Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Comprehensive Assessment of the Antigenic Impact of Human Papillomavirus Lineage Variation on Recognition by Neutralizing Monoclonal Antibodies Raised against Lineage A Major Capsid Proteins of Vaccine-Related Genotypes.

Human papillomavirus (HPV) is the causative agent of cervical and other epithelial cancers. Naturally occurring variants of HPV have been classified into lineages and sublineages based on their whole-genome sequences, but little is known about the impact of this diversity on the structure and function of viral gene products. The HPV capsid is an icosahedral lattice comprising 72 pentamers of the major capsid protein (L1) and the associated minor capsid protein (L2). We investigated the potential impact of this genome variation on the capsid antigenicity of lineage and sublineage variants of seven vaccine-relevant, oncogenic HPV genotypes by using a large panel of monoclonal antibodies (MAbs) raised against the L1 proteins of lineage A antigens. Each genotype had at least one variant that displayed a ≥4-fold reduced neutralizing antibody sensitivity against at least one MAb, demonstrating that naturally occurring variation can affect one or more functional antigenic determinants on the HPV capsid. For HPV16, HPV18, HPV31, and HPV45, the overall impact was of a low magnitude. For HPV33 (sublineages A2 and A3 and lineages B and C), HPV52 (lineage D), and HPV58 (lineage C), however, variant residues in the indicated lineages and sublineages reduced their sensitivity to neutralization by all MAbs by up to 1,000-fold, suggesting the presence of key antigenic determinants on the surface of these capsids. These determinants were resolved further by site-directed mutagenesis. These data improve our understanding of the impact of naturally occurring variation on the antigenicity of the HPV capsid of vaccine-relevant oncogenic HPV genotypes.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and some other epithelial cancers. HPV vaccines generate functional (neutralizing) antibodies that target the virus particles (or capsids) of the most common HPV cancer-causing genotypes. Each genotype comprises variant forms that have arisen over millennia and which include changes within the capsid proteins. In this study, we explored the potential for these naturally occurring variant capsids to impact recognition by neutralizing monoclonal antibodies. All genotypes included at least one variant form that exhibited reduced recognition by at least one antibody, with some genotypes affected more than others. These data highlight the impact of naturally occurring variation on the structure of the HPV capsid proteins of vaccine-relevant oncogenic HPV genotypes.

Multilaboratory Assessment of Epstein-Barr Virus Serologic Assays: the Case for Standardization.

IgA antibodies targeting Epstein-Barr virus (EBV) have been proposed for screening for nasopharyngeal carcinoma (NPC). However, methods differ, and the antigens used in these assays differ considerably between laboratories. To enable formal comparisons across a range of established EBV serology assays, we created a panel of 66 pooled serum samples and 66 pooled plasma samples generated from individuals with a broad range of IgA antibody levels. Aliquots from these panels were distributed to six laboratories and were tested by 26 assays measuring antibodies against VCA, EBNA1, EA-EBNA1, Zta, or EAd antigens. We estimated the correlation between assay pairs using Spearman coefficients (continuous measures) and percentages of agreement (positive versus negative, using predefined positivity cutoffs by each assay developer/manufacturer). While strong correlations were observed between some assays, considerable differences were also noted, even for assays that targeted the same protein. For VCA-IgA assays in serum, two distinct clusters were identified, with a median Spearman coefficient of 0.41 (range, 0.20 to 0.66) across these two clusters. EBNA1-IgA assays in serum grouped into a single cluster with a median Spearman coefficient of 0.79 (range, 0.71 to 0.89). Percentages of agreement differed broadly for both VCA-IgA (12% to 98%) and EBNA1-IgA (29% to 95%) assays in serum. Moderate-to-strong correlations were observed across assays in serum that targeted other proteins (correlations ranged from 0.44 to 0.76). Similar results were noted for plasma. We conclude that standardization of EBV serology assays is needed to allow for comparability of results obtained in different translational research studies across laboratories and populations.

[Assessment of immunogenic activity of the cloned human rotavirus A WA strain.]

