Antigenic Competition Research Articles

Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer.

Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy. PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings. We report an automated PD-L1 immunohistochemistry (IHC) assay, which was developed to detect cell surface PD-L1 in formalin-fixed paraffin-embedded human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal anti-human PD-L1 antibody, clone 28-8. The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines. The specificity of the PD-L1 IHC assay was further evaluated in a collection of 30 normal human tissues. The PD-L1 IHC assay was optimized for high sensitivity and precision in routine application. A pathology scoring and interpretation method specific to nivolumab clinical studies was adopted for the assay. The analytical performance of the assay was validated for application in the determination of PD-L1 status in human NSCLC specimens. The clinical application of the assay and scoring method was further validated in 3 Clinical Laboratory Improvement Amendments certified labs. The assay is currently being investigated in a variety of clinical studies for use as an in vitro diagnostic to select and stratify patients for treatment with the anti-PD-1 therapeutic antibody, nivolumab.

A novel tetravalent formulation combining the four aggregated domain III-capsid proteins from dengue viruses induces a functional immune response in mice and monkeys.

Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed.

Synchrony in serum antibody response to conserved proteins of Streptococcus pneumoniae in young children

Conserved Streptococcus pneumoniae (Spn) proteins are currently under investigation as vaccine candidates. We recently identified a subset of children prone to frequent acute otitis media (AOM) that we refer to as stringently-defined otitis prone (sOP). We investigated the synchrony of serum antibody responses against 5 Spn protein vaccine antigens, PhtD, LytB, PcpA, PhtE, and PlyD1 resulting from nasopharyngeal colonization and AOM in sOP children (49 observations) and non-otitis prone (NOP) children (771 observations). Changes in serum IgG and IgM were quantitated with ELISA. IgG antibody concentrations against PhtD, PcpA, and PlyD1 rose in synchrony in sOP and NOP children; that is, the proteins appeared equally and highly immunogenic in children at age 6 to 15 months and then leveled off in their rise at 15 to 25 months. In contrast, rises in concentrations to PhtE and LytB were significantly slower and had not peaked in children even at 25 months of age, consistent with lower immunogenicity. Serum IgM responses against PhtD and PlyD1 were in synchrony in children at age 6–25 months old. PcpA did not induce a significant increase of serum IgM response in children, suggesting that primary responses to PcpA occurred prior to children attaining age 6 months old. PhtD, PcpA, and Ply elicit a synchronous natural acquisition of serum antibody in young children suggesting that a trivalent Spn protein vaccine combining PhtD, PcpA, and PlyD1 would be less likely to display antigen competition when administered as a combination vaccine in young children.

Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses.

Aluminum salts gels (alum) are TLR-independent adjuvants and have been used to boost antibody responses in alum-based vaccines such as diphtheria, pertussis, and tetanus toxoid (DPT) triple vaccine. However, the pro-Th2 activity of alum-based vaccine formulations has not been fully appreciated. Here we found that alum-based tetanus toxoid (TT) vaccine was biased toward a Th-2 profile as shown by TT-induced airway eosinophilic inflammation, type 2 cytokine production, and high levels of IgE anaphylactic antibodies. The adsorption into alum of prototypic TLR4 agonists such as lipopolysaccharides (LPS) derived from Escherichia coli consistently dampened TT-induced Th2 activities without inducing IFNγ or Th1-like responses in the lung. Conversely, adsorption of monophosphoryl lipid A (MPLA) extracted from Salmonella minnesota, which is a TIR-domain-containing adapter-inducing interferon-β- (TRIF-) biased TLR4 agonist, was less effective in decreasing Th-2 responses. Importantly, in a situation with antigenic competition (OVA plus TT), TT-specific IgG1 or IgG2a was decreased compared with TT sensitization. Notably, LPS increased the production of IgG1 and IgG2a TT-specific antibodies. In conclusion, the addition of LPS induces a more robust IgG1 and IgG2a TT-specific antibody production and concomitantly decreases Th2-cellular and humoral responses, indicating a potential use of alum/TLR-based vaccines.

