Antigenic Competition Research Articles

Co-Administration Therapy Modulates Immunity to FVIII In Hemophilia A Mice

Abstract 1219Our long-term objective is the development of novel therapies for the safe and effective treatment of hemophilia. In particular a safe and economic prophylactic modality to prevent inhibitors development is therefore highly desirable. To this end we have explored strategies to modulate inhibitor development based on the co-administration of FVIII with other proteins, specifically albumin and IVIG. We hypothesise that such co-administration may modulate immunity to FVIII and result in a safe and cost-effective therapy. Based on some published evidence that FIX and vWF may have a modulatory effect on FVIII, we reasoned that perhaps albumin might also play an immunomodulatory effect on FVIII. We have developed a protocol for infusing recombinant FVIII in hemophilia A mice modelled on the current therapy of hemophiliacs, and in response to this protocol hemophilia mice develop high titre of inhibitors. Thus, this model can be used to assess potential immununomodulatory strategies.Groups of hemophilia A mice (n=5) were infused weekly IV with 2IU rFVIII for a total of 4 weeks. Three of these groups of mice were also administered human albumin (either 25mg IV, 25mg IP, or 500mg IV) at the same time as FVIII. Mice were bled before the treatment and weekly for the 4 weeks, and total antibodies to FVIII were detected by ELISA. Importantly, while mice receiving FVIII had high antibody titer and were in good health, there was significant mortality in the mice treated with FVIII and albumin. Interestingly, the surviving mice had a very low antibody titre, statistically significant from mice receiving only rFVIII.Given the mortality, another experiment was designed to test the effect of reduced doses of albumin. Groups of hemophilia A mice (n=5) were infused IV with a weekly injection of 2IU rFVIII for a total of 4 weeks supplemented with either 25μg, 50μg or 100μg of human albumin. Mice were bled at weekly intervals until the end of the experiment (day 28), and the titre of anti-FVIIII antibodies was determined. Most All mice survived the entire treatment and were found in good health. Further, the FVIII antibody titre in the group that received albumin was detectable, but significantly reduced with respect to the mice that received only FVIII. Marked variability among individual mice was observed.A group of hemophilia A mice (n=5) received weekly IP injections of 2IU rFVIII. Another group of mice received the same regimen of FVIII in addition to human IVIG (1g/kg). A third group of hemophilia mice received 2IU rFVIII and a higher dose of human IVIG (2g/kg). These doses mimic the established human IVIG regimen. Mice were bled before treatment and at weekly intervals thereafter. The titre of anti-FVIII antibodies in mice was determined by ELISA. By day 21, the antibody titre in mice injected with FVIII was high, as expected. In contrast, the titre in mice receiving FVIII and IVIG was low, and comparable to that of all the mice prior to the treatment (na•ve mice), strongly suggesting an immune modulatory effect by IVIG (Figure 1). Our data supports the limited use of IVIG to treat inhibitors. [Display omitted] Our findings suggest that co-administration of FVIII with albumin and IVIG can modulate the humoral immune response to recombinant human FVIII in hemophilia mice. Further, the observed effect appears to be dependent on the dose of albumin co-administered. It is hypothesized that the observed modulation is due to antigen competition, as previously speculated. Importantly, while IVIG was well tolerated by mice, very serious adverse effects were observed in mice treated with high doses of albumin. Interestingly, even the high dose used represents a small fraction of the physiological levels of albumin in human plasma, which is 3–5 g/dL. IVIG, in contrast, is a known modulator of the immune response in humans, and the study of this modulatory effect in a hemophilia mouse may lead to novel therapeutic strategies, relevant for the management of hemophilia. Disclosures:No relevant conflicts of interest to declare.

Abstract PL04-04: Synthetic long peptide vaccines against established disease caused by high-risk human papillomavirus.