INTRODUCTION: Rotovirus infection (RVI) caused by the dsRNA-containing virus from genus Rotavirus, Reoviridae family, belonging to group A (RVA), is the cause of severe diarrhea in human and other mammalian species. Vaccination is the most effective way to reduce the incidence of RVI. At present, the effectiveness of using gnotobiotic piglets as a universal model for reproducing human rotavirus infection and assessing the quality of RVI vaccine preparations has been experimentally proven. OBJECTIVES: Evaluation of immunogenic activity of the cloned RVA Wa strain in the new-born Vietnamese potbellied piglets trial. MATERIAL AND METHODS: Development of viral preparations of the cloned human Wa strain PBA, development of human RVA rVP6, ELISA, polymerase chain reaction with reverse transcription, immunization and experimental infection of newborn piglets. RESULTS: The article presents the results of the experiment on double immunization of newborn piglets with native virus preparations with the infection activity 5.5 lg TCID50/ml, 3 cm3 per dose, HRV with adjuvant 500 µg per dose and mock preparation (control group) followed with experimental inoculation of all animals with virulent virus strain Wa G1P[8] human RVA with infectious activity of 5.5 lg TCID50/ml in 5 cm3 dose. Development of clinical signs of disease and animal death were observed only in control group. RT-PCR system to detect RVA RNA in rectal swabs, samples of small intestine and peripheral lymph nodes was developed. ELISA based on obtained human RVA rVP6 was developed and results on RVA-specific IgG-antibodies in serum samples of experimental piglets are presented. CONCLUSION: In the course of the research, a high immunogenic activity of the native and purified virus of the cloned Wa RVA strain Wa was established and the possibility of its use as the main component of the RVI vaccine was confirmed. The possibility of using conventional newborn pigs instead of gnotobiotic piglets as an experimental model was demonstrated.

Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies.

Spherical particles (SPs) generated by thermally denatured tobacco mosaic virus (TMV) coat protein can act as an adjuvant, as they are able to enhance the magnitude and longevity of immune responses to different antigens. Here, the toxicity of TMV SPs was assessed prior to it being offered as a universal safe adjuvant for the development of vaccine candidates. The evaluation included nonclinical studies of a local tolerance following the single administration of TMV SPs, and of the local and systemic effects following repeated administrations of TMV SPs. These were conducted in mice, rats and rabbits. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Also, reproductive and development toxicity were studied. No significant signs of toxicity were detected following single or repeated administrations of the adjuvant (TMV SPs). The absence of toxicological effects following the injection of TMV SPs is promising for the further development of recombinant vaccine candidates with TMV SPs as an adjuvant.

Characterization of an Escherichia coli-derived human papillomavirus type 16 and 18 bivalent vaccine.

Human papillomavirus (HPV) types 16 and 18 account for approximately 70% of cervical cancer worldwide. Neutralizing HPV prophylactic vaccines offer significant benefit, as they block HPV infection and prevent subsequent disease. However, the three licensed HPV vaccines that cover these two genotypes were produced in eukaryotic cells, which is expensive, particularly for low-income countries where HPV is highest. Here, we report a new HPV16 and -18 bivalent candidate vaccine produced from Escherichia coli. We used two strategies of N-terminal truncation of HPV L1 proteins and soluble non-fusion expression to generate HPV16 and HPV18 L1-only virus-like particles (VLPs) in a scalable process. Through comprehensive characterization of the bivalent candidate vaccine, we confirm lot consistency in a pilot scale-up of 30L, 100L and 500L. Using cryo-EM 3D reconstruction, we found that HPV16 and -18VLPs present in a T=7 icosahedral arrangement, similar in shape and size to that of the native virions. This HPV16/18 bivalent vaccine shares comparable immunogenicity with the licensed vaccines. Overall, we show that the production of a HPV16/18 bivalent vaccine from an E. coli expression system is robust and scalable, with potentially good accessibility worldwide as a population-based immunization strategy.

Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus-like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative.

Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.

Functional assessment and structural basis of antibody binding to human papillomavirus capsid.

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.

Performance of the architect EBV antibody panel for determination of Epstein-Barr virus infection stage in immunocompetent adolescents and young adults with clinical suspicion of infectious mononucleosis.