The Interplay of the Local Microbiome with Oral Mucosal Immune Compartment: Minireview

The oral microbiome is formed and functioned in balanced state during health and in an unbalanced state in the disease conditions. Microbiome in the oral compartment combats the weapons of the invading microbial pathogens .Now , there are a specific microbiome signature in each of the cases of health and disease. Oral microbiome expressed; probiotic, antigenic competition and immunomodulatory effects locally in the oral compartment .In the immunologic sense ,oral microbiome is constituting an element that is able to interact in a bidirectional fashion with the components of the oral immune compartment with the net result of homiostasis , immune response ,tolerance ,anergy and hypersensitivity and/or immune mediated diseases .Thus it has played an important roles in oral immune mechanisms.

Vaccines and the looming threat of syphilis

During the past several decades, syphilis has increased in the US and explosively so in China and Russia. While most current cases can be cured by penicillin or second line antibiotics, strains of the syphilis spirochete (Treponema pallidum) resistant to the latter have emerged. And the potential of strains to acquire resistance to penicillin via plasmids has recently been recognized. The 36 million-plus undertreated cases of syphilis in the world represent metaphorically a vast global 37°C incubator allowing exchange of antibiotic resistant genes among the human-borne bacteria and theoretically permitting the emergence of penicillin resistant strains. None has yet been identified but they seem inevitable along with the threat of multi-resistant strains. There is now concern over the lack of new antibiotics to succeed the penicillins. Since such new drugs would likely require a decade or so of research and for federal approval, vaccine-based protection is of immediate consideration here. This approach may exploit the new knowledge about the biology of T. pallidum strains their many complex antigens, serotypes, and the latter’s distribution in populations. Equally important are recent insights into immunology, such as cellular aspects and antigenic competition.

Immunization Against Influenza but Not MMR Modulates the Anti-Factor VIII Immune Response in Hemophilia Α Mice

Introduction: The etiology of inhibitory factor VIII (FVIII) antibodies in 25-30% of hemophilia A (HA) patients remains poorly understood. It is possible that concurrent exposure to inflammatory stimuli, or 'danger signals’, with FVIII increases the risk of inhibitor formation. HA patients generally begin FVIII replacement therapy around 12 months of age, and the median age of inhibitors onset is between 15-21 months. During this time frame, patients may be exposed to vaccines, such as the mumps-measles-rubella (MMR) and seasonal influenza vaccines. Our investigation is the first to address the concern that these vaccines may serve as danger signals that augment FVIII immunogenicity.Methods: Our studies used 8-12 week-old FVIII E17KO C57Bl6/S129 HA mice, which carry a complete knockout of all murine MHC class II molecules, and instead express a chimeric human-mouse HLA-DRB1*1501 allele associated with increased inhibitor risk. Our preliminary studies show that 40-80% of these animals develop FVIII inhibitors following treatment.HA mice were immunized with 10x the standard human dose of live-attenuated MMR vaccine (Priorix) subcutaneously or intravenously 24 hrs prior to 4 weekly intravenous infusions of 2 IU recombinant human FVIII (rhFVIII, Advate). Mice were subsequently re-challenged with MMR 24 hrs prior to 4 biweekly infusions of 6 IU rhFVIII. Blood samples were collected retro-orbitally or via cardiac puncture. Plasma from weeks 5 and 9 was assessed for anti-FVIII IgG by ELISA; inhibitor concentrations were assessed by a Bethesda assay on week 9.The inactivated influenza vaccine (Agriflu) was administered at standard human doses either intramuscularly or intravenously 24 hrs before, after, or concurrently with the first of 7 biweekly infusions of 6 IU rhFVIII. Week 5 plasma samples were subjected to anti-FVIII and anti-influenza IgG ELISA and Bethesda assays. Statistical comparisons were made using the Fisher’s exact Mann-Whitney U tests, as appropriate.Results: Subcutaneous MMR vaccination exhibited no significant differences in the incidence or titres of anti-FVIII IgG compared to HBSS-injected controls at weeks 5 and 9 (n=13). Similarly, no differences in the incidence or concentration of inhibitors were detected at week 9. We next evaluated the effects of intravenous MMR immunization on FVIII immunogenicity. Surprisingly, we again observed no differences in the incidence or magnitude of the anti-FVIII immune response at weeks 5 and 9 (n=28-30).Importantly, we found a significant decrease in the incidence of FVIII-specific IgG in mice that were immunized intramuscularly with the influenza vaccine 24 hrs after and at the same time as the first infusion of rhFVIII (t=-24 hrs: 30%, t=0 hrs: 20%, t=+24 hrs: 26% vs control: 67%; p=0.11, 0.06, 0.04; n=10-15). Similarly, there was a significant decrease in the incidence of inhibitors at all immunization time points (t=-24 hrs: 30%, t=0 hrs: 43%, t=+24 hrs: 11% vs control: 80%; p=0.03, 0.02, 0.0022). No differences in IgG titres or inhibitor concentrations were detected. When immunized intravenously with the influenza vaccine, we observed an increase in the presence of FVIII-specific IgG, but no differences in titres. However, these differences were not statistically significant and need confirmation (t=-24 hrs: 80%, t=0 hrs: 90%, t=+24 hrs 40% vs control: 50%; p=0.58, 0.14, 1.00; n=5-10).Conclusion: These are the first experimental studies to address vaccination as a potential danger signal in the development of an anti-FVIII immune response. Our results suggest that both subcutaneous and intravenous immunization of HA mice with the MMR vaccine do not influence the incidence or magnitude of the anti-FVIII immune response. In contrast, our data suggest that intramuscular immunization with the inactivated influenza vaccine modulates the anti-FVIII immune response and may enhance tolerance induction to FVIII, possibly through antigen competition. However, this proposal awaits further confirmation. Finally, a trend in increased antibody and inhibitor incidence in intravenously immunized mice suggests that the influenza vaccine can serve as a danger signal, but is dependent on the route of administration. Our findings contradict current vaccination concerns in the treatment of young HA patients, and instead suggest an inhibitor-protective effect from influenza immunization. DisclosuresMoorehead:Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria. Steinitz:Baxter: Employment. Reipert:Baxter: Employment. Hough:Bayer: Research Funding. Lillicrap:Baxter: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding.

