Evade Host Immunity Research Articles

The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a typeI interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

The Genetics of Enteropathogenic Escherichia coli Virulence.

In many parts of the world, enteropathogenic Escherichia coli (EPEC) are a leading cause of death in children with diarrhea. Much of what we know about the pathogenesis of EPEC infections is based on the study of one or two prototypic strains that have provided deep insight into the precise mechanisms by which EPEC colonizes the intestine, evades host immunity, and spreads from person to person. In some cases, defining the biochemical activity of the host-interacting effector proteins from these prototypic strains has led to the discovery of novel post-translational protein modifications and new understandings of biology and host-pathogen interactions. However, genomic analysis of recent EPEC isolates has revealed that the EPEC pathotype is more diverse than previously appreciated. Although by definition all strains carry the locus of enterocyte effacement, the effector repertoires of different clonal groups are quite divergent, suggesting that there is still a great deal to learn about the genetic basis of EPEC virulence.

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems in bacteria and archaea provide adaptive immunity against invading foreign nucleic acids. Previous studies suggest that certain bacteria employ their Type II CRISPR-Cas systems to target their own genes, thus evading host immunity. However, whether other CRISPR-Cas systems have similar functions during bacterial invasion of host cells remains unknown. Here we identify a novel role for Type I CRISPR-Cas systems in evading host defenses in Pseudomonas aeruginosa strain UCBPP-PA14. The Type I CRISPR-Cas system of PA14 targets the mRNA of the bacterial quorum-sensing regulator LasR to dampen the recognition by toll-like receptor 4, thus diminishing the pro-inflammatory responses of the host in cell and mouse models. Mechanistically, this nuclease-mediated RNA degradation requires a "5'-GGN-3'" recognition motif in the target mRNA, and HD and DExD/H domains in Cas3 of the Type I CRISPR-Cas system. As LasR and Type I CRISPR-Cas systems are ubiquitously present in bacteria, our findings elucidate an important common mechanism underlying bacterial virulence.

High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC.

The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 – group 1, mAB 305-78-7 – group 2, and mAB 305-101-8 – group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.

Suppression of Host Innate Immune Response by Hepatitis C Virus via Induction of Autophagic Degradation of TRAF6.

HCV can cause severe liver diseases and is one of the most important human pathogens. It establishes chronic infections in the great majority of patients that it infects, indicating that it has evolved sophisticated mechanisms to evade host immunity. TRAF6 is an important signaling molecule that mediates activation of NF-κB and expression of proinflammatory cytokines and interferons. In this study, we found that HCV infection suppressed the host innate immune response through the induction of autophagic degradation of TRAF6. This finding provided important information for further understanding how HCV evades host immunity to establish persistence.

Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation.

ABSTRACTThe chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; however, challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Here, we use a genetic and proteomic approach to identify authentic CPAF targets. Human epithelial cells infected with CPAF-sufficient and CPAF-deficient chlamydiae were lysed using known CPAF-denaturing conditions. Their protein profiles were analyzed using isobaric mass tags and liquid chromatography-tandem mass spectrometry. Comparative analysis of CPAF-sufficient and CPAF-deficient infections identified a limited number of CPAF host and chlamydial protein targets. Host targets were primarily interferon-stimulated gene products, whereas chlamydial targets were type III secreted proteins. We provide evidence supporting a cooperative role for CPAF and type III secreted effectors in blocking NF-κB p65 nuclear translocation, resulting in decreased beta interferon and proinflammatory cytokine synthesis. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). We propose that CPAF and T3SEs cooperate in the inhibition of host innate immunity.

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable and thus difficult to analyze using short-read sequencing technologies. Here, we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, indel and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that indels are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and indel markers to analyze patterns of meiotic recombination, confirming a high rate of crossover events and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of meiotic recombination within copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired.

Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq.

An RNA chaperone of Escherichia coli, called host factor required for phage Qβ RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity.

Introduction to Biofilms Thematic Minireview Series

The biofilms that many bacteria and fungi produce enable them to form communities, adhere tightly to surfaces, evade host immunity, and resist antibiotics. Pathogenic microorganisms that form biofilms are very difficult to eradicate and thus are a frequent source of life-threatening, hospital-acquired infections. This series of five minireviews from the Journal of Biological Chemistry provides a broad overview of our current understanding of biofilms and the challenges that remain. The structure, biosynthesis, and biological function of the biofilms produced by pathogenic fungi are the subject of the first article, by Sheppard and Howell. Gunn, Bakaletz, and Wozniak focus on the biochemistry and structure of bacterial biofilms, how these structures enable bacteria to evade host immunity, and current and developing strategies for overcoming this resistance. The third and fourth articles present two of the best understood cell signaling pathways involved in biofilm formation. Valentini and Filloux focus on cyclic di-GMP, while Kavanaugh and Horswill discuss the quorum-sensing (agr) system and the relationship between quorum sensing and biofilm formation. Mechanisms of antibiotic resistance, particularly the role of efflux pumps and the development of persister cells, are the topics of the final article by Van Acker and Coenye. The advances described in this series guarantee that ongoing interdisciplinary and international efforts will lead to new insights into the basic biology of biofilm formation, as well as new strategies for therapeutic interventions.

