Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.     

Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis

The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas'' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas'' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas'' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.Immunological methods are nowadays the elective procedure to diagnose Chagas'' disease (http://whqlibdoc.who.int/trs/WHO_TRS_905.pdf). This illness, caused by infection with the parasite Trypanosoma cruzi, has been estimated to affect between 16 and 18 million people in Latin America alone, with a further 100 million considered at risk (http://www.globalhealthprogress.org/issues/ntds_who.php). Chagasic infection is diagnosed mostly when specific antibodies (Abs) against T. cruzi antigens (Ags) are detected in a patient''s blood, by use of conventional serological methods, such as enzyme-linked immunosorbent assay (ELISA) and indirect hemmagglutination (IHA). Total homogenate of the parasite at the epimastigote stage provides Ags for serological tests, since it was proved to render the appropriate sensitivity to detect even very low Ab levels (19). However, when using this complex mixture of variable, largely undefined Ags, not only do specificity problems appear but also difficulties in standardizing the method (12, 44, 45). The current trend is to use recombinant proteins as sensitizing elements, since large amounts of them can be obtained in a highly purified form, and additionally, they can be synthesized from DNA sequences engineered to encode peptide fragments in which the specific regions responsible for cross-reactivity have been excised (2, 12, 40, 48). Thus, a number of recombinant peptides have been used for serological diagnosis, based upon their capability to improve the test performance in different aspects compared to the total parasite homogenate (TPH) (http://whqlibdoc.who.int/trs/WHO_TRS_905.pdf) (1-3, 8, 9, 15, 16, 18, 25, 26, 30, 34-38, 41-44, 50-53). Bearing in mind the sensitivity loss when using single recombinant peptides, several authors who have evaluated the performance of recombinant Ags in separate assays have suggested that a peptide mixture would reach a sensitivity equal to the sum of those of the individual Ags (3, 31, 34, 42, 50). Under this assumption, different peptide assortments have been used, improving in different ways the assay performance (3, 5, 8, 16, 31, 42, 51, 52). Alternatively, the use of multiepitope proteins expressing several unrelated antigenic determinants has also been proposed to enhance sensitivity (2, 25, 26). An argument supporting the use of chimeric molecules instead of the assortment of the epitopes expressed separately is that unique molecules facilitate the standardization procedure by lowering purification and immobilization steps and by balancing the number of epitopes on the surface of the immunoassay microplate (4, 12, 13). Recombinant DNA technology to obtain hybrid molecules has been used largely to obtain immunogens for vaccine preparation, but only a small number of authors have taken advantage of this approach in T. cruzi infection diagnosis. To the best of our knowledge, no work reporting on recombinant constructions compares systematically the performance of a multiepitope chimeric Ag with that of the mixture containing all the individual peptides that constitute the chimeric protein under study, and this work aims to cover this aspect.For this purpose, we focused first on the rational selection of the antigenic peptides from those that have already proved to have diagnostic utility. We evaluated them alone, and we synthesized new multiepitope chimeric constructions by fusing the Ags that rendered better signal-to-noise ratios. Afterward, we analyzed the Ags'' utility for T. cruzi infection diagnosis by comparing the ELISA performances displayed by the chosen synthetic peptides alone, their assortments, the new chimeric proteins with the selected peptides fused, and TPH. Finally, considering the permanent need to improve the selectivity and sensitivity displayed by T. cruzi Ags, the new, best-performing chimeric protein was preliminarily evaluated as a diagnostic tool with a 141-Chagas'' disease-positive and 164-Chagas'' disease-negative serum sample panel, and the results are discussed.     

Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.     

Complement-Fixing Antigens of Herpes Simplex Virus Types 1 and 2: Reactivity of Capsid, Envelope, and Soluble Antigens

Capsid, envelope, and nonvirion-associated soluble components of type 1 and type 2 herpes simplex virus (HSV) were obtained from infected monolayer cell cultures and used as complement fixation (CF) antigens. Capsids were prepared by treatment of cells with the nonionic detergent Nonidet-P40, envelope material by treatment of virions with ether and high pH, and soluble components were obtained from culture fluids of untreated cells. Serological studies with experimental anti-herpesvirus sera indicate that these serotypes share cross-reacting envelope, capsid, and soluble antigens with each other and with herpesvirus B but not with varicella virus. In addition, animals immunized with crude HSV preparations contain high levels of CF antibody (1:32 to 1:64) to soluble antigens, whereas sera from humans who have experienced natural infection contain low levels of antibody (相似文献   

Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.     