Competition for Antigen between Th1 and Th2 Responses Determines the Timing of the Immune Response Switch during Mycobaterium avium Subspecies paratuberulosis Infection in Ruminants

Johne's disease (JD), a persistent and slow progressing infection of ruminants such as cows and sheep, is caused by slow replicating bacilli Mycobacterium avium subspecies paratuberculosis (MAP) infecting macrophages in the gut. Infected animals initially mount a cell-mediated CD4 T cell response against MAP which is characterized by the production of interferon (Th1 response). Over time, Th1 response diminishes in most animals and antibody response to MAP antigens becomes dominant (Th2 response). The switch from Th1 to Th2 response occurs concomitantly with disease progression and shedding of the bacteria in feces. Mechanisms controlling this Th1/Th2 switch remain poorly understood. Because Th1 and Th2 responses are known to cross-inhibit each other, it is unclear why initially strong Th1 response is lost over time. Using a novel mathematical model of the immune response to MAP infection we show that the ability of extracellular bacteria to persist outside of macrophages naturally leads to switch of the cellular response to antibody production. Several additional mechanisms may also contribute to the timing of the Th1/Th2 switch including the rate of proliferation of Th1/Th2 responses at the site of infection, efficiency at which immune responses cross-inhibit each other, and the rate at which Th1 response becomes exhausted over time. Our basic model reasonably well explains four different kinetic patterns of the Th1/Th2 responses in MAP-infected sheep by variability in the initial bacterial dose and the efficiency of the MAP-specific T cell responses. Taken together, our novel mathematical model identifies factors of bacterial and host origin that drive kinetics of the immune response to MAP and provides the basis for testing the impact of vaccination or early treatment on the duration of infection.

Identification and validation of protein-protein interactions by combining co-immunoprecipitation, antigen competition, and stable isotope labeling.

Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions.

