Major Antigenic Region Research Articles

Human antibody signatures towards the Chlamydia trachomatis major outer membrane protein after natural infection and vaccination

Chlamydia trachomatis (CT) Major Outer Membrane Protein (MOMP) holds a neutralising epitope in the Variable Domain 4 (VD4), and this region's immune dominance during infection is well known. This study aimed to assess the antibody response induced after infection and compare it for specificity and functionality to the response following vaccination with the vaccine CTH522, which contains VD4's from serovars D, E, F, and G. We assessed the antibody epitopes in MOMP by a high density peptide array. Furthermore, the role of the VD4 epitope in neutralisation was explored by competitive inhibition experiments with a fusion protein holding the neutralising VD4 linear epitope. This was done in two independent groups: 1) MOMP seropositive individuals infected with CT (n=10, from case-control study) and 2) CTH522/CAF®01-vaccinated females (n=14) from the CHLM-01 clinical trial. We identified the major antigenic regions in MOMP as VD4 and the conserved region just before VD3 in individuals infected with CT. The same regions, with the addition of VD1, were identified in vaccine recipients. Overall, the VD4 peptide responses were uniform in vaccinated individuals and led to inhibition of infection invitro in all tested samples, whereas the VD4 responses were more heterogenous in individuals infected with CT, and only 2 out of 10 samples had VD4-mediated neutralising antibody responses. These data provide insights into the role of antibodies against MOMP VD4 induced after infection and vaccination, and show that their functionality differs. The induction of functional VD4-specific antibodies in vaccine recipients mimics previous results from animal models. This work was supported by the European Commission through contract FP7-HEALTH-2011.1.4-4-280873 (ADITEC) and Fonden til Lægevidenskabens Fremme.

Complete genome characterization of foot-and-mouth disease virus My-466 belonging to the novel lineage O/ME-SA/SA-2018

Foot-and-mouth disease virus (FMDV), the causative agent of the foot-and-mouth disease of cattle population possesses a rapid evolutionary rate. In Bangladesh, the first circulation of the O/ME-SA/SA-2018 lineage as a novel sublineage, MYMBD21 was reported from our laboratory. The first whole genome sequence of an isolate, BAN/MY/My-466/2021 (shortly named My-466) of the SA-2018 lineage is characterized and represented in this study. The genome is 8216 nucleotides long with 6996 nucleotides open reading frame flanked by 5ꞌ UTR (1–1100) and 3ꞌ UTR (8097–8216). VP1 was found to be highly variable among the structural proteins with crucial mutations in the major antigenic region, G-H loop. Structural variations of the VP1 against both field and proposed local vaccine strains were evidenced by the G-H loop displacement in a superimposed 3D model. The complete genome information of the isolate would be valuable for undertaking proper control measures needed to limit the spread of the newly emerged FMDV strain.

Identification and Characterization of a Novel Epitope of ASFV-Encoded dUTPase by Monoclonal Antibodies.

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100–120 aa, 120–140 aa, and 140–165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149–RGEGRFGSTG–158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.

Identification of novel B-cell epitopes on the capsid protein of type 1 porcine astrovirus, using monoclonal antibodies

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes (120-GNNTFG-125, 485-RISDPTWFSA-494) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120-GNNTFG-125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods.

Interleukin-12 receptor β2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation

Interleukin-12 receptor β2 (IL-12Rβ2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rβ2 in teleost fish. In this work, we have cloned and characterized IL-12Rβ2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rβ2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rβ2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rβ2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rβ2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rβ2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rβ2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rβ2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rβ2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rβ2 is involved in the regulation of intestinal inflammation.

Microtubule-associated protein 1B: Novel paraneoplastic biomarker.

