The Functions of Hepatitis B Virus Encoding Proteins: Viral Persistence and Liver Pathogenesis.

About 250 million people worldwide are chronically infected with Hepatitis B virus (HBV), contributing to a large burden on public health. Despite the existence of vaccines and antiviral drugs to prevent infection and suppress viral replication respectively, chronic hepatitis B (CHB) cure remains a remote treatment goal. The viral persistence caused by HBV is account for the chronic infection which increases the risk for developing liver cirrhosis and hepatocellular carcinoma (HCC). HBV virion utilizes various strategies to escape surveillance of host immune system therefore enhancing its replication, while the precise mechanisms involved remain elusive. Accumulating evidence suggests that the proteins encoded by HBV (hepatitis B surface antigen, hepatitis B core antigen, hepatitis B envelope antigen, HBx and polymerase) play an important role in viral persistence and liver pathogenesis. This review summarizes the major findings in functions of HBV encoding proteins, illustrating how these proteins affect hepatocytes and the immune system, which may open new venues for CHB therapies.

Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target.

Preparation and functional evaluation of monoclonal antibodies targeting Hepatitis B Virus Polymerase.

HBV pol plays a critical role in the replication of hepatitis B virus (HBV). Previous studies conducted on HBV pol have produced limited evidence on HBV pol expression due to the lack of effective detection methods. The present study used the HBV pol (159-406 aa) protein as a target to screen for specific monoclonal antibodies that recognize HBV pol and subsequently evaluate their diagnostic and therapeutic value. Four antibodies (P3, P5, P12, P20) against HBV pol were obtained. Among them, the P20 antibody indicated optimal binding with HBV pol as demonstrated by Western blotting (WB) in a cell model transfected with the HBV genome. We also expressed P5 and P12 antibodies in mouse liver cells by transfection and the results indicated significant antiviral effects caused by these two antibodies especially P12. In summary, the present study established an antibody which was denoted P20. This antibody can be used to detect HBV pol expression by four HBV genomes via WB analysis. In addition, the antibody denoted P12 could exert antiviral effects via intracellular expression, which may provide a promising approach for the treatment of chronic hepatitis B.

Tet DNA demethylase is required for plasma cell differentiation by controlling expression levels of IRF4.

Antibodies produced by plasma cells are critical for protection from infection. It has been demonstrated that global epigenetic modification, such as changes in DNA methylation, occurs during differentiation of plasma cells from B cells. However, the precise mechanisms by which DNA methylation controls plasma cell differentiation are not fully understood. We examined the effect of deficiency of DNA demethylases, Tet2 and Tet3, on B-cell activation and plasma cell differentiation, by generating conditional Tet2/3 double-KO (Tet dKO) B cells. We found that Tet dKO B cells failed to differentiate into plasma cells upon immunization with antigens. Tet dKO B cells proliferated normally and were capable of generating cells with IRF4int, but not with IRF4hi, the majority of which were CD138+ plasma cells. IRF4 overexpression rescued the defect of Tet dKO B cells in plasma cell differentiation, suggesting that Tet2/3-dependent high IRF4 expression is required for plasma cell differentiation. We identified CpG sites in the Irf4 locus that were demethylated specifically in plasma cells and in a Tet2/3-dependent manner. Our results suggest that Tet2/3-dependent demethylation of these CpG sites is dispensable for initial IRF4 expression but is essential for high IRF4 expression which is prerequisite for plasma cell differentiation.

Immune cell infiltrate-associated dysregulation of DNA repair machinery may predispose to papillary thyroid carcinogenesis.

BACKGROUND: An altered immune microenvironment may contribute to papillary thyroid cancer development, as immune infiltrates are identified postoperatively in many papillary thyroid cancer cases with or without diagnosed thyroiditis. Oxygen radicals, endogenous or inflammation-induced, can generate DNA damage, which causes mutations when repaired incorrectly. We hypothesized that infiltrating immune cells might promote aberrant DNA repair, predisposing thyrocytes to papillary thyroid cancer. METHODS: Quantitative reverse-transcriptase polymerase chain reaction assays measured gene expression in fresh-frozen samples (n = 55). RNA-seq data was obtained for papillary thyroid cancer and normal thyroid samples from the Cancer Genome Atlas (n = 564), and Hashimoto's-affected and normal thyroids from the Genotype-Tissue Expression project (n = 279). Immune cell marker expression levels were compared to histological estimates and to selected DNA repair genes. Immunohistochemistry localized gene expression to specific cell types. RESULTS: DNA polymerase theta expression by quantitative reverse-transcriptase Polymerase chain reaction was higher in papillary thyroid cancer and papillary thyroid cancer-adjacent samples than in benign normal thyroid (P < .001). Immune markers including CD4 correlated with DNA polymerase theta expression (r = 0.50) but not other DNA repair genes examined. Benign tissue with Hashimoto's exhibited increased DNA polymerase theta (P < .0001) and CD3E (P < .0001) expression. DNA polymerase theta localized to thyrocytes, not lymphocytes. CONCLUSION: We identified a strong correlation between immune cell infiltrate and dysregulated thyrocyte DNA repair genes, likely reflecting a pathway to papillary thyroid cancer development.