Formulation and immunological evaluation of a trivalent vaccine comprising emulsified submicron particles and inactivated virions of H5N1/EV71/JEV

Combination vaccines can reduce the number of injections and simplify the immunization schedule required to prevent different diseases. Here we assessed the immunogenicity in a mouse model of a vaccine composition comprising inactivated influenza viruses (H5N1/H1N1), enterovirus 71 (EV71), and/or Japanese encephalitis virus (JEV) and investigated whether the vaccine formulations can overcome the immunologic interference between the individual vaccine components. We demonstrated that the antigenic competition happens between H5N1/H1N1 or H5N1/EV71 inactivated virions when the vaccine combinations either formulated with Alum suspensions or without adjuvant. In the presence of PELC emulsified particles, EV71-specific immune responses before and after incorporating H5N1 virus into EV71 vaccine were detected of no significant difference; in addition, H5N1- and EV71-specific immune responses were found at the same level when H5N1/EV71/JEV consolidating into combination vaccine. Emulsified vaccine formulation was represented as a potential tool that is found to reduce the number of injections required to prevent multiple infectious strains causing the same disease (H5N1/H1N1) and/or that protect against different diseases (H5N1/EV71). Combination vaccines can also include a third component to protect against H5N1/EV71/JEV at the same time.

A comparison of methods for classifying samples as truly specific with confirmatory immunoassays

Biotechnology-derived therapeutics may induce an unwanted immune response leading to the formation of anti-drug antibodies (ADAs) which can result in altered efficacy and safety of the therapeutic protein. Anti-drug antibodies may, for example, affect pharmacokinetics of the therapeutic protein or induce autoimmunity. It is therefore crucial to have assays available for the detection and characterization of ADAs. Commonly, a screening assay is initially used to classify samples as either ADA positive or negative. A confirmatory assay, typically based on antigen competition, is subsequently employed to separate false positive samples from truly positive samples. In this manuscript we investigate the performance of different statistical methods classifying samples in competition assays through simulation and analysis of real data. In our evaluations we do not find a uniformly best method although a simple t-test does provide good results throughout. More crucially we find that very large differences between uninhibited and inhibited measurements relative to the assay variability are required in order to obtain useful classification results questioning the usefulness of competition assays with high variability.

Competition for antigen selects T cell responses that undergo memory inflation and maintains clonal dominance during MCMV infection (P6091)

Abstract Cytomegalovirus (CMV) establishes a life-long, persistent/latent infection. Continuous immune surveillance by CMV-specific CD8+ T cells results in their accumulation over time, a process called “memory inflation”. These “inflationary” T cells migrate systemically and comprise the largest T cell populations in humans, making CMV-based vaccines attractive. Using recombinant murine (M)CMV expressing the SIINFEKL peptide, we found that SIINFEKL-specific T cells inflated at the expense of T cells specific for other MCMV-derived peptides. However, co-infecting mice with viruses expressing and lacking SIINFEKL enabled T cells with multiple specificities to inflate, implying T cell competition for antigen at the level of the infected target. Such competition was also observed among SIINFEKL-specific T cells. Adoptively transferred OT-I T cells, which have a high affinity for the SIINFEKL peptide, were preferentially enriched shortly after infection and inhibited the inflation of host-derived T cells specific for SIINFEKL. Importantly, we observed sporadic, late rejection of OT-Is as a result of minor histocompatibility differences, and only such rejection enabled host-derived T cells to inflate robustly. These data imply that relative clonal abundance is actively maintained by competition. Together, these data show that T cell competition for antigen during MCMV infection dictates which T cell responses inflate and even which clones dominate within a response.

Abstract SY27-01: Synthetic vaccine for immunotherapy of high risk HPV infections.

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 competition from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes of 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. 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 this 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 months after the last vaccination, an objective response was observed in 12/20 (60%). Five patients showed a complete and durable regression of the lesions. 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 a preclinical mouse model we have shown marked synergism in eradication of established HPV16 E6/E7+ tumors between platinum-based chemotherapy and HPV16 SLP vaccination, associated with a decrease in myeloid cells in the blood and in the tumor. In a pilot clinical study, end stage cervical cancer patients, treated with carbotaxol, showed no T cell immunodeficiency and showed robust T cell effector responses to a single dose of HPV16 SLP® vaccine. These findings set the stage for a randomized phase II study of chemo-immunotherapy in this category of patients, in which chemotherapy alone will be compared with chemo-immunotherapy. In conclusion, treatment with the HPV16 SLP vaccine is 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 chemotherapy and with immunomodulatory drugs such as type I interferon and checkpoint-control blocking monoclonal antibodies. Citation Format: Cornelis J. M. Melief. Synthetic vaccine for immunotherapy of high risk HPV infections. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY27-01. doi:10.1158/1538-7445.AM2013-SY27-01