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3 coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5 end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.     

Diagnostic Potential and Antigenic Properties of Recombinant Tick-Borne Encephalitis Virus Subviral Particles Expressed in Mammalian Cells from Semliki Forest Virus Replicons

The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibodies against European and Far-Eastern subtype strains of TBEV, indicating proper folding of the expressed glycoproteins. The prME glycoproteins were secreted into the cell culture supernatant, forming TBEV subviral particles of 20 to 30 nm in diameter. IgM μ-capture and IgG monoclonal antibody (MAb)-capture enzyme immunoassays (EIAs) were developed based on prME Karelia-94 (Siberian subtype) particles. Altogether, 140 human serum samples were tested using these assays, and the results were compared to those obtained with a commercial IgM EIA, an in-house μ-capture IgM assay based on baculovirus-expressed antigen, a commercial IgG EIA, and a hemagglutination inhibition test. Compared to reference enzyme-linked immunosorbent assays (ELISAs), the sensitivities of the generated μ-capture IgM SFV-prME and IgG MAb-capture SFV-prME EIAs were 97.4 to 100% and 98.7%, respectively, and the specificities of the two assays were 100%. IgM and IgG immunofluorescence assays (IFAs) were created based on Vero E6 cells transfected with the recombinant plasmid carrying the TBEV Karelia-94 prME glycoproteins. The IgM IFA was 100% concordant with the μ-capture IgM bac-prME ELISA. The IgG IFA sensitivity and specificity were 98.7% and 100%, respectively, compared to those of the commercial ELISA. In conclusion, the tests developed based on SFV replicon-driven expression of TBEV glycoproteins provide safe and robust alternatives for conducting TBEV serology.     

Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.     

Production of a Recombinant Major Inner Capsid Protein for Serological Detection of Epizootic Hemorrhagic Disease Virus

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.     

Serodiagnosis of Epstein-Barr Virus Infection by Using Recombinant Viral Capsid Antigen Fragments and Autologous Gene Fusion

Using recombinant 15- to 30-kDa fragments and fusion with glutathione S-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a micro-capture (microc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The microc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and microc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.     

Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1⁻ strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1⁻ mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1⁻ CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.     

Highly Efficient Expression of Proteins Encoded by Recombinant Vaccinia Virus in Lymphocytes

Using a recombinant vaccinia virus (VV) that expresses E. coli beta galactosidase (beta-Gal) to infect lymphocytes, we show that enzymometrically or immunologically detectable beta-Gal expression is less pronounced among T cells than among B cells. VV infection caused growth inhibition of B cells, but barely affected T-cell proliferation in vitro. Moreover, the production of infectious viral particles was less pronounced in T lymphocytes. Kinetic studies revealed that after an initial dose-dependent growth inhibition, T cells continued to proliferate without the doubling time being affected by VV infection. Nonetheless, the T cells do express proteins encoded by recombinant VV, such as beta-Gal, or secrete soluble proteins such as interleukin-4, though at a lower efficiency at the per cell level than B lymphocytes. In conclusion, the physiology of T cells appears to be less perturbed by VV than that of B cells, although the virus is capable of directing expression of recombinant genes to T lymphocytes.     

Screening of Predicted Secreted Antigens from Mycobacterium bovis Identifies Potential Novel Differential Diagnostic Reagents

To date, the most promising vaccination strategies for the control of bovine tuberculosis (TB) focus on improving the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG). However, vaccination with BCG results in sensitization of animals to bovine tuberculin and compromises tests currently used for diagnosis of bovine TB infection. Thus, the development of specific diagnostic reagents capable of discriminating between infected and uninfected vaccinated animals (DIVA) is of high priority. To test the hypothesis that M. bovis-secreted proteins are likely to contain immunogenic antigens that can be used to increase the specificity of diagnostic tests, we screened 379 pools of overlapping peptides representing 119 antigens for their ability to stimulate a gamma inferferon (IFN-γ) response in vitro using whole blood from both TB reactor and BCG-vaccinated animals. Peptide pools representing antigens Rv3020c and Rv2346c induced responses in 61% and 57% of the TB reactor animals, respectively, without inducing responses in any BCG-vaccinated animal studied. Furthermore, individual peptides contained within pools recognized by BCG vaccinates were identified that were specific and induced IFN-γ responses in TB reactor animals. From these results, we constructed a cocktail of nine peptides representing multiple antigen targets that was recognized by 54% of TB reactor animals but also failed to induce responses in any BCG-vaccinated animal studied. In summary, we have identified three peptide cocktails for prioritization in larger trials to discriminate between M. bovis infection and BCG vaccination.Despite the current “test and slaughter” control policy, the incidence of bovine tuberculosis (BTB), a zoonotic infection in cattle caused by Mycobacterium bovis, has been steadily rising in Great Britain over the last 20 years (10). Thus, the British government has acknowledged the urgent need for an effective cattle vaccine. To date, the only available vaccine for bovine tuberculosis is M. bovis bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis which has shown various levels of efficacy in cattle (4, 6, 23). More recent studies have shown that by utilizing a heterologous prime-boost approach, the efficacy of BCG vaccination can be significantly improved following boosting with DNA (17), protein (24), or viral (19-21) subunit vaccines. However, vaccination with BCG results in sensitization of animals to bovine tuberculin and compromises the single intradermal comparative tuberculin test (SICCT) currently used for diagnosis of bovine TB infection. Thus, to ensure the continuation of testing and slaughter-based control strategies, it is imperative that a complementary diagnostic test capable of discriminating between infected and uninfected vaccinated animals (DIVA) is developed in parallel with vaccine initiatives.Different but complementary approaches have been used to identify antigens showing potential as a DIVA reagent. Genetic analysis has revealed regions of difference (RDs) deleted during the evolution of BCG (3, 5, 11), and several of the antigens (including ESAT-6 [Rv3875] and CFP-10 [Rv3874]) located in these RDs have been shown to possess outstanding diagnostic potential for the detection of mycobacterial infection in both cattle and humans (1, 2, 7, 13, 22). Furthermore, some of these antigens (e.g., Rv3875, Rv3874, Rv1986, Rv3872, and Rv3878) are differentially recognized by M. bovis-infected cattle compared to BCG-vaccinated animals (8, 22). Alternatively, microarray analysis that quantified the level of M. bovis gene expression revealed the “abundant invariome,” a population of gene products that were consistently expressed at high levels under a variety of different culture conditions (16). One member of the abundant invariome, Rv3615c, stimulated gamma interferon (IFN-γ) responses in M. bovis-infected cattle but not in BCG-vaccinated animals (15). In addition, Rv3615c induced responses in a proportion of animals that failed to recognize an ESAT-6-CFP-10 peptide cocktail, suggesting that target antigens identified through different approaches have the potential to complement each other in the detection of M. bovis-infected cattle.In tuberculosis research, it has long been held that active secretion of antigenic proteins by mycobacteria induces strong cellular immune responses in the host. Indeed, we have often observed that, in general, secreted proteins are among the most frequently recognized antigens in M. bovis-infected cattle. Thus, the objective of this present study was to screen a panel of potential M. bovis secreted antigens in order to identify immunogenic targets and to formulate peptide cocktails that distinguish between M. bovis-infected and BCG-vaccinated animals in blood-based screening assays.     

Novel Immunoblot Assay Using Four Recombinant Antigens for Diagnosis of Epstein-Barr Virus Primary Infection and Reactivation

A new immunoblot assay, composed of four Epstein-Barr virus (EBV)-encoded recombinant proteins (virus capsid antigen [VCA] p23, early antigen [EA] p138, EA p54, and EBNA-1 p72), was compared with an immunofluorescence assay on a total of 291 sera. The test was accurate in 94.5% of cases of primary EBV infection, while an immunoglobulin G anti-VCA p23 band with strong intensity correlated with reactivation.     

Diagnostic Potential of Toscana Virus N Protein Expressed in Escherichia coli

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.     

Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced by Spodoptera frugiperda Cells for Use as Vaccine and Diagnostic Antigens

New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda-derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.Dengue virus types 1 to 4 (DENV1-4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are globally important human pathogens (10) for which new or improved vaccines are urgently required. DENV1-4 cause dengue fever and dengue hemorrhagic fever in tropical areas and many subtropical areas. An estimated 50 million to 100 million dengue cases occur annually, with 2.5 billion people at risk of infection (11). However, there is no approved vaccine for dengue diseases, and the development of such a vaccine is urgently needed (12). JEV is the single largest cause of childhood viral encephalitis in the world, with an estimated 50,000 cases annually. Mortality rates can reach 30% among confirmed cases, and as many as one-third of survivors suffer from permanent and severe psychoneurological sequelae (13, 39). Although inactivated vaccines are used internationally for JE, they are too expensive for widespread use in most developing countries (3), and therefore, more cost-effective alternatives are needed.Neutralizing antibodies are important in host protection against dengue diseases and JE (10, 34). For JE, previously used mouse brain-derived (16, 44) and more recently used Vero cell-derived (20, 25, 35) inactivated vaccines can efficiently induce neutralizing antibody responses. However, these protein-based vaccines are produced from infectious agents, and their production therefore requires biosafety level 2 or 3 containment facilities and complex purification protocols, thus increasing the cost of the vaccine. Vaccine production without infective procedures can be achieved using genetic engineering techniques (29, 55).DENV1-4 and JEV are members of the genus Flavivirus in the family Flaviviridae (37). The envelope (E) protein is the major component of the envelopes of flavivirus virion particles and possesses most of the neutralizing epitopes (46). The other protein on the envelopes of mature virions is the membrane (M) protein, which is synthesized as the precursor membrane (prM) protein in infected cells. Cells expressing flavivirus prM and E proteins are known to secrete nucleocapsid-free subviral extracellular particles (EPs), which are similar to slowly sedimenting hemagglutinin (SHA) particles secreted from flavivirus-infected cells (47). EPs of JEV synthesized in mammalian expression systems have been evaluated for their immunogenicity and/or protective efficacy in mice (15, 24, 43). Two of these studies (24, 43) demonstrated that the EPs induced neutralizing antibodies at levels comparable to those induced by an inactivated JE vaccine. In our laboratory, mammalian cell lines continuously expressing EPs of dengue type 2 virus (DENV2) (26) or JEV (27) have been generated and designated D cells and F cells, respectively. The EPs contained an E protein that was antigenically and biochemically equivalent to the authentic E protein, and the EPs were immunogenic and protective in mice. However, the yields of viral antigens produced from D and F cells were low and would not meet the requirements for commercial vaccine production. Increasing the levels of viral antigen production from transfected cells would reduce the cost of vaccine preparation.Recently, insect cell expression systems have been increasingly used in various fields of medical sciences (1, 6, 17, 53). In general, insect cells are easier to cultivate than mammalian cells, because they often do not require serum supplementation in the culture medium or incubation under CO₂. In addition, insect cells can be adapted to suspension culture, allowing cultures to be simply scaled up. Furthermore, various techniques have been developed for high-density culture of insect cells; for example, in one study, the immobilization of insect cells within biomass support particles achieved a density of approximately 3 × 10⁷ cells/cm³ (50). Thus, the insect cell expression system can be a simple and inexpensive strategy for vaccine antigen production.In addition to their use as vaccine antigens, EPs derived from mammalian cells could be used as serodiagnostic antigens (27, 32). The production of serodiagnostic antigens may also encounter problems when the antigens are sourced directly from infectious agents. Currently, numerous commercial assays utilizing several different formats, such as the immunochromatography test and the IgM capture enzyme-linked immunosorbent assay (ELISA), are available for the diagnosis of DENV and JEV infections (4, 49). These commercial tests use viral antigens derived from transfected or infected cultured cells. Thus, the application of insect cell-derived EPs as diagnostic antigens would be an attractive alternative.In this study, we produced EPs of DENV2 and JEV in a transient expression system using the Sf9 cell line, which was derived from the pupal ovarian tissue of the fall armyworm, Spodoptera frugiperda. These proteins were evaluated for vaccine and diagnostic antigens, mainly by direct comparison with mammalian-cell-derived EPs. The EPs produced from Sf9 cells were immunogenic in mice and useful as antigens for ELISA. In addition, Sf9 cells produced larger amounts of antigen than CHO cells, suggesting the potential applicability of insect cells for the production of DENV2 and JEV antigens for vaccines and serodiagnostic tests.