The Architect EBV antibody panel is a new chemiluminescence immunoassay system used to determine the stage of Epstein-Barr virus (EBV) infection based on the detection of IgM and IgG antibodies to viral capsid antigen (VCA) and IgG antibodies against Epstein-Barr nuclear antigen 1 (EBNA-1). We evaluated its diagnostic accuracy in immunocompetent adolescents and young adults with clinical suspicion of infectious mononucleosis (IM) using the RecomLine EBV IgM and IgG immunoblots as the reference standard. In addition, the use of the antibody panel in a sequential testing algorithm based on initial EBNA-1 IgG analysis was assessed for cost-effectiveness. Finally, we investigated the degree of cross-reactivity of the VCA IgM marker during other primary viral infections that may present with an EBV IM-like picture. High sensitivity (98.3% [95% confidence interval {CI}, 90.7 to 99.7%]) and specificity (94.2% [95% CI, 87.9 to 97.8%]) were found after testing 162 precharacterized archived serum samples. There was perfect agreement between the use of the antibody panel in sequential and parallel testing algorithms, but substantial cost savings (23%) were obtained with the sequential strategy. A high rate of reactive VCA IgM results was found in primary cytomegalovirus (CMV) infections (60.7%). In summary, the Architect EBV antibody panel performs satisfactorily in the investigation of EBV IM in immunocompetent adolescents and young adults, and the application of an EBNA-1 IgG-based sequential testing algorithm is cost-effective in this diagnostic setting. Concomitant testing for CMV is strongly recommended to aid in the interpretation of EBV serological patterns.

Prophylactic papillomavirus vaccines.

Human papillomaviruses (HPV) are the causative agents of cervical cancer, the third most common cancer in women. The development of prophylactic HPV vaccines Gardasil® and Cervarix® targeting the major oncogenic HPV types is now the frontline of cervical cancer prevention. Both vaccines have been proven to be highly effective and safe although there are still open questions about their target population, cross-protection, and long-term efficacy. The main limitation for a worldwide implementation of Gardasil® and Cervarix® is their high cost. To develop more affordable vaccines research groups are concentrated in new formulations with different antigens including capsomeres, the minor capsid protein L2 and DNA. In this article we describe the vaccines' impact on HPV-associated disease, the main open questions about the marketed vaccines, and current efforts for the development of second-generation vaccines.

Immunogenic assessment of plant-produced human papillomavirus type 16 L1/L2 chimaeras.

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus-like particle (VLP) type-restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second-generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV-16 L1-based chimaeras, containing cross-protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV-16 L2 amino acid 108-120, 56-81 or 17-36 substituted into the C-terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium-mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108-120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti-L1 and anti-L2 responses in mice, and antisera neutralized homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.

Assessment of a passive immunity mouse model to quantitatively analyze the impact of neutralizing antibodies on adeno-associated virus-mediated gene transfer.

Adeno-associated viruses (AAVs) are common infective agents of primates. As such, healthy primates carry a large pool of AAV-specific neutralizing antibodies (NAbs), which inhibit AAV-mediated gene transfer therapeutic strategies. Thus, a clinical method to screen patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, will facilitate individualized treatment design and enhance therapeutic efficacy. In this study, we evaluated the efficacy and sensitivity of a passive immunity mouse model to quantitatively assess anti-AAV8 NAb titers, as compared to an in vitro immunoassay. The passive transfer model was established in C57BL/6 mice by tail vein injection of pre-defined sera from 23 male rhesus monkeys. The mice were then administered low dose (3e10 GC/mouse) self-complementary (sc) AAV8. The in vitro NAb assay indicated that 69.57% of the rhesus donors had pre-existing anti-AAV8 NAb. The in vivo NAb assay, however, was better able to detect low NAb titer (≤ 1:5), which can mediate neutralization in vivo. Indeed, 17 rhesus donors (74.0%) had pre-existing anti-AAV8 neutralization by in vivo NAb assay. Our findings indicated that the in vivo NAb assay is superior to the in vitro assay for detecting low NAb titers.

An integrated approach to extreme thermostabilization and affinity maturation of an antibody.

Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.

Assessment of the cross-protective capability of recombinant capsid proteins derived from pig, rat, and avian hepatitis E viruses (HEV) against challenge with a genotype 3 HEV in pigs.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is primarily transmitted via the fecal-oral route through contaminated water supplies, although many sporadic cases of hepatitis E are transmitted zoonotically via direct contact with infected animals or consumption of contaminated animal meats. Genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species, whereas genotypes 1 and 2 HEV are restricted to humans. There exists a single serotype of HEV, although the cross-protective ability among the animal HEV strains is unknown. Thus, in this study we expressed and characterized N-terminal truncated ORF2 capsid antigens derived from swine, rat, and avian HEV strains and evaluated their cross-protective ability in a pig challenge model. Thirty, specific-pathogen-free, pigs were divided into 5 groups of 6 pigs each, and each group of pigs were vaccinated with 200 µg of swine HEV, rat HEV, or avian HEV ORF2 antigen or PBS buffer (2 groups) as positive and negative control groups. After a booster dose immunization at 2 weeks post-vaccination, the vaccinated animals all seroconverted to IgG anti-HEV. At 4 weeks post-vaccination, the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV.

