2, and the HLA-A2- control target SKOV3 of 28.8+/-3.7% (P<.01), 29.5+/-4.0% (P<.01), and 16.9+/-2.7%, respectively. When compared with E75, GP2-stimulated CD8+ T cells lysed HER2/neu+ targets at 43.8+/-5.2% versus 44.2+/-5.7% for E75 (P=.87). When combined, an additive effect was noted with 58.6+/-5.4% lysis (P=.05). GP2-stimulated CD8+ T cells specifically recognized both GP2-loaded (19.6+/-5.7%) and 2VGP2-loaded T2 targets (17.7+/-5.2%). GP2 is a clinically relevant HER2/neu-derived peptide with immunogenicity comparable to that of E75. Importantly, GP2-specific effectors recognize 2VGP2-expressing targets; therefore, a GP2 vaccine should be effective in patients carrying this polymorphism. GP2 may be most beneficial used in a multiepitope vaccine.

993. Optical Bioluminescence and Immunologic Analyses of Lentiviral Vectors Administered In Vivo: Transcriptional Targeting through an MHCII Promoter Yields Persistent Transgene Expression and Lack of CTL Stimulation in Immunocompetent Mice Indicating Tolerance

In vivo administration of lentiviral vectors is a promising novel approach for genetic vaccination and for gene replacement therapy. Bio-distribution studies have shown that i.v. systemic administration of lentiviral vectors produces conspicuous gene marking of antigen presenting cells (APCs) in the liver and spleen. Expression and presentation of immunogenic antigens by APCs may explain the potential of lentiviral vectors for vaccination and the poor persistent expression in immunocompetent animals. Based on this rationale, we attempted to improve the biosafety and immunization potential of lentiviral vectors by targeting the transgene expression to APCs through the Major Histocompatibility Complex class II (MHCII) promoter. We produced third generation self-inactivating vectors with the MHCII or with the CMV promoter as ubiquitous expression control. Ex vivo transduction of MHCII+ and MHCII- cell lines and primary cells using GFP and luciferase as reporter genes confirmed the expression and selectivity of vectors with the MHCII promoter. Nu/Nu nude and C57BL/6 mice (n=3 per group) were injected i.v. with 2 mcg p24 equivalent of vectors expressing firefly luciferase (LV-CMV-LUC or LV-MHCII-LUC) and were analyzed by optical imaging on weeks 1, 2, 4, 8, 12, 16 and 24 after injections. Statistical analyses used a linear regression model comparing the overall differences (bioluminescence signal detected in liver and spleen) between the LV-CMV-LUC and LV-MHCII-LUC injected groups in early (1-8 weeks) and late (8-24 weeks) periods. Nude mice injected with LV-CMV-LUC and LV-MHCII-LUC showed increase in bioluminescence signal during the first 8 weeks, which maintained stable for the late period. Notably, a significant drop in bioluminescence signal occurred for LV-CMV-LUC expression in C57BL/6 mice throughout the observation periods (slope= -21+/-8, p=0.0112), whereas for LV-MHCII-LUC a much lower drop was observed (slope= -8+/-7.4, p=0.2827) and the signal persisted for 24 weeks. Subcutaneous administration of vectors produced similar results. Additionally, we performed immunologic studies with vectors expressing the melanoma-associated antigen TRP2. I.v. vaccination of C57BL/6 mice with LV-CMV-TRP2 produced high frequencies of TRP-2 specific CTL responses (4.5% - 12.5% of circulating CD8+ cells, measured by flow cytometry after anti-TRP-2 tetramer staining or intracellular IFN-gamma staining) and significant (p=0.0365) protection against B16 melanoma challenge. Vaccination with LV-MHCII-TRP2 yielded baseline results comparable to the placebo vaccination group. In summary, these results indicate that transgene expression regulated through the MHCII promoter may induce a tolerogenic effect that would be unsuitable for use in vaccination strategies, but might allow more persistent expression in gene replacement therapy.

Induction of a strong HIV-specific CD8+ T cell response in mice using a fowlpox virus vector expressing an HIV-1 multi-CTL-epitope polypeptide.

Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.

Cellular immunity to the Her-2/neu protooncogene

Her-2/neu (HER-2) is a 185-kDa receptor-like glycoprotein that is overexpressed by a variety of tumors such as breast, ovarian, gastric, and colorectal carcinomas. Overexpression of this oncogene is directly associated with malignant transformation of epithelial cells. The frequency of HER-2 overexpression varies among the different types of cancers, but universally represents a marker of poor prognosis. The critical role of HER-2 in epithelial oncogenesis as well as its selective overexpression on malignant tissues makes it an ideal target for immunotherapy. Antibodies and T cells reactive to HER-2 are known to naturally occur in patients with HER-2 positive tumors, confirming the immunogenicity of the molecule. Both antibodies as well as T cells reactive to HER-2 have been utilized for immunotherapy of HER-2 positive tumors. The "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and found to be an effective adjuvant therapy for HER-2 positive breast and ovarian cancer patients. However, the frequency of patients responding to Herceptin is limited and a majority of patients initially responding to Herceptin develop resistance within a year of treatment. The use of vaccination strategies that generate T cell responses with or without accompanying antibody responses may serve to mitigate the problem. Various strategies for generating T cell-mediated responses against HER-2 are currently being examined in animal models or in clinical trials. The potential advantages of the various approaches to immunotherapy, their pitfalls, and the mechanisms by which HER-2 positive tumors can evade immune responses are discussed in this review.

The role of antibacterial therapy of acute otitis media in promoting drug resistance.

Treatment of acute otitis media (AOM) is the leading cause of antibacterial use in children in most developed countries. Rates of Streptococcus pneumoniae strains resistant to many classes of antibacterial agents have risen dramatically in many countries over the past 20 years. While more restricted use of antibacterial agents for AOM would almost certainly slow the rise in resistance, AOM is a potentially painful disease and may have suppurative complications such as mastoiditis. In this review, we discuss the prudent use of antibacterial agents for AOM and provide an overview of the epidemiology of S. pneumoniae resistance worldwide. Data from 10 placebo-controlled studies in patients with AOM show that antibacterial treatment is generally associated with a significantly higher cure rate than placebo. Of the three studies which analysed children <2 years of age, cure rates were 28 to 48% for placebo and 41 to 74% with antibacterial agents. Of the studies purporting to show no difference in cure rates between placebo and antibacterial therapy, the diagnostic criteria defining entry into the study were poor; therefore, the studies may have included many children without bacterial disease. Accurate diagnosis of AOM is the key element in reducing unnecessary antibacterial usage. Either pneumatic otoscopy or tympanometry can provide evidence of an effusion and the presence of an opaque, yellow or creamy white bulging eardrum will confirm AOM. Finally, the selection of appropriate antibacterial agents will reduce the rise in resistance. Low dosages of antibacterial agents used for prophylaxis select for resistance, and certain classes of drugs such as the sulfonamides and macrolides appear to do the same even at therapeutic doses. Amoxicillin at high dosages should remain the first-line antibacterial agent. In the future, use of vaccination strategies against pneumococci, influenza, respiratory syncytial virus and non-typeable Haemophilus influenzae will also decrease antibacterial use.

Bone Marrow and Peripheral Blood Dendritic Cells From Patients With Multiple Myeloma Are Phenotypically and Functionally Normal Despite the Detection of Kaposi’s Sarcoma Herpesvirus Gene Sequences

Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi's sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P =.01), CD86 (P =. 0003), and CD14 (P =.04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.