Competition for Antigen at the Level of the APC Is a Major Determinant of Immunodominance during Memory Inflation in Murine Cytomegalovirus Infection

The unique ability of CMV to drive the expansion of virus-specific T cell populations during the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor Ags, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine CMVs (MCMVs) encoding a SIINFEKL-enhanced GFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for nonrecombinant (i.e., MCMV-derived) Ags. Moreover, when the virus encoded SIINFEKL, T cells specific for nonrecombinant Ags displayed a phenotype indicative of less frequent exposure to Ag. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV Ags. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled coinflation of T cells specific for both SIINFEKL and nonrecombinant Ags. Because coinfection allows presentation of SIINFEKL and MCMV-derived Ags by different cells within the same animal, these data reveal that competition for, or availability of, Ag at the level of the APC determines the composition of the inflationary response to MCMV. SIINFEKL's strong affinity for H-2K(b), as well as its early and abundant expression, may provide this epitope's competitive advantage.

949 IDENTIFICATION OF TWO CLINICALLY DISTINCT SUBTYPES IN AUTOIMMUNE HEPATITIS BY COMPARATIVE FUNCTIONAL GENOMICS

Conclusion: Incidence of autoimmune hepatitis is increasing, as other autoimmune diseases in developed countries. The main recognised hypothesis is the ‘hygienist theory’ (improvement of hygien leading to a decrease in infections) with few putative non exclusive mechanisms involving antigenic competition, extension of immune regulation induced by exogenous bacterial antigen or Toll Like Receptors.

Outbreak-specific monovalent/bivalent vaccination to control and eradicate virulent ovine footrot

Footrot is a contagious disease of small ruminants which is caused by the bacterium Dichelobacter nodosus. In its virulent form there are severe economic losses and a very significant animal welfare issue. Sheep and goats can be vaccinated for treatment and prevention of the disease. There are 10 different serogroups of D. nodosus (A–I and M) and immunity is serogroup-specific. When all 10 serogroups are presented together in a vaccine, protection persists for only a few months due to “antigenic competition”. Consequently we evaluated the use of sequential monovalent or bivalent vaccines to control/eliminate/eradicate virulent footrot in a longitudinal intervention study on 12 commercial farms in southeast Australia with flock sizes of approximately 1200–4200 sheep. Overall, virulent footrot was eradicated from 4 of the flocks, 2 of which had 2 serogroups, and the others 4 or 5 serogroups. Where there were only 1 or 2 serogroups (3 farms) the clinical response was rapid and dramatic; prevalence was reduced from 45 to 50% before vaccination to 0% (2 farms) or 0.4% (1 farm) after one round of vaccination. In the remaining 9 flocks there were more than 2 serogroups and successive bivalent vaccines were administered leading to eradication of virulent footrot on 2 farms over 4 years and control of the disease on all but 3 of the others. Of the latter farms, 1 discontinued, and 2 initially had poor response to vaccine due to misdiagnosis of serogroup ‘M’, which was previously unknown in Australia. Control was achieved after administration of a serogroup M vaccine. These results provide clear evidence for control, elimination and eradication of virulent footrot by outbreak-specific vaccination in Australia.