Abstract Therapeutic vaccination of persistent virus infections and associated diseases including (pre-)cancer so far has largely evaded success, mainly due to the fact that insufficiently consistent and robust effector T cell responses were induced by the commonly used vaccine constructs and formulations such as recombinant viruses and bacteria, recombinant proteins, DNA constructs, Dendritic Cell (DC) vaccines and exact HLA class I binding peptides (short peptides). Problems have been severe antigenic competion from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes injected DC and antigen presentation of short peptides by non-professional antigen presenting cells in vivo, causing tolerance instead of immunity. Much more robust and consistent T cell responses can be obtained by vaccination with long (28–35 amino acid long) synthetic peptides. Such immunogens are more efficiently processed and presented than intact proteins by DC and only DC can efficiently perform this task. Moreover only concentrated antigen of choice is offered and antigenic competition therefore plays no role. In earlier work we showed that therapeutic vaccination with a synthetic long peptide (SLP®) vaccine mediated the eradication of established human papilloma virus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papilloma virus. Subsequent phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. We then tested the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses. The vaccine was safe. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions compared to non-responders. Patients with large lesions were less likely to experience a complete clinical response than patients with small lesions and we ascribe this to a larger proportion of vaccine induced HPV-specific regulatory cells in the patients with large lesions. In conclusion, treatment with the HPV16 SLP vaccine is clearly effective in patients with established VIN disease. The SLP platform lends itself for development of therapeutic vaccines against many other chronic infections and non-viral cancers. In patients with cancer, it is attractive to combine this type of vaccination with immunogenic forms of cancer chemotherapy and with immuno-modulatory drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-04.

Delivery of a multivalent scrambled antigen vaccine induces broad spectrum immunity and protection against tuberculosis

The development of effective anti-Tuberculosis (TB) vaccines is an important step towards improved control of TB in high burden countries. Subunit vaccines are advantageous in terms of safety, particularly in the context of high rates of HIV co-infection, but they must contain sufficient Mycobacterium tuberculosis antigens to stimulate immunity in genetically diverse human populations. We have used a novel approach to develop a synthetic scrambled antigen vaccine (TB-SAVINE), comprised of overlapping, recombined peptides from four M. tuberculosis proteins, Ag85B, ESAT-6, PstS3 and Mpt83, each of which is immunogenic and protective against experimental TB. This polyvalent TB-SAVINE construct stimulated CD4 and CD8T cell responses against the individual proteins and M. tuberculosis in C57BL/6 and Balb/c mice, when delivered as DNA, Fowl Pox Virus or Vaccinia Virus vaccines. In addition, the DNA-TBS vaccine induced protective immunity against pulmonary M. tuberculosis infection in C57BL/6 mice. Co-immunization of Balb/c mice with virally expressed TBS and HIV1-SAVINE vaccine stimulated strong T cell responses to both the M. tuberculosis and HIV proteins, indicating no effects of antigenic competition. Further development of this TB-SAVINE vaccine expressing components from multiple M. tuberculosis proteins may prove an effective vaccine candidate against TB, which could potentially form part of a safe, combined preventative strategy together with HIV immunisations.

Orbital fibrosis in a mouse model of Graves' disease induced by genetic immunization of thyrotropin receptor cDNA

The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered by in vivo skeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. Female BALB/c mice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD.

Broad Humoral and Cellular Immunity Elicited by a Bivalent DNA Vaccine Encoding HA and NP Genes from an H5N1 Virus

Influenza A virus is highly variable and a major viral respiratory pathogen that can cause severe illness in humans. Therefore it is important to induce a sufficient immune response specific to current strains and to heterosubtypic viruses with vaccines. In this study, we developed a dual-promoter-based bivalent DNA vaccine that encodes both hemagglutinin (HA) and nucleoprotein (NP) proteins from a highly pathogenic A/Chicken/Henan/12/2004 (H5N1) virus. Our results show that the expression levels of HA and NP genes from the dual-promoter plasmid are similar to those seen when they are expressed individually in independent plasmids. When the bivalent DNA vaccine was inoculated via intramuscular injection and in vivo electroporation, high levels of both humoral and cellular immune responses were elicited against homologous H5N1 virus and heterosubtypic H9N2 virus. Furthermore, no obvious antigenic competition was observed between HA and NP proteins in the dual-promoter-based bivalent vaccine compared to monovalent vaccines. Our data suggest that a combination of influenza surface and internal viral genes in a dual-promoter-expressing plasmid may provide a new approach for developing a DNA vaccine that may protect not only specifically against a currently circulating strain, but also may cross-protect broadly against new heterosubtypic viruses.