DNA polymerase kappa counteracts inflammation-induced mutagenesis in multiple organs of mice.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52-/- B cells show increased CSR, fewer intra-Sµ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52-/- versus Rad52+/+ B cells on Ku86 knockdown.

Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures.

For 30 years two general mechanisms have competed to explain somatic hypermutation of immunoglobulin (Ig) genes. The first, the DNA-based model, is focused only on DNA substrates. The modern form is the Neuberger "DNA Deamination Model" based on activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA Polymerase-η operating around AID C-to-U lesions. The other is an RNA-based mechanism or the "Reverse Transcriptase Model" of SHM which produces strand-biased mutations at A:T and G:C base pairs. This involves error-prone cDNA synthesis via an RNA-dependent DNA polymerase copying the Ig pre-mRNA template and integrating the now error-filled cDNA copy back into the normal chromosomal site. The modern form of this mechanism depends on AID dC-to-dU lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA Polymerase-η acting as a reverse transcriptase. The evidence for and against each mechanism is critically evaluated. The conclusion is that all the SHM molecular data gathered since 1980 supports directly or indirectly the RNA/RT-based mechanism. All the data and critical analyses are systematically laid out so the reader can evaluate this conclusion for themselves. Recently we have investigated whether similar RNA/RT-based mutator mechanisms explain how de novo mutations arise in somatic tissues (cancer genomes). The data analyses indeed suggest that cancers arise via dysregulated "Ig-like SHM responses" involving rogue DNA and RNA deaminations coupled to genome-wide RT events. Further, Robyn Lindley has recently shown that the strand-biased mutations in cancer genome genes are also in "codon-context." This has been termed Targeted Somatic Mutation (TSM) to highlight that mutations are far more targeted than previously thought in somatic tissues associated with disease. The TSM process implies an "in-frame DNA reader" whereby DNA and RNA deaminases at transcribed regions are guided in their mutagenic action, by the codon reading frame of the DNA.

Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner.

Demethylation of CpG motifs in the Foxp3 intronic element, conserved noncoding sequence 2 (CNS2), is indispensable for the stable expression of Foxp3 in regulatory T cells (Tregs). In this study, we found that vitamin C induces CNS2 demethylation in Tregs in a ten-eleven-translocation 2 (Tet2)-dependent manner. The CpG motifs of CNS2 in Tregs generated in vitro by TGF-ß (iTregs), which were methylated originally, became demethylated after vitamin C treatment. The conversion of 5-methylcytosin into 5-hydroxymethylcytosin was more efficient, and the methyl group from the CpG motifs of Foxp3 CNS2 was erased rapidly in iTregs treated with vitamin C. The effect of vitamin C disappeared in Tet2(-/-) iTregs. Furthermore, CNS2 in peripheral Tregs in vivo, which were demethylated originally, became methylated after treatment with a sodium-dependent vitamin C transporter inhibitor, sulfinpyrazone. Finally, CNS2 demethylation in thymic Tregs was also impaired in Tet2(-/-) mice, but not in wild type mice, when they were treated with sulfinpyrazone. Collectively, vitamin C was required for the CNS2 demethylation mediated by Tet proteins, which was essential for Foxp3 expression. Our findings indicate that environmental factors, such as nutrients, could bring about changes in immune homeostasis through epigenetic mechanisms.

Class-switched memory B cells remodel BCRs within secondary germinal centers.

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice.

OBJECTIVE: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). METHODS: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. RESULTS: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. CONCLUSIONS: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

Antibody to Epstein-Barr virus deoxyuridine triphosphate nucleotidohydrolase and deoxyribonucleotide polymerase in a chronic fatigue syndrome subset.

BACKGROUND: A defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar "clinical" conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥ 12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbach's alpha. METHODS: Antibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13-16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS. RESULTS: Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls. CONCLUSIONS: There is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.

Multiple distinct forms of CD8+ T cell cross-reactivity and specificities revealed after 2009 H1N1 influenza A virus infection in mice.