Safety and Clinical Efficacy of Rapidly-Generated Trivirus-Directed T Cells After Allogeneic Hematopoietic Stem Cell Transplant

We have previously demonstrated that small numbers of ex vivo-expanded, trivirus-specific T cells targeting EBV, CMV, and Adv are safe, effective and protective in vivo. However, broader implementation is limited by the need for infectious virus/vector, and prolonged (8-12wks) and complex manufacture, while antigenic competition limits extension to additional viruses. We now evaluate whether T-cell lines manufactured using methods that exclude viral components and utilize simplified manufacturing technology can be clinically effective. With NHLBI-PACT support, 29 clinical-grade rCTL lines have been generated. From an initial 15x106 PBMCs, we prepared a median of 214±88x106 T-cells (range 100-420x106) over 9-11 days using DCs nucleofected with DNA plasmids encoding immunogenic EBV (LMP2, EBNA1 and BZLF1), Adv (Hexon and Penton), and CMV (pp65 and IE1) antigens, and expansion with IL4+7 in G-Rex devices. The rCTL lines were polyclonal, comprising both CD4+ (33±3%) and CD8+ (60.5±3%) cells, that expressed activation and memory markers. Twenty lines generated from donors that were seropositive for all viruses demonstrated activity against all 3 targets - CMV (IE1: 359±100; pp65: 637±177 SFC/2x105), EBV (LMP2: 217±60, EBNA1: 67±19 and BZLF1: 111±31) and Adv (Hexon: 265±74, Penton: 191±53) - while 9 lines generated from CMV seronegative donors demonstrated activity exclusively against EBV (LMP2: 197±70, EBNA1: 145±51 and BZLF1: 239±84) and Adv (Hexon: 271±96, Penton: 254±90). None of the T-cell lines reacted against unmodified recipient cells. To date we have administered these lines to 10 allogeneic HSCT recipients at doses ranging from 0.5-2x107/m2 as treatment for CMV (n=3), Adv (n=2), EBV (n=2), EBV+Adv (n=1), and CMV+Adv (n=2). One patient developed a skin rash 2 weeks post-rCTLs but no other toxicity have been observed. Eight treated patients, including one with a biopsy-proven EBV lymphoma and the 3 patients with double reactivations, had complete clinical responses to rCTL, which corresponded with an increase in the frequency of virus-specific T-cells detected in peripheral blood. For CMV we saw an increase from a median of 0.5 to 96 and 1 to 277 SFC/4x105 IE1 and pp65-specific T cells, respectively 3-6wks post-infusion; for Adv an increase from mean 0.5 to 137 and 0.5 to 99 SFC/4x105 Hexon and Penton-specific cells, respectively, and for EBV an increase from 2.8 to 227, 1.5 to 39, and 1 to 188.5 SFC/4x105 EBNA1, LMP2, and BZLF1-specific T-cells, respectively. Two patients failed to respond to rCTLs; one with a 3 year history of persistent CMV colitis and one with elevated EBV DNA; both had high pre-existing virus-specific T-cell precursors. rCTLs have been safe and effective in 80% of treated patients and have the potential to increase the availability of cell products for HSCT recipients. We are currently extending this platform to additional viruses.

Rational Design of DNA Vaccines for the Induction of Human Papillomavirus Type 16 E6- and E7-Specific Cytotoxic T-Cell Responses

Many DNA vaccine candidates have been developed for the treatment of human papillomavirus type 16 (HPV16)-induced malignancies. Most of these vaccines consist of a fusion of E7 with a "carrier-protein" that functions to increase the potency of the vaccine. The nature of these carrier-proteins varies widely, and the mechanisms proposed to explain the enhanced immunogenicity of such fusions are often linked to the biological function of the carrier-protein. However, the potentiating effect of these carrier-proteins might also be explained by more general mechanisms, such as the provision of CD4+ T-cell help, increased antigen stability, or altered subcellular localization of the antigen. To assess whether these more generic mechanisms could suffice to generate highly immunogenic DNA vaccines, we evaluated a series of modular HPV16 E7 DNA vaccines in which the presence of CD4+ T-cell help, the presence of an endogenous carrier-protein, and the subcellular localization of the antigen could be systematically altered. Using this approach, we demonstrate that the addition of an element that provides CD4+ T-cell help, elements that enforce endoplasmic reticulum (ER) localization/retention are both necessary and sufficient to create markedly effective HPV16 E7-directed DNA vaccines. Importantly, the resulting design rules also apply to an HPV16 E6-directed DNA vaccine. The developed "HELP(ER)" HPV DNA vaccines encode only very limited additional sequences besides the antigen, thereby reducing the risk of antigenic competition and/or autoimmunity.

Rapidly Generated Multivirus-specific Cytotoxic T Lymphocytes for the Prophylaxis and Treatment of Viral Infections

Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein–Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4+ and CD8+) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.

Generation of Multi-Antigen Specific T Cells for Adoptive Immunotherapy of Myeloid Leukemia and Identification of MHC Class I and II-Restricted Peptides for WT1, Proteinase 3 and Human Neutrophil Elastase

Abstract 2985Generating cytotoxic T lymphocytes (CTL) against malignant cells using defined leukemia associated antigen (LAA) vaccines, or ex vivo adoptively transferred T cells, is a promising approach to prevent or treat leukemia. Vaccine or T cell therapies have largely focused on CD8 responses to defined HLA-A2 restricted LAA peptides. Strategies targeting single peptides are limited to the recipient’s HLA type, lack of CD4 help, and risk of tumor immune escape. To overcome these constraints and develop T cell immunotherapy for patients with myeloid malignancies, we used entire peptide libraries of several LAA to generate multi-specific CD4+ and CD8+ leukemia–reactive CTL. We obtained 15mer peptide libraries overlapping by 11 aa for WT1, Proteinase 3 (Pr3), MAGE-A3, PRAME and human neutrophil elastase (HNE). PBMC of 10 healthy donors were stimulated with DC pulsed with peptide libraries of one antigen or the mix of 5 LAA (LAA mix). Cultures were re-stimulated weekly with peptide-pulsed DCs and peptide-recognition was tested by IFN γ-ELIspot. Positive cultures were further characterized by performing intracellular cytokine staining, measuring degranulation by CD107a release, and assessing in vitro cytolytic activity using peptide-pulsed autologous PHA blasts as targets in a flow cytometric killing assay.Nine out of 10 healthy donors responded to the LAA mix as assessed by IFN γ-ELIspot (mean: 471.2 SFU/2×105 cells, range: 296–968.5). The expanded CTL specifically recognized 3 antigens (mean, range 1–5), and PRAME was most frequently recognized (in 9 of 10 donors screened). Within donors we consistently saw recognition of the same profile of antigens irrespective of whether the CTL were induced with individual antigen or LAA mix, indicating that antigenic competition between LAA was not a limiting factor in our cultures. Correlating with ELIspot results, LAA CTL were cytotoxic to PHAB pulsed with the LAA mix (E:T=40:1; mean: 33.1%, range: 12.4–80.0%; negative control: PHAB: mean: 4.2%, range: 0–13.4%; n=7), as well as individual antigens (WT1: mean :12.2%, range: 0–26.1%; Pr3: mean: 9.7%, range: 5.6–13.7%; HNE: mean: 35.4%, range: 0–84.9%; MAGE-A3: mean: 19.9%, range: 15.2–28.2%; PRAME: mean: 16.7%, range: 3.8–37.5%). Cytotoxicity against the peptide library or the LAA mix was greater than against targets pulsed with the immunodominant peptide, demonstrating the benefit of a broad immune response. Cytolytic activity was also seen in donors with predominantly CD4+ response, suggesting a role for T helper cells in tumor lysis.The approach of stimulation with LAA mix was also feasible for epitope mapping. In addition to known peptides, we identified 16 new MHC class I and II-restricted peptides (WT1: 8, HNE: 6 and Pr3: 2). Thereby, up to 4 immunogenic peptides of two different antigens could be identified in one LAA mix culture. WT1 peptides showed class I and class II-restriction by blocking experiments with HLA antibodies. HNE peptides were mainly class I-restricted, with a dominant response against RRLACLFLACVLPAL in 5 of 7 donors.Thus, stimulation of healthy donor PBMC with LAA mix generates CTL without depending on known HLA-restrictions and may be a practical approach for adoptive immunotherapy of myeloid leukemia after stem cell transplantation. Moreover, the strategy enabled the identification of several new class I and II-restricted peptides. Importantly, in vitro cytolytic activity could also be shown for MHC class II-restricted peptides, indicating the importance of CD4+ T cells for anti-leukemia responses. Disclosures:No relevant conflicts of interest to declare.