An efficacy analysis for nasopharyngeal carcinoma screening of different screening intervals.

OBJECTIVE: To evaluate the impact of different screening intervals on screening for nasopharyngeal carcinoma (NPC). METHODS: A Markov model was constructed, based on the natural history of NPC. The 5-year mortality rate of NPC was the major measurement to evaluate the efficacies of 16 screening strategies. Parameters for the model were derived from published literature. RESULTS: Screening reduced the 5-year mortality rate for NPC by 20.4 - 43.3%, compared with the equivalent rate without screening. The 5 year mortality rate and the NPC pick-up rate with strategy A1 (annual screening) were 23.6% and 83.9%, respectively. Compared with strategy A1, strategy B1 (annual screening for seropositive subjects; biennial screening for seronegative subjects) had a similar 5-year mortality rate (24.0%) and a slightly smaller NPC pick-up rate (81.7%), but led to a 39.3% reduction in total screenings. Compared with all other strategies excluding strategy A1, strategy B1 achieved the lowest 5-year mortality rate and the largest NPC pick-up rate. CONCLUSIONS: Strategy B1 had the highest efficacy for NPC screening.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic rainbow papaya.

The virus-resistant, transgenic commercial papaya Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland United States and Canada since their release to planters in Hawaii in 1998. These papaya are derived from transgenic papaya line 55-1 and carry the coat protein (CP) gene of papaya ringspot virus (PRSV). The PRSV CP was evaluated for potential allergenicity, an important component in assessing the safety of food derived from transgenic plants. The transgene PRSV CP sequence of Rainbow papaya did not exhibit greater than 35% amino acid sequence homology to known allergens, nor did it have a stretch of eight amino acids found in known allergens which are known common bioinformatic methods used for assessing similarity to allergen proteins. PRSV CP was also tested for stability in simulated gastric fluid and simulated intestinal fluid and under various heat treatments. The results showed that PRSV CP was degraded under conditions for which allergenic proteins relative to nonallergens are purported to be stable. The potential human intake of transgene-derived PRSV CP was assessed by measuring CP levels in Rainbow and SunUp along with estimating the fruit consumption rates and was compared to potential intake estimates of PRSV CP from naturally infected nontransgenic papaya. Following accepted allergenicity assessment criteria, our results show that the transgene-derived PRSV CP does not pose a risk of food allergy.

Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines.

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T0, T1 and T2 generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.

Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein-Barr virus serologic status.

We have compared a multiplexed bead-based assay (BBA) with an enzyme immunoassay (EIA) and immunofluorescence assay (IFA) for the assessment of the Epstein-Barr virus (EBV) serostatus. Three hundred and ninety-three sera, classified according to IFA results as seronegative (n=100), acute infection (n=100), past infection (n=100) and indeterminate (n=93), were tested by BBA and EIA. Overall, the three methods gave similar results with a relatively high (75.2%) concordance with the consensus interpretation of the serostatus. The most significant discordances were: (i) 58 samples had uninterpretable results for BBA, in majority due to the detection of non-antigen specific antibody binding by control beads. (ii) almost half the samples positive for anti-Epstein-Barr nuclear antigen (EBNA) IgG by BBA or EIA were negative by IFA. Among the latter, only a minority had a history of immunocompromise or treatment, or detectable anti-early antigen antibody. This discrepancy probably reflects a poor sensitivity of IFA for anti-EBNA IgG detection. EIA and BBA had a similar performance and had substantial practical advantages over IFA with respect to testing for EBV serostatus.

Evaluation of Epstein-Barr virus antibodies, anti-VCA avidity by immunofluorescence and immunoblot assays for assessment of Epstein-Barr virus immunologic state.

Three hundred twenty two serum samples were evaluated by indirect immunofluorescence assay (IFA) and immunoblotting for the assessment of the immunologic status of Epstein-Barr virus (EBV). Serological profiles of 322 serum samples were classified by the IgM and IgG antibodies by the immunofluorescence assay as seronegative, acute infection, reactivation and past infection. Higher rates of seropositivity were determined in serum samples belonging to old age groups. Routine tests used for EBV diagnosis were efficient.