What's on the Outside Matters: The Role of the Extracellular Polymeric Substance of Gram-negative Biofilms in Evading Host Immunity and as a Target for Therapeutic Intervention

Biofilms are organized multicellular communities encased in an extracellular polymeric substance (EPS). Biofilm-resident bacteria resist immunity and antimicrobials. The EPS provides structural stability and presents a barrier; however, a complete understanding of how EPS structure relates to biological function is lacking. This review focuses on the EPS of three Gram-negative pathogens: Pseudomonas aeruginosa, nontypeable Haemophilus influenzae, and Salmonella enterica serovar Typhi/Typhimurium. Although EPS proteins and polysaccharides are diverse, common constituents include extracellular DNA, DNABII (DNA binding and bending) proteins, pili, flagella, and outer membrane vesicles. The EPS biochemistry promotes recalcitrance and informs the design of therapies to reduce or eliminate biofilm burden.

Pilot trial of intratumoral (IT) G100, a toll-like receptor-4 (TLR4) agonist, in patients (pts) with Merkel cell carcinoma (MCC): Final clinical results and immunologic effects on the tumor microenvironment (TME).

3021Background: MCC is an aggressive skin cancer with suboptimal therapies. Despite persistent expression of Merkel cell polyomavirus in ~80% of pts, MCC tumors are able to evade host immunity. G10...

Genetic diversity of immune-related antigens in Region of Difference 2 of Mycobacterium tuberculosis strains

Region of Difference 2 (RD2) was lost during the ongoing propagation of BCG between 1927 and 1931, a time that coincides with reports of the ongoing attenuation of the vaccine. Some data demonstrate that RD2 plays a role in mycobacterial virulence, and that its deletion from Mycobacterium tuberculosis leads to a decrease in bacterial growth in both a macrophage and a murine model. Human T-cell epitopes of M. tuberculosisare evolutionarily hyperconserved and thus it was deduced that M.tuberculosis lacks antigenic variation and immune evasion. However, two antigens, Rv1986 and MPT64, encoded by RD2 harbored more than one amino acid changes. In this study, we used same set of clinical M.tuberculosis complex (MTBC) isolates from China, amplified the five genes containing T and B cell epitopes other than MPT64 encoded by RD2, and compared the sequences. It turned out that proteins in RD2 region, especially Rv1980c, Rv1985 and Rv1986 may be a special region that undergo antigenic variation in response to host immune pressure and may be involved in ongoing immune evasion. The dN/dS value of all six genes (including MPT64) were 2.33, much higher than 1, which means T cell antigens in RD2 region appeared to be under diversifying selection. Our data support the view that RD2 regions tend to be more variable than we expected to evade host immunity and the immune-related antigens in RD2 were more variable than we expected, especially in T-cell epitope regions.

Bait and Switch: A strategy to avoid host defense by the ticks

Abstract Ticks are obligate haematophagous ectoparasites of animals and humans. In the livestock industry, economic losses due to ticks and tick borne diseases (TBD) amount to million of dollars. In public health, ticks are second to mosquitoes in terms of impact of human diseases. CDC has listed 14 TBD causative agents of which, 9 are transmitted by Amblyomma americanum and Ixodes scapularis. With few exceptions, the transmission of TBD agents requires successful tick feeding. Thus understanding tick-feeding mechanism is a critical step in developing novel methods to stop TBD agent transmission. We know that ticks secrete numerous tick saliva proteins that suppress host immunity to facilitate feeding and disease agent colonization. However, mechanisms of how the tick suppresses host immunity remain unanswered. We investigated the effect of 15 A. americanum and I. scapularis recombinant tick saliva proteins (rTSPs) on murine macrophage function as revealed by expression of pro-inflammatory co-stimulatory markers (CD40, C80, and CD86). Additionally, the effect of rTSP on macrophage secretion of pro- and anti-inflammatory cytokines (TNFa, IL1, IL6, IL-10, TGF beta) was also investigated. Our analysis identified five pro-inflammatory and two anti-inflammatory tick saliva proteins. The two anti-inflammatory proteins appear to reverse the expression of co-stimulatory markers activated by LPS and rTSPs. These data collectively demonstrate the presence of a “bait and switch” mechanism through which the tick may evade host immunity. We propose that ticks secrete two types of immune-modulating proteins: those that stimulate host immune cells and those that dampen the activated cells.