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G2/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G1 cells. Its amount increased upon entry into the S phase and remained high during the G2/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G2/M phase regulation in plant cells is discussed.

Germinal Center Dynamics Revealed by Multiphoton Microscopy with a Photoactivatable Fluorescent Reporter

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

Abrogation of antigenic competition phenomenon by a low dose of cyclophosphamide.

Summary Using the antigenic competition system, the effect of low doses of cyclophosphamide on the suppressor cell responsible for the phenomenon was evaluated. Inbred RF mice were injected with different doses of sheep red blood cells as a competitor antigen 24 hours before giving rat red blood cells, the antigen against which the response is measured. The results show that the suppressor effect elicited by the first antigen depends on the amount of immunogen injected and can be selectively abrogated by low doses of cyclophosphamide (25 mg./kg. body weight) given between the two immunogens. The most probable agent for this effect is the splenic suppressor cell, an immature, spleen-seeking and T-derived lymphocyte. Zusammenfassung Hemmung des Antigenwettbewerb-Phänomens durch niedrige Dosen von Cyclophosphamid In einem Versuchssystem, bei dem zwei verschiedene Antigene in Wettbewerb traten, wurde der Einfluß niedriger Dosierungen von Cyclophosphamid auf die relevanten Suppressorzellen untersucht. Ingezüchtete RF-Mäuse erhielten zunächst verschiedene Dosierungen Schaferythrocyten, nach 24 Stunden dann Rattenerythrocyten. Gegenüber letzteren wurde die Immunantwort gemessen. Das erste Antigen übte einen Suppressoreffekt in Abhängigkeit von der injizierten Dosis aus. Wurden geringe Dosen Cyclophosphamid (25 mg/kg Kpgw.) zwischen beiden Antigenen appliziert, so verschwand der Suppressoreffekt. Es wird angenommen, daß es sich bei den Suppressorzellen um unreife Milz-suchende Zellen mit T-Zellcharakter handelt. Résumé Inhibition du phénomène de concurrence antigénique par de faibles doses de cyclophosphamide On a examiné l'influence de faibles doses de cyclophosphamide sur les cellules suppressives dans un système de recherche dans lequel entraient en concurrence deux antigènes différents. Des souris RF ont reçu tout d'abord différentes doses d'érythrocytes de moutons suivies à heures d'érythrocytes de rats. On a mesuré la réponse immunitaire vis-à-vis de ces derniers. Le premier antigène a manifesté un effet suppressif en fonction de la dose injectée. L'effet suppressif a disparu en appliquant de faibles doses de cyclophosphamide (25 mg./kg. de poids). On suppose que les cellules suppressives sont des cellules immatures provenant de la rate avec caractère de cellules T. Resumen Inhibición del fenómeno de competencia antigénica mediante dosis reducidas de ciclofosfamida En un sistema de ensayo, en el cual entraron en competencia dos antígenos diferentes, se examinó el influjo de dosificaciones bajas de ciclofosfamida sobre las células supresoras relevantes. Ratones RF, de consanguinidad estrecha, recibieron por de pronto dosificaciones diversas de eritrocitos de oveja, 24 horas más tarde eritrocitos de rata. La respuesta inmunológica se midió frente a estos últimos. El primer antígeno ejercía un efecto supresor en dependencia de la dosis inyectada. Al aplicar dosis escasas de ciclofosfamida (25 mg./kg. peso vivo) entre ambos antígenos, desaparecía el efecto supresor. Se supone que en las células supresoras se trata de células inmaduras del bazo con carácter celular T.

PrP Conformational Transitions Alter Species Preference of a PrP-specific Antibody

The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109-112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106-110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.

Neutrophils markedly influence the immune response to protein antigens in adjuvant (130.45)

Abstract Neutrophils (PMN) have a crucial role in innate immunity against microbial infections. In vitro experiments indicated that PMN dampened T cell proliferation. However, it is unclear if PMN affect adaptive immunity in vivo. Here we used protein immunization in various adjuvants as a model system to study how PMN modulate T cell priming. After immunization with Freund’s adjuvants or alum, PMN rapidly entered the draining lymph nodes (dLN), localizing to the cortical sinus where antigen accumulated. In mice lacking PMN, due to antibody-mediated depletion or genetic ablation, CD4 T cell priming was enhanced by 2-3 fold. We observed enhanced CD4 T cell responses to three different protein antigens (hen-egg white lysozyme, ovalbumin and listeriolysin-O); this effect was independent of the adjuvant and antigen dose. PMN depletion in mice lacking NADPH oxidase or inducible NO synthase still showed enhanced CD4 T cell responses. Dendritic cells (DCs) and macrophages in the dLN contained more antigen and presented more peptide-MHC complexes in the absence of PMN, suggesting antigen competition between PMN and antigen presenting cells (APCs). Finally, PMN-depletion enhanced CD4 T cell responses in mice immunized with antigen-loaded DCs, indicating that PMN migrating to the dLN had a negative effect on antigen bearing APCs. Together, these experiments showed that PMN influenced adaptive immune responses via modulation of antigen presentation.

Vaxfectin ® enhances both antibody and in vitro T cell responses to each component of a 5-gene Plasmodium falciparum plasmid DNA vaccine mixture administered at low doses

We previously reported the capacity of the cationic lipid-based formulation, Vaxfectin ®, to enhance the immunogenicity and protective efficacy of a low dose plasmid DNA vaccine against Plasmodium yoelii malaria in mice. Here, we have extended this finding to human Plasmodium falciparum genes, evaluating the immune enhancing effect of Vaxfectin ® formulation on a mixture, designated CSLAM, of five plasmid DNA vaccines encoding antigens from the sporozoite ( PfCSP, PfSSP2/TRAP), intrahepatic ( PfLSA1), and erythrocytic ( PfAMA1, PfMSP1) life cycle stages of P. falciparum administered at 2, 10 or 50 μg doses. Vaxfectin ® formulation enhanced both antibody and cellular immune responses to each component of the multi-antigen vaccine mixture, as assessed by ELISA, IFAT, and IFN-γ ELIspot, respectively. There was no apparent antigenic competition, as indicated by comparison of responses induced in mice immunized with PfCSP vs. CSLAM. These data showing that Vaxfectin ® can enhance the immunogenicity of plasmid DNA vaccines administered at low doses per body weight, and in combinations, has important clinical implications for the development of a vaccine against malaria, as well as against other public health threats.

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot ( Dichelobacter nodosus) vaccines in sheep

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.

Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice

AbstractThe use of plasma-derived factor VIII (pdFVIII) concentrates in hemophilia A has been reported to result in reduced anti-FVIII antibody formation. In this study, we have investigated whether the cytokine microenvironment induced by pdFVIII has an influence on reducing anti-FVIII antibody titers in hemophilic mice. Microarray and confirmatory quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments show that pdFVIII infusion causes a different transcriptional profile in dendritic cells than recombinant FVIII (rFVIII). Both treatments caused up-regulation of proinflammatory gene expression, but rFVIII and pdFVIII treatments promote expression of genes that induce Th1 and Th2 responses, respectively. Moreover, administration of rFVIII or pdFVIII concentrates resulted in distinct T-cell splenic cytokine microenvironments. rFVIII induced the release of Th1 cytokines and IL-10, whereas pdFVIII induced the release of Th2 cytokines and transforming growth factor-β. We have also observed high titers of anti–human von Willebrand factor (VWF) antibodies in the pdFVIII-treated mice and propose that this results from antigenic competition. We further investigated the role of this phenomenon using infusions of FVIII and increasing concentrations of recombinant human factor IX (FIX). These studies show an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies report new mechanisms that contribute to reduced anti-FVIII antibody development in hemophilia A after pdFVIII infusions.

Immunological Usefulness of Semen Manipulation for Artificial Insemination Homologous (AIH) in Subjects with Antisperm Antibodies Bound to Sperm Surface*/Immunologische Nützlichkeit der Spermaaufbereitung für eine homologe artifizielle msemination bei Män

The authors report trials carried out in the preparation of semen for homologous artificial insemination in subjects with antisperm autoantibodies. The tests have demonstrated that swim-up is not able to select spermatozoa free of antibodies bound to the sperm surface. All the experiments of migration, dilution, antigenic competition and cryoconservation, carried out to evaluate the theoretical possibility of a better recovery of non "immunologically compromised" spermatozoa, have confirmed such negative results both with the Immunobead Test and Cytofluorimetric analysis.