To report the identification of microtubule-associated protein (MAP) 1B as the antigen of the previously described Purkinje cell cytoplasmic antibody type 2 (PCA-2) antibody, its frequency, and clinical, oncological, and serological associations. Archival serum or cerebrospinal fluid (CSF) specimens were available from 96 of 118 consecutive PCA-2-IgG-seropositive patients identified during 1993-2016. The autoantigen, defined in mouse brain lysate by Western blot and mass spectrometry, was confirmed by dual immunohistochemical staining using commercial antibodies. The major antigenic region was defined by Western blot using recombinant protein fragments. IgG in 95 of 96 patients' serum or CSF (but in none of 98 healthy or disease control subjects' serum specimens) bound to recombinant MAP1B. A minority (17.5%) of patients' IgG also bound to MAP1A. PCA-2 was often accompanied by additional neural autoantibody markers of small-cell carcinoma, including collapsin response-mediated protein 5 (CRMP5) IgG (26%) or antineuronal nuclear antibody type 1 (ANNA-1) IgG (also known as anti-Hu; 13%). Neurological manifestations in 95 patients were (in decreasing frequency): peripheral neuropathy, 53%; cerebellar ataxia, dysmetria, or dysarthria, 38%; and encephalopathy, 27%. Cancer (majority small-cell lung carcinoma [SCLC]) was detected in 66 of 84 evaluated patients (79%). The MAP1B (PCA-2) autoantibody detection rate, among approximately 70,000 patients undergoing service neural autoantibody evaluation in 2015, was 0.024%, equaling amphiphysin IgG (0.026%) and more common than ANNA-2 (also known as anti-Ri; 0.016%) and PCA-Tr (also known as delta/notch-like epidermal growth factor-related receptor [DNER]; 0.006%). MAP1B, the PCA-2 autoantigen, represents a novel target in paraneoplastic neurological disorders and has high predictive value for SCLC. Its relatively high prevalence, compared with other recognized paraneoplastic neural autoantibodies, justifies its testing in comprehensive paraneoplastic neural autoantibody evaluation. Ann Neurol 2017;81:266-277.

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA

As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.

Molecular identification of a G2 rotavirus that provided a G1P[4] mono-reassortant with a DS-1-like genotype constellation.

The segmented nature of the rotavirus genome provides an opportunity for the virus to reassort upon co-infection of more than one rotavirus strain. Previously, two G1P[4] strains isolated from children with diarrhoea, AU64, and AU67, were shown by RNA-RNA hybridization to be a VP7 mono-reassortant possessing a DS-1 genogroup background. However, the origin of the parental G2 strain was not sought for at that time. The aim of this study, therefore, was to identify the G2 strain that provided AU64/AU67 with the DS-1-like genotype constellation. By carefully comparing the genomic RNA migration patterns on polyacrylamide gels of strains circulating at the same and nearby geographic locations around the time of the mono-reassortant detection (1989), a G2 strain designated 88H449 was shown to possess 10 genome segments co-migrating with those of AU64. Sequencing of all genome segments of AU64 and 88H449 showed that those two strains were 99.6-100% identical at the nucleotide level in all except the VP7 gene, indicating that the parental G2 strain that provided AU64 with the 10 genome segments was a sibling of 88H449. Sequencing of the VP7 genes of 36 G1P[8] strains circulating locally between 1981 and 1990 revealed the presence of strains bearing diverse VP7 sequences among which the closest nucleotide identity to AU64 was 98.6% and the closest amino acid identity in the major antigenic regions was 100%. This unusual mono-reassortant was concluded to be the result of a contemporary reassosrtment event occurring between locally co-circulating strains.

Cloning and expression of hexon protein of human respiratory adenovirus of three serotypes and analysis of antigenicity of the recombinant proteins

Objective To clone and express the hexon protein of three prevalent human adenovi -rus strains causing respiratory disease and analyze the antigenic characteristics of the recombinant proteins . Methods The full length genes encoding hexon protein of human adenovirus serotype 3（HAdV3）, serotype 4（HAdV4） and serotype 7（HAdV7） were cloned by PCR and sequenced , respectively.The alignment anal-ysis was performed by using hexon gene sequences from GenBank .The major antigenic regions of hexon pro-tein of the three serotypes were expressed in E.coli and purified.The antigenicity, immunogenicity and cross reactivity of the recombinant proteins were determined by ELISA and Western blot assay .Results The full length gene sequence encoding hexon protein of human adenovirus serotype 4 was firstly reported in China , which showed more than 99%homology in both nucleotide and amino acid sequences with the human adeno-virus type 4 NHRC3 strain.The partial hexon protein sequence of HAdV 3, HAdV4 and HAdV7 containing all of the 7 hyperviriable regions （ HVRs） were expressed in E.coli, respectively .The purified recombinant proteins could be recognized by antiserum of the three serotypes of adenovirus .The antiserum samples against the three recombinant proteins could cross-react with particles of the three serotypes of adenovirus . The possible type-and species-specific epitopes were predicted .Conclusion The major antigenic regions of hexon protein of the three serotypes were successfully expressed .The purified recombinant proteins contai-ning both intertypes and type-specific epitopes showed a strong immunogenicity . Key words: Human respiratory adenovirus; Hexon; Recombinant expression; Antigenicity; Epitope

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Investigating the impact of hepatitis B virus surface gene polymorphism on antigenicity using ex vivo phenotyping

The hepatitis B virus (HBV) surface antigen (HBsAg) is a complex protein, and understanding accurately the impact of amino acid changes on the antigenicity of the immunodominant a determinant must take this complexity into consideration. Epitope mapping with four mAbs was used to phenotype HBsAg directly from patients' sera to investigate the effect of mutations in their native genetic backbone. The expected mAb reactivity was established initially for samples harbouring 'wild-type' HBsAg sequences across genotypes A-E. The alteration of HBsAg antigenicity, defined by mAb epitope loss, was demonstrated in a number of samples with sequence-inferred amino acid changes. Individual mutations within the mapped epitopes to which the mAbs were directed usually affected their binding. However, the loss of more than one epitope was observed as the number of mutations within a sequence increased. Conversely, not all mutations occurring in the a determinant altered the HBsAg conformation. The genotype backbone, the specific amino acid substitution and amino acid changes occurring outside the major antigenic region appeared to be important in determining expression of the predicted epitope loss. These data clearly demonstrate that sequence-based methods alone may not accurately define HBsAg phenotype. This phenotyping methodology allows for the rapid and accurate identification of antigenically altered viruses and will greatly enhance current HBV surveillance, research and diagnostic activities. The data generated can be used to inform on public health issues relating to prevalence, transmission and impact of HBsAg mutants in HBV-infected populations.

Regions of recognition by blocking antibodies on the light chain of botulinum neurotoxin A: Antigenic structure of the entire toxin

The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1–453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed.

Antigenic structure of the hepatitis C virus envelope 2 protein

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.

Sequential autoantigenic determinants of the small nuclear ribonucleoprotein Sm D shared by human lupus autoantibodies and MRL lpr/lpr antibodies.

Autoantibodies directed against the Sm proteins of the spliceosome complex are found in approximately 25% of systemic lupus erythematosus (SLE) patients sera. To determine which regions of the Sm D polypeptide are involved in the lupus autoimmune response, binding to overlapping octapeptides of Sm D has been evaluated with sera from nine Sm D-positive patients, six patients with other autoimmune serology, and five normal human sera. Lupus patient sera which are Sm precipitin-positive bind various combinations of five regions of the peptide. The major antigenic region, Epitope 5 (REAVA(GR)10GGPRR), is bound by eight of nine Sm precipitin-positive sera tested. This region of Sm D shows significant sequence homology with Epstein-Barr nuclear antigen-1. To determine the fine specificity of the murine Sm response, four unique Sm D MoAbs derived from MRL lpr/lpr mice and three adult anti-Sm-positive MRL lpr/lpr mouse sera have been analysed. Two of these monoclonals, KSm 4 and Y12, as well as the MRL lpr/lpr sera tested, show binding with Epitope 5. Another of these monoclonals, KSm 2, binds octapeptides 84-91, DVEPKVKSKKREAVAG, which corresponds to Epitope 4 of this study. Antibodies from SLE patients with autoimmune serology other than anti-Sm bind the carboxyl glycine-arginine repeat (GR)10 peptides of Sm D. However, none of the antibodies tested from patients who do not have lupus and who have different autoimmune serology binds any of the Sm D octapeptides. Normal controls did not significantly bind any of the Sm D octapeptides. These results describe two major regions of shared antigenicity of Sm D between sera from SLE patients and MRL lpr/lpr mice, thereby establishing a basis for the cross-species similarity of autoimmunity to the Sm autoantigen in SLE.

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region

Numerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed. Based on 45 sequences, representing HCV genotypes 1 - 6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182 - 2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1 - 6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis. The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182 - 2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182 - 2211 (R1), 2272 - 2301 (R7) and 2302 - 2331 (R9): the highly variable 2212 - 2241 (R3) and 2257 - 2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis. The major antigenic regions in HCV NS5A region were at 2212 - 2241 (R3), 2272 - 2301 (R7) and 2302 - 2331 (R9). Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

Breakthrough of hepatitis B virus escape mutants after vaccination and virus reactivation

Escape mutants of hepatitis B virus (HBV) carry mutations in the major antigenic region of the hepatitis B surface antigen (HBsAg). They are able to grow in the presence of antibodies against HBsAg (anti-HBs) and may escape detection by immuno assays for HBsAg. Antibodies against HBV core antigen (anti-HBc) are considered to be a universal marker for active or resolved HBV infections but they may appear very late. Highly sensitive detection of HBV DNA is the most universal reliable marker of infectivity, but in the blood donation setting even the most sensitive assay for HBV DNA may not detect all infectious donations. This brief report describes three cases of HBV infections caused by HBV escape mutants. The implications of the findings for the prevention of HBV transmission through donor blood are discussed.

Recombinant newcastle disease virus capsids displaying enterovirus 71 VP1 fragment induce a strong immune response in rabbits

The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.

Unravelling Selection Shifts among Foot-and-Mouth Disease virus (FMDV) Serotypes

FMDV virus has been increasingly recognised as the most economically severe animal virus with a remarkable degree of antigenic diversity. Using an integrative evolutionary and computational approach we have compelling evidence for heterogeneity in the selection forces shaping the evolution of the seven different FMDV serotypes. Our results show that positive Darwinian selection has governed the evolution of the major antigenic regions of serotypes A, Asia1, O, SAT1 and SAT2, but not C or SAT3. Co-evolution between sites from antigenic regions under positive selection pinpoints their functional communication to generate immune-escape mutants while maintaining their ability to recognise the host-cell receptors. Neural network and functional divergence analyses strongly point to selection shifts between the different serotypes. Our results suggest that, unlike African FMDV serotypes, serotypes with wide geographical distribution have accumulated compensatory mutations as a strategy to ameliorate the effect of slightly deleterious mutations fixed by genetic drift. This strategy may have provided the virus by a flexibility to generate immune-escape mutants and yet recognise host-cell receptors. African serotypes presented no evidence for compensatory mutations. Our results support heterogeneous selective constraints affecting the different serotypes. This points to the possible accelerated rates of evolution diverging serotypes sharing geographical locations as to ameliorate the competition for the host.

Fulminant B Hepatitis in a Hepatitis B Surface Antigen-Negative Patient after Rituximab Therapy for B-CLL.

Concerns about safety of rituximab in hepatitis B virus (HBV) carriers have risen at the light of reported cases of HBV reactivation following the administration of this agent. We recently observed a patient affected by B-cell Chronic Lymphocytic Leukemia (B-CLL) who developed this complication without any serological evidence of HBsAg expression.Case report: the patient, a 51 old year's male, was diagnosed as having B-CLL in November 1998 and followed until his death due to acute liver failure due to HBV reactivation of a mutant HBV strain in September 2004 (Table). At diagnosis of B-CLL no risk factors regarding liver diseases or viral infections were reported; serum HBsAg and HBeAg, antibodies against the B-core antigen (HBcAb), HBe (HBeAB), HBsAg (HBsAb), HCV, hepatitis A and Delta virus, Cytomegalovirus, and Epstein Barr virus were negative while HBcAb alone were positive. He presented stable disease until November 2001, when fludarabine was started because of progressive disease. A good partial response (GPR) was achieved so that he received four weekly standard doses (375 mg/m2) of rituximab as consolidation therapy (July 2002). A complete remission (CR) was obtained and he was then closely followed-up until about two years later (February 2004) when, for a disease recurrence, rituximab was given again. At that time all the above hepatitis markers remained unmodified. After four weekly standard doses of rituximab a GPR was recorded, and then six monthly courses (150 mg/m2) of the same agent were administered as maintenance therapy. In September 2004, being the patient in CR, he was admitted with acute hepatitis. HBV DNA strains (200,000 copies/mL) and HbcAb IgM were detected, whereas HbsAg-negative status persisted. The viral form was characterized as having D genotype and ayw3 serotype respectively.Sequence analysis of polymerase chain reaction (PCR) products amplified from the S region revealed 5 remarkable mutations in the major antigenic regions (C124Y, A128V, G130D, P135R and G145R). These mutations were associated with the lack of HbsAg production and were compatible with an escape of a rare HBV strain from the patient's own HbsAb. Despite treatment with lamivudine, the patient died of fulminant hepatic failure.Conclusion: our experience indicates that patients receiving rituximab who are negative for HBsAg but positive for anti-HBc are still at risk for reactivation of latent HBV and should be considered for HBV DNA testing and prophylaxis with lamivudine.Patient clinical featuresClinical featuresDiagnosis (November 1998)Disease Progression (November 2001)Disease Recurrence (February 2004)B Hepatitis onset (September 2004)Rai Clinical StageIIIII0CD38NegativeNegativeNegativeNegativeZap 70PositivePositivePositiveNegativeCD4/CD80.950.900.850.80Beta 2 microglobulin (mcg/L)1503182518441325Serum Ig level (gr/L)0,9800,7900,6600,620HBsAgNegativeNegativeNegativeNegativeHBsAbNegativeNegativeNegativeNegativeHBcAbPositivePositivePositivePositiveHBeAgNegatriveNegativeNegativePositiveHBeAbPositivePositivePositivePositiveHBV DNA*NANANA200.000/μL*D genotype, ayw3 serotype; C124Y, A128V, G130D, P135R and G145R mutations in the S region. NA:not available.