Influenza primed mice are protected against lethal infection with H1N1 A/CA/04/E3/09 virus, and T depletion and serum transfer studies suggest a T-dependent mechanism. We therefore set out to investigate the quality of the cross-reactive T cell response to CA/E3/09 in mice primed with H3N2 influenza A/Hong Kong/X31 virus. Sequences of the immunodominant nucleoprotein (NP) NP366-374 and acid polymerase (PA) PA224-233 CD8 epitopes from X31 each differ from the CA/E3/09 virus by one amino acid: an M371V substitution at position 6 of the NP peptide, and an S224P substitution at position 1 of the PA peptide, raising questions about the role of these epitopes in protection. PA224-233 peptides from either virus could elicit IFN-γ spot forming cells from mice infected with X31, indicating cross-reactivity of these two peptides. However, no T cell responses to either PA224-233 peptide were detectable after primary CA/E3/09 infection, suggesting it is cryptic in this virus. In contrast, primary responses to the NP366 peptides were detectable after infection with either virus, but did not cross-react in vitro. Similarly, H2-D(b) tetramers of each NP epitope stained CD8+ T cells from each respective virus infection, but did not obviously cross-react. Early after lethal CA/E3/09 challenge, X31 primed mice had enhanced IFN-γ responses toward both NP366 peptides, as well as recall responses to a set of subdominant NP and PA peptides not detectable after primary X31 infection alone. Furthermore, dual-tetramer staining revealed an expanded population of CD8 T cells reactive to both NP366 variant peptides also not seen after the priming infection alone. These observations demonstrate unusual CD8+ T cell cross-reactivity and specificity are elicited after primary and secondary CA/E3/09 influenza virus infections.

Sindbis virus replicase-based DNA vaccine construct encoding FMDV-specific multivalent epitope gene: studies on its immune responses in guinea pigs.

Foot-and-mouth disease (FMD) is still a perennial global menace affecting livestock health and production. It is imperative to figure out new ways to curb this disease. In this study, a sindbis virus replicase-based DNA vaccine, pSinCMV-Vac-MEG990, encoding a multivalent epitope gene (representing tandemly linked VP1 C-terminal halves of three foot-and-mouth disease virus (FMDV) serotypes) was constructed. In vitro transfection studies in BHK-21 cells revealed that the construct was able to express FMDV-specific antigen but does not overproduce the antigen. Immunization of guinea pigs with the construct at dose rate of 10, 5, 2 and 1 µg per animal through intramuscular route showed significant neutralizing antibody induction at all doses against all serotype tested as compared to non-immunized controls. On viral challenge of guinea pigs 4 week post-immunization with 1000 GPID(50) of FMDV serotype A, it was observed that the immunization not only delayed the appearance and reduced the severity of FMD lesions significantly (P < 0.05) but also provided complete protection in several guinea pigs. In fact, two of six and one of six guinea pigs were completely protected in 10 and 5 µg immunized groups, respectively. These results suggest that the development of the replicase-based DNA vaccine may provide a promising approach as an alternative vaccine strategy for controlling FMD.

The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between -54 and -43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

Isolation and characterization of high affinity aptamers against DNA polymerase iota.

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

Immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis B virus proteins.

Viruses employ various means to evade immune detection. Reduction of CD8(+) T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8(+) T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.

Characterization and epitope mapping of monoclonal antibodies recognizing N-terminus of Rep of porcine circovirus type 2.

Two genotypes of porcine circovirus (PCV) have been described, the non-pathogenic PCV1 and the pathogenic PCV2 in pigs. PCV-ORF1 encodes Rep and Rep' proteins which have identical N-terminal sequence (RepN) within each PCV strain, but RepN has only 88% similarity between PCV1 and PCV2. Purified RepN of PCV2 was used as an immunogen to produce monoclonal antibodies (mAbs). 11 mAbs were screened out and established, and they were divided into two groups according to Western blot and IFA results. One group, including 1C1, bound only PCV2-RepN, while the other, including 3D10, had cross reactivity with RepN of both PCV1 and PCV2. Epitope mapping indicated that 1C1 and 3D10 recognized the linear epitopes L(39)FDYFIVG(46) and K(99)EGNLLIE(106) in PCV2-RepN, respectively. Protein sequence alignment of RepN indicated L(39)FDYFIVG(46) is conserved in all PCV2 in NCBI database, whereas the PCV1 has amino acid substitutions V(41)C(42) in this region. mAb 3D10 could recognize all PCV because all natural mutations in its epitope did not affect its binding. The information about characteristics and epitope of monoclonal antibodies may be useful for the development of diagnostic methods for PCV2 and for analyzing the function of Rep and Rep' of PCV.