Overcoming Antigenic Competition to Produce Multispecific Cytotoxic T Lymphocyte Lines for Adoptive Transfer

Overcoming Antigenic Competition to Produce Multispecific Cytotoxic T Lymphocyte Lines for Adoptive Transfer

Multivirus-Specific T Cell Immunotherapy to Prevent or Treat Infections of Stem Cell Transplant Recipients.

Viral infections caused by community viruses frequently cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Antiviral drugs are costly, often have severe adverse effects, and are frequently ineffective. Treatment of the underlying problem, namely lack of antigen-specific T cells, should offer effective and longterm protection. Our group has produced trivirus-reactive T cells targeting EBV, CMV, and adenoviruses (Adv) using monocytes and EBV-transformed lymphoblastoid cell lines (EBV-LCL) expressing pp65 from an adenoviral vector as antigen-presenting cells to present CMV, Adv and EBV antigens. As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs post-infusion into HSCT recipients and appeared to protect the recipients against all three viruses. Despite these encouraging clinical results, the broader implementation of the approach is limited by (i) the infectious virus material (EBV/Adv) required for CTL generation and (ii) the prolonged culture required to produce the EBV-LCL, increase viral specificity and reduce alloreactivity (3 months) which means T cells must be produced “speculatively” for all patients. Finally, (iii) “antigenic competition” between multiple viruses limits the extension of the approach to additional problematic pathogens. To overcome these limitations we have developed an approach to rapidly produce multivirus-specific CTL with broad spectrum specificity without using adenoviral vectors or EBV-LCL. Using the Amaxa system to nucleofect monocyte-derived DCs we consistently detected GFP transgene expression in 39% (median; range 30–58%) of cells 24hrs posttransfection. Viability was ~70% and the DC maturation state, as measured by CD80, 83, 86, and HLA-DR expression, was unaffected by the transfection. To show that nucleofected DCs reactivated virus-specific T cells in vitro, we cocultured p-Shuttle-pp65-GFP-transfected DCs from CMV seropositive donors with PBMCs at a responder:stimulator ratio of 20:1. After nine days, phenotypic and functional characterization of the responder T cell lines showed higher or comparable frequencies of pp65-specific T cells in IFN-g ELIspot and minimal alloreactivity when compared to pp65-specific T cells lines generated from the same donors using our standard protocol with Ad5f35pp65-transduced DCs as APCs. Pentamer analysis of pShuttle-pp65-generated CTL lines also showed a higher frequency of pp65 pentamer-directed T cells than the Ad5f35pp65-transduced counterparts (median 2.05 fold higher frequency of HLA-A2 NLV-directed T cells; range 1.34–3.35 fold) (n=4 donors). Importantly, this protocol could also be used to reactivate T cells against multiple viruses for which high (EBV), intermediate (BK), and low (Adv) frequencies of reactive memory T cells circulate. Using a panel of p-Shuttle plasmids encoding LMP2 and BZLF1 (EBV), Large T (BK), and Hexon and Penton (Adv), we amplified CTLs from seropositive donors, using as stimulators DCs transfected with each construct. This modification overcomes the need for EBV-LCL generation. Furthermore, we demonstrated that by pooling transfected DCs prior to coculture with PBMC, we could reproducibly generate multivirus-specific CTL lines with specificity for all the stimulating antigens, irrespective of the circulating memory T cell frequency. To further shorten the CTL production process, we established that virus-activated T cells could be specifically selected by IFN-g capture 24 hours after DC stimulation and that the selected cells were highly specific for the stimulating antigens as measured by IFN-g ELIspot, proliferation and cytotoxicity assay. In summary, we have established a GMP-applicable protocol for the rapid generation (<10 days) of two different CTL products without using infectious viral material. In 10 days we can generate virus-specific CTLs with broad specificity which can be administered prophylactically to high risk SCT recipients. However by combining DC transfection with IFN-g selection we can also rapidly generate mono- or multivirus-specific CTL products for treatment of acute infection. We demonstrate the feasibility of generating CTL lines targeting 6 different antigens from 4 common viruses without using infectious viral material. Future studies will extend our approach to additional viral, fungal, and bacterial antigens.

Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.

Epaxal®: a virosomal vaccine to prevent hepatitis A infection

Over the last few decades, different types of inactivated hepatitis A virus (HAV) vaccines have been developed: several aluminum-adjuvanted vaccines and an aluminum-free, virosome-formulated vaccine. Both types of vaccines are whole-virus preparations that are produced by growth of HAV strains in human diploid cell cultures and are subsequently inactivated with formaldehyde. This review summarizes all published papers on a virosome-formulated vaccine, Epaxal®, based on formalin inactivated HAV (strain RG-SB) adsorbed to the surface of special liposomes (virosomes), that replace aluminum hydroxide as the adjuvant principle. A single injection of virosomal HAV vaccine is well tolerated and highly immunogenic, with 88–97% of seroprotection 2 weeks after a first dose. HAV virosomal vaccine can be administered concomitantly with other vaccines, without inducing antigenic competition. Direct comparison with aluminum-adsorbed vaccine has shown that the immunogenicity was similar, but fewer local reactions were reported with Epaxal. Recent studies in children have demonstrated that Epaxal Junior is also an excellent HAV vaccine for mass vaccination programs.

Pilot trials in Australia on eradication of footrot by flock specific vaccination

Footrot is a contagious disease of ruminants requiring strains of Dichelobacter nodosus that possess virulence factors including proteases and fimbriae. Sheep can be immunised against footrot using vaccine-containing fimbriae, either native or recombinant. The fimbriae are responsible for the serological K-agglutination reaction, which has been used to classify field isolates into nine major serogroups. The range of protection conferred by vaccination is largely restricted to the serogroup involved, but antigenic competition precludes effective vaccination with multivalent vaccines that contain all serogroups. However, vaccination with specific bivalent recombinant fimbrial vaccine led to eradication of virulent footrot from small ruminants in Nepal and the same result was obtained in Bhutan using a specific whole cell vaccine. In the study reported here two pilot trials have been conducted in Australian sheep flocks, one with a virulent form of footrot caused by a single serogroup F, and the other with an intermediate form also caused by a single serogroup C. In trial 1 pre-vaccination prevalence of clinical footrot in a group of randomly selected animals was 44%. This reduced to 2% at 3 months and 0.5% at 4 months, and there were no clinical cases at 5 months or at 16 months post-vaccination in the whole flock. Similarly in trial 2 pre-vaccination whole flock prevalence was 8.5%, while it was 2% at 3 months, 0.3% at 6 months and zero at 18 months post-vaccination. Use of flock specific monovalent whole cell vaccines over whole flocks for only one season and culling of the few non-responders has been a successful approach in eradication of the disease from both these flocks. This is the first study to report the successful use of specific vaccine for the intermediate form of footrot.

Modulation of lung environment and new T cell responses by flu‐primed CD4 memory T cells

Infection of mice with influenza (flu) viruses generates CD4 memory T cells that reduce the accumulation of newly primed CD4 and CD8 T cells in the lungs during subsequent infections, while allowing normal T cell priming in lymphoid organs. Since the modulation of new T cell responses by CD4 memory T cells is not due to competition for antigen, we assayed for classical regulatory T cell populations within the CD4 memory T cell pool. While there was an increase in classical Treg and Tr1 phenotype T cells after flu infection, the CD4 memory T cells did not need to express CD25, GITR, or IL‐10 to carry out their modulatory functions. Gene expression in the lungs of flu infected mice adoptively transferred with CD4 memory T cells, versus naïve CD4 T cell transfer, revealed a reduced expression of genes associated with type‐1 inflammation and increased expression of genes associated with type‐2 inflammation. Thus, we are exploring whether CD4 memory T cells prevent accumulation of newly primed T cells in the lungs due to a reduced expression of type‐1 chemoattractants or active suppression of newly primed T cell survival in a type‐2 biased lung environment. However, we have not ruled out the possibility that the modulatory CD4 memory T cells may be recruiting classical regulatory T cells to the lungs during flu infection. Determining the mechanism by which CD4 memory T cells modulate newly primed T cell responses at sites of infection, and the impact this has on the generation of subsequent memory T cell responses, will yield important insights into methods for inducing protective T cell memory to a broad range of pathogens. Funding provided by NBC‐AI057158‐Lipkin.