Immune checkpoint inhibition in ovarian cancer.

Recent studies have shown that tumor cells acquire escape mechanisms to evade host immunity in the tumor microenvironment. Two key immune checkpoint pathways mediated by immunosuppressive co-signaling, the first via programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-1/PD-L1) and the second via CTLA-4 and B7 (CTLA-4/B7), have been previously described. Several clinical trials have revealed an outstanding anti-tumor efficacy of immune checkpoint inhibitors (anti-CTLA-4 antibody, anti-PD-1 antibody and/or anti-PD-L1 antibody) in patients with various types of solid malignancies, including non-small cell lung cancer, melanoma, renal cell cancer and ovarian cancer. In this review, we examine pre-clinical studies that described the local immune status and immune checkpoint signals in ovarian cancer, highlight recent clinical trials that evaluated immune checkpoint inhibitors against ovarian cancer and discuss the clinical issues regarding immune checkpoint inhibitors.

PD20-06 PRESENCE OF SEROTYPE H EXHIBITS MILDER SYMPTOMATIC CHARACTERISTICS IN AGED MEN THAN OTHER SEROTYPES

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Prostate & Genitalia I1 Apr 2016PD20-06 PRESENCE OF SEROTYPE H EXHIBITS MILDER SYMPTOMATIC CHARACTERISTICS IN AGED MEN THAN OTHER SEROTYPES Yu Seo and Gil Lee Yu SeoYu Seo More articles by this author and Gil LeeGil Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1419AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The rate of Chlamydia trachomatis infection has been steadily increasing over the past decade. Extant research on genital chlamydia screening focuses primarily on women. Furthermore, current clinical practices do not recommend routine chlamydia screening for middle aged heterosexual men showing little or no symptoms. Consequently, these men are often omitted from studies on genital chlamydia infection, resulting in limited availability of their epidemiological data. Chlamydia ompA gene has unique regions for evading host immunity. Therefore, certain ompA genotype may have a selective advantage for its survival. In this study, we evaluated the patterns of urinary chlamydia ompA genotypes in men who visited the clinic with or without symptoms and signs of urethritis and investigated the effects of different genotypes on clinical features. METHODS We routinely collected 685 urine samples from participants from January 2009 to April 2012. In addition, we included four stored urine specimens from four urethritis patients collected in 2008. A multiplex PCR was performed using KL1-KL2 primers for chlamydia infection and HO1-HO3 primers for Neisseria gonorrhoeae, and the amplified ompA products were sent to determine the nucleotide sequences. RESULTS The mean age (Yrs ± SD) is 37.38±11.97. The serotypes, in order of prevalence, are serotype H (69.23%, n=27), followed by E (12.82%, n=5), F (7.69%, n=3), D (7.69%, n=3), and J (2.56%, n=1). We find a substantial difference in age between serotype H and the other serotypes (D, E, F, and J) (p=0.004, nonparametric analysis) (Table 1). In addition, men with serotype H showed milder symptom (urethral discharge, P=0.001) and sign (pyuria, P=0.001) than the other serotypes (Table 1). CONCLUSIONS The presence of serotype H was associated reduced symptomatic characteristics in middle aged men compared to other serotypes. This mild nature of senotype H may facilitate the spread of infection as the infected persons do not visit the clinic and hence clinicians cannot evaluate the infection. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e452-e453 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yu Seo More articles by this author Gil Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Discovery and expression of 3 siglecs-like in Oreochromis niloticus neutrophil, and their interaction with group B streptococcal sialylated capsular polysaccharides.

Sialic acid - binding immunoglobulin - like lectins (Siglecs) are members of the largest superfamily of immune receptors; they recognize sialic acid and are mainly expressed in immune cells. Studies on mammals indicate that Streptococcus agalactiae (GBS) evade immune reactions by interacting with the host immune cells via the sialic acid of sialylated capsular polysaccharides. However, it is currently unknown if fish-derived GBS can interact with Siglecs to evade host immunity. In this study, we examined the binding of FITC-GBS with neutrophils to determine the presence of receptors that binds with GBS. Furthermore, 3 Siglec-like genes, (OnSiglec-1-like/-4b-like/-14-like) from the neutrophils cDNA were screened by PCR. All the genes had specific domains (immunostimulation and immunosuppression domains), conserved amino acid residues, and sialic acid polysaccharide binding sites that are found in mammalian Siglecs. Flow cytometry of Siglecs-like/COS-7 cells and ELISA of Siglecs/Ex-Fc fusion proteins confirmed that 3 Siglecs-like have high binding activity with GBS. Erythrocytes adhesion assays and sialylated glycans binding assay confirmed that 3 Siglecs-like bind to sialic acid polysaccharides. Siglecs-like had high expression levels in the spleen, gill, and kidney in Oreochromis niloticus by qPCR. After experimental infection, Siglec-1-like/-14-like showed a significant upregulated initially and later downregulated in liver, spleen, kidney, and gill. However, Siglec-4b-like was downregulated in most tissues, except that in liver. The results indicate that 3 OnSiglecs-like may recognize GBS sialylated capsular polysaccharides. GBS infections led to significant changes in Siglecs-like expression in immune-related tissues. However, immunostimulation or immunosuppression via the recognition of GBS by different Siglecs-like molecules requires additional studies.

Abstract A064: Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers

Abstract Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of development in immunosuppressed patients. Some however evade host immunity, and establishing the mechanism of immune evasion could assist with squamous cancer immunotherapy. Method: We have used a model of squamous epithelial pre malignancy, in which HPV16 E7 transgenic and hyperproliferative mouse skin is transplanted to naïve mice, and have explored local regulation of effector T cell function by E7 transgenic epithelial cells through manipulation of the graft donor immune system. Results: E7 transgenic skin is not rejected by naïve recipient mice, or by mice immunised with E7 protein and expressing E7 specific cytotoxic T cells. E7 expressing hyper proliferative epithelium demonstrates caspase-1 activation, production of IL-1β, IL-18, and CCL2, and attraction and activation of NKT cells and mast cells. Rejection of E7 transgenic skin grafts can be observed following depletion from the donor skin of bone marrow derived cells, of NKT cells or of mast cells, whereas adjacent E7 transgenic grafts, otherwise unmanipulated, are not rejected. E7 graft rejection can also be induced by suppression in the donor skin or recipient animal of NKT cell secreted IL-17 or IFN-γ, or by depletion of the soluble IL-1 receptor IL-1R1 or blockade of Arginase activity in the recipient. Inhibition of graft rejection is not overcome by antibody to PD-1, PD-L1, or PD-L2. Hyperproliferative cervix epithelium in patients with CIN3 expresses the factors identified as inhibitory to E7 transgenic skin graft rejection, whereas skin from E7 transgenic mice with a mutated Rb gene preventing E7 induced epithelial proliferation lacks the inhibitory factors. Conclusion: Epithelial hyper proliferation induces immunosuppressive mechanisms that locally prevent cytotoxic T cell function through mechanisms independent of checkpoint blockade. We hypothesise from our data that the local balance of production of IL-1β and Il-18 locally determines cytotoxic T cell effector function targeted at epithelial antigens. Citation Format: Stephen Mattarollo, Graham Leggatt, James Wells, Paul Lambert, Ian H. Frazer. Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A064.

Blockade of PD-1 in Combination with Dendritic Cell/Myeloma Fusion Cell Vaccination Following Autologous Stem Cell Transplantation Is Well Tolerated, Induces Anti-Tumor Immunity and May Lead to Eradication of Measureable Disease

Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. DisclosuresRichardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion.

Previous studies have demonstrated that the Pro‑Glu/Pro‑Pro‑Glu (PE/PPE) genes in strains of Mycobacterium tuberculosis exhibit high sequence variation and may be involved in antigenic variation and immune evasion. Region of Difference 1 (RD1), encoding genes from Rv3871 to Rv3879, was observed to be lost during the original derivation of Bacillus Calmette‑Guérin between 1908 and 1921. It has been previously demonstrated that two PE/PPE proteins, PE35 (Rv3872) and PPE68 (Rv3873), are encoded by RD1 and exhibit immunodominance. To explore the genetic diversity of PE35 and PPE68, and to evaluate the impact of sequence variation on the immune recognition of these proteins, 161 clinical M. tuberculosis strains were selected from China and comparative sequence analysis of PE35 and PPE68 was performed. The results indicated that polymorphisms in PE35 and PPE68 may lead to alterations in the function of these proteins, which may potentially affect strain virulence. In addition, the human T‑cell epitopes of PE35 and PPE68 were highly variable, suggesting that the two antigens may be involved in diversifying selection to evade host immunity. The prevalence of strains with PE35 mutations in the non‑Beijing family was significantly greater compared with the Beijing family, indicating that Beijing strains may be more conservative than non‑Beijing strains in this gene.

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) Identifies Immune-Selected HIV Variants.

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations of mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. With well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines.