Sixteen capillary electrophoresis applications for viral vaccine analysis.

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.

Targeting Viral Surface Proteins through Structure-Based Design.

The emergence of novel viral infections of zoonotic origin and mutations of existing human pathogenic viruses represent a serious concern for public health. It warrants the establishment of better interventions and protective therapies to combat the virus and prevent its spread. Surface glycoproteins catalyzing the fusion of viral particles and host cells have proven to be an excellent target for antivirals as well as vaccines. This review focuses on recent advances for computational structure-based design of antivirals and vaccines targeting viral fusion machinery to control seasonal and emerging respiratory viruses.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

Rational design of multimeric based subunit vaccine against Mycoplasma pneumonia: Subtractive proteomics with immunoinformatics framework.

Mycoplasma pneumoniae is the prevalent cause of acquired respiratory infections around the globe. A multi-epitope vaccine (MEV) must be developed to combat infections of M. pneumoniae because there is no specific disease-modifying treatment or vaccination is present. The objective of this research is to design a vaccine that targets M. pneumoniae top five highly antigenic proteins using a combination of immunological techniques and molecular docking. T-cell (HTL & CTL), B-cell, and IFN-γ of target proteins were forecasted and highly conservative epitopes were chosen for further study. For designing of final vaccine, 4LBL, 7CTL, and 5HTL epitopes were joined by linkers of KK, AAY, and GPGPG. The N-end of the vaccine was linked to an adjuvant (Cholera enterotoxin subunit B) with a linker named EAAAK to enhance immunogenicity. After the addition of adjuvants and linkers, the size of the construct was 395 amino acids. The epitopes of IFN-γ and B-cells illustrate that the model construct is optimized for cell-mediated immune or humoral responses. To ensure that the final design is safer and immunogenic, properties like non-allergens, antigenicity, and various physicochemical properties were evaluated. Molecular docking of the vaccine with the toll-like receptor 4 (TLR4) was conducted to check the compatibility of the vaccine with the receptor. Besides, in-silico cloning was utilized for validation of the credibility and proper expression of the vaccine. Furthermore, to confirm that the multi-epitope vaccine created is protective and immunogenic, this research requires experimental validation.

Two Dimensional Anion Exchange-Size Exclusion Chromatography Combined with Mathematical Modeling for Downstream Processing of Foot and Mouth Disease Vaccine.

The production of high-quality purified virus particles in high quantities for vaccine preparation requires a scalable purification procedure in the downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D-AEC-SEC) was developed for the production of non-structural protein-free foot and mouth disease vaccine, and the whole procedure was accomplished with 77.9% virus yield. Additionally, a mathematical modeling and a simulation approach based on a plate model of chromatography were developed and matched with the experimental chromatography data to improve prediction of retention behavior and save time in the development of the downstream scale-up method. The purified pooled virus fraction obtained from the final polishing step had a purity higher than 85% based on analytical size exclusion analysis. Moreover, more than 90.1% of residual DNA (rDNA) was removed from the purified vaccine. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM) provided clear evidence of purity and demonstrated that the final product is structurally spherical, intact particles qualified for formulation as a vaccine product.

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis.

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.

Isolation and phylogenetic analysis of Getah virus from a commercial modified live vaccine against porcine reproductive and respiratory syndrome virus.

In the present study, Getah virus (GETV) isolate, GETV-V1, was isolated from a commercial PRRSV attenuated live vaccine (MLV), which has been widely used to immunize pigs against porcine reproductive and respiratory syndrome virus (PRRSV). Further analysis demonstrated that nine batches of the PRRSV MLV vaccine (three batches per year from 2017 to 2019) from the same manufacturer were all positive for GETV. Genomic analyses indicated that the GETV-V1 isolate shared the highest sequence identity with the GETV strain, 16-I-674, which was isolated from horses in Japan. The phylogenetic analysis based on the genomic sequences showed that the GETV-V1 strain was clustered with the Japanese GETV strains. Taken together, this is the first report of GETV contamination in live swine vaccines in China. Our findings demonstrate that immunization with commercial live vaccines might be a potential novel route of GETV transmission in swine. This highlights the need for more extensive monitoring of commercial live vaccines.

Multiple recombination events between field and vaccine strains resulted in the emergence of a novel infectious bronchitis virus with decreased pathogenicity and altered replication capacity.

In this study, we isolated and identified 2 infectious bronchitis virus (IBV) strains from layer chickens soon after vaccination with the Massachusetts-Connecticut bivalent vaccine (Conn) and H120 and 4/91 booster vaccines in China in 2011. The results of cross-virus-neutralization tests and phylogenetic analysis of the S1 subunit of spike gene of these vaccine strains and other reference strains showed that strain LJL/110302 was of GI-19 lineage, whereas LLN/111169 was of the GI-1 lineage of the Conn serotype. Further comparative genomic analysis revealed that LLN/111169, an IBV strain with novel traits, originated from multiple recombination events (at least 3 recombination sites) between GI-19 and the Conn and 4/91 vaccine strains. LLN/111169 was pathogenic to specific pathogen-free (SPF) chickens. This is of prime importance because while IBV prevention measures worldwide are mainly dependent on modified live vaccine strains, our results showed that recombination between field and vaccine strains has produced a novel pathogenic IBV strain. In addition, LLN/111169 showed relatively broad tissue tropism (trachea, lungs, kidneys, and cecal tonsils) in infected SPF chickens. These results emphasize the importance of IBV surveillance in chicken flocks.

Comparison of chemical extraction methods for determination of 146S content in foot-and-mouth disease oil-adjuvanted vaccine.

AIMS: To compare antigen extraction efficiency of chemical methods such as benzyl alcohol, chloroform, sodium citrate, extraction buffer with Tween-20 (EBT) and isopropyl myristate for determination of 146S content in the fresh and stored FMD oil-adjuvanted vaccines. METHODS AND RESULTS: Standard vaccine with antigen payload of 10, 5 and 5 µg per cattle dose (2 ml) for serotypes O, A and Asia1, respectively, was used to compare the antigen extraction efficiency of five chemical methods: benzyl alcohol, chloroform, sodium citrate, EBT buffer and isopropyl myristate. The purity of the extracted 146S antigen was quantified by caesium chloride (CsCl) ultracentrifugation. Serotype-specific sandwich ELISA (sELISA) was developed to identify the serotype and to compare the 146S in aqueous phase and ultrafractions. The antigen recovery was also tested in stored trivalent vaccine. Coefficient of regression was calculated to assess the predictive power of the benzyl alcohol extraction method. Of the five methods, benzyl alcohol showed consistent antigen recovery of >90% in monovalent as well as trivalent vaccines. Ultrafraction showed a 1·4 ratio at A259/239 nm in UV spectrophotometry indicating the presence of 146S. sELISA revealed that the antigen recovery was significantly less in ultrafractions than that of aqueous phase. Further, there was no significant difference in antigen recovery from stored trivalent vaccine for 12 months, indicating the usefulness of the benzyl alcohol method. Linear regression model revealed R2  = 0·99 with a narrow band of predictive interval. CONCLUSIONS: The benzyl alcohol method was efficient in extracting 146S from the monovalent and trivalent fresh and stored FMD vaccines. CsCl density gradient precisely quantified the 146S, while sELISA identified the serotype of the vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: When the benzyl alcohol method is coupled with CsCl density gradient and sELISA, it has the potential to determine the 146S content of FMD vaccine.

Removal of bovine viral diarrhea virus (BVDV) from lumpy skin disease virus (LSDV) vaccine stocks by passage on chorioallantoic membranes of fertilized hens' eggs.

Bovine viral diarrhea virus (BVDV) is a common contaminant of Madin-Darby bovine kidney (MDBK) cells as well as fetal calf serum (FCS). It is pathogenic to cattle and regulatory authorities require that veterinary vaccine stocks are free from BVDV. MDBK cells are used in the generation of recombinant lumpy skin disease virus (LSDV) and have been used for the growth of LSDV vaccines. This paper describes how vaccine stocks can be cleared of BVDV by passage through an avian host, nonpermissive to BVDV, but permissive to LSDV. LSDV vaccine stocks were shown to be cleared of BVDV after passage on the chorioallantoic membranes (CAMs) of fertilized 7-day old hens' eggs. Vaccines were passaged a second time on CAMs before being grown in primary lamb testes (LT) cells. Vaccines retained BVDV-negative status after passage on LT cells.

Development and application of a combined molecular and tissue culture-based approach to detect latent infectious laryngotracheitis virus (ILTV) in chickens.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens. ILTV can establish latency and reactivate later in life, but there have been few investigations of ILTV latency. This study aimed to contribute to the methodologies available to detect latent ILTV. A nested PCR was developed which was more sensitive than three other molecular methods investigated in this study. This nested PCR was then used in conjunction with in vitro reactivation culture methods that were optimized and applied to trigeminal ganglia (TG) and tracheal samples from ILTV-vaccinated commercial layer birds (n = 30). ILTV DNA was detected by nested PCR in the upper respiratory tract (URT) or eye of 22 birds. Of the remaining 8 birds, ILTV could be detected by co-culture in TG of 5 birds, with reactivated virus mostly detected 6 days post-explant (dpe). ILTV was also detected in tracheal cultures by 6 dpe. In the ILTV-positive URT samples, the virus could be characterised as vaccine strains SA2 (n = 9) or A20 (n = 5). This study provides evidence for reactivation and shedding of vaccine ILTV in commercial layer birds. Moreover, this study produced a molecular and in-vitro culture method to detect latent viral infection.

Current use and development of fish vaccines in China.

In the past decades, the aquaculture industry made great progress in China, which contributes more than 70% yield of the world's farmed fish. Along with the rapid growth of fish production, increased emergence and outbreak of numbers of diseases pose harm to the aquaculture industry and food safety. From the efficient, safe, environmental and ethical aspects, vaccines is definitely the most appropriate and focused method to control different kinds of fish diseases. In China, researchers have done huge works on the fish vaccines, and so far six domestic aquatic vaccine products along with one imported aquatic vaccine have obtained the national veterinary medicine certificate. More critically, some new vaccines have also entered the field experiment stage and showed broad market prospects. In the present review, authors summarize seven aquatic vaccines, including the live vaccine against grass carp hemorrhagic disease, the inactivated vaccine against Aeromonas hydrophila sepsis in fish, the live vaccine against Edwardsiella tarda in turbot, the anti-idiotypic antibody vaccine against Vibrio alginolyticus, V. parahaemolyticus, and E. tarda in Japanese flounder, the cell-cultured inactivated vaccine against grass carp hemorrhagic disease, the inactivated vaccine against fish infectious spleen and kidney necrosis virus (ISKNV), and the genetically engineered live vaccine against V. anguillarum in turbot. Moreover, different delivery routes of fish vaccines are also compared in this review, along with differential fish immune response after vaccination. All these efforts will ultimately benefit the healthy and sustainable development of aquaculture industry in China.

West Nile Virus: Veterinary Health and Vaccine Development.

West Nile virus (WNV) (Flaviviridae: Flavivirus) was discovered in Africa more than 80 yr ago and became recognized as an avian pathogen and a cause of neurologic disease in horses largely during periodic incursions into Europe. Introduction of WNV into North America stimulated great anxiety, particularly in the equine industry, but also for pet owners and livestock producers concerned about the effect of WNV on other domestic animals. Numerous subsequent studies of naturally occurring and experimentally induced disease greatly expanded our understanding of the host range and clinical consequences of WNV infection in diverse species and led to rapid development and deployment of efficacious vaccines for horses. In addition to humans, horses are clearly the animals most frequently affected by serious, sometimes lethal disease following infection with WNV, but are dead-end hosts due to the low-magnitude viremia they develop. Dogs, cats, and livestock species including chickens are readily infected with WNV, but only occasionally develop clinical disease and are considered dead-end hosts for the virus.

A New Strategy for the Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Live Vaccine by Droplet Digital PCR.

Chicken infectious anemia virus (CIAV) causes the atrophy of bone marrow hematopoietic and lymphoid tissues in chicks, leading to huge economic losses all over the world. The using of attenuated vaccine contaminated with CIAV increased the mortality and the pathogenicity of other diseases in many farms. However, it is difficult to detect the CIAV contamination by general detection technology due to the extremely low dose of CIAV in vaccines. In this study, we established a new method called droplet digital Polymerase Chain Reaction (ddPCR) to detect CIAV contamination of vaccines more sensitively and accurately. The lowest detection limitation of this method is 2.4 copies of CIAV plasmid or CIAV contamination at 0.1 EID50/1000 feathers in vaccines without any positive signals of other viruses. Besides, the sensitivity of ddPCR is 100 times greater than that of conventional PCR and 10 times greater than that of real-time PCR. The ddPCR technique is more sensitive and more intuitive. Therefore, it could be valuable for the detection of CIAV contamination in vaccines.

Recent advances on the role of host factors during non-poliovirus enteroviral infections.

Non-polio enteroviruses are emerging viruses known to cause outbreaks of polio-like infections in different parts of the world with several cases already reported in Asia Pacific, Europe and in United States of America. These outbreaks normally result in overstretching of health facilities as well as death in children under the age of five. Most of these infections are usually self-limiting except for the neurological complications associated with human enterovirus A 71 (EV-A71). The infection dynamics of these viruses have not been fully understood, with most inferences made from previous studies conducted with poliovirus.Non-poliovirus enteroviral infections are responsible for major outbreaks of hand, foot and mouth disease (HFMD) often associated with neurological complications and severe respiratory diseases. The myriad of disease presentations observed so far in children calls for an urgent need to fully elucidate the replication processes of these viruses. There are concerted efforts from different research groups to fully map out the role of human host factors in the replication cycle of these viral infections. Understanding the interaction between viral proteins and human host factors will unravel important insights on the lifecycle of this groups of viruses.This review provides the latest update on the interplay between human host factors/processes and non-polio enteroviruses (NPEV). We focus on the interactions involved in viral attachment, entry, internalization, uncoating, replication, virion assembly and eventual egress of the NPEV from the infected cells. We emphasize on the virus- human host interplay and highlight existing knowledge gaps that needs further studies. Understanding the NPEV-human host factors interactions will be key in the design and development of vaccines as well as antivirals against enteroviral infections. Dissecting the role of human host factors during NPEV infection cycle will provide a clear picture of how NPEVs usurp the human cellular processes to establish an efficient infection. This will be a boost to the drug and vaccine development against enteroviruses which will be key in control and eventual elimination of the viral infections.

Implementation of at-line capillary zone electrophoresis for fast and reliable determination of adenovirus concentrations in vaccine manufacturing.

A CZE method was validated and implemented for fast and accurate in-process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector-based vaccines. An analytical-quality-by-design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.

Nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies - a comprehensive review.

Nipah (Nee-pa) viral disease is a zoonotic infection caused by Nipah virus (NiV), a paramyxovirus belonging to the genus Henipavirus of the family Paramyxoviridae. It is a biosafety level-4 pathogen, which is transmitted by specific types of fruit bats, mainly Pteropus spp. which are natural reservoir host. The disease was reported for the first time from the Kampung Sungai Nipah village of Malaysia in 1998. Human-to-human transmission also occurs. Outbreaks have been reported also from other countries in South and Southeast Asia. Phylogenetic analysis affirmed the circulation of two major clades of NiV as based on currently available complete N and G gene sequences. NiV isolates from Malaysia and Cambodia clustered together in NiV-MY clade, whereas isolates from Bangladesh and India clusterered within NiV-BD clade. NiV isolates from Thailand harboured mixed population of sequences. In humans, the virus is responsible for causing rapidly progressing severe illness which might be characterized by severe respiratory illness and/or deadly encephalitis. In pigs below six months of age, respiratory illness along with nervous symptoms may develop. Different types of enzyme-linked immunosorbent assays along with molecular methods based on polymerase chain reaction have been developed for diagnostic purposes. Due to the expensive nature of the antibody drugs, identification of broad-spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs). High pathogenicity of NiV in humans, and lack of vaccines or therapeutics to counter this disease have attracted attention of researchers worldwide for developing effective NiV vaccine and treatment regimens.

Immunoinformatic and systems biology approaches to predict and validate peptide vaccines against Epstein-Barr virus (EBV).

Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is a member of the Herpesviridae family and causes infectious mononucleosis, Burkitt's lymphoma, and nasopharyngeal carcinoma. Even in the United States of America, the situation is alarming, as EBV affects 95% of the young population between 35 and 40 years of age. In this study, both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted by using the ElliPro and NetCTL.1.2 webservers for EBV proteins (GH, GL, GB, GN, GM, GP42 and GP350). Molecular modelling tools were used to predict the 3D coordinates of peptides, and these peptides were then docked against the MHC molecules to obtain peptide-MHC complexes. Studies of their post-docking interactions helped to select potential candidates for the development of peptide vaccines. Our results predicted a total of 58 T-cell epitopes of EBV;  where the most potential were selected based on their TAP, MHC binding and C-terminal Cleavage score. The top most peptides were subjected to MD simulation and stability analysis. Validation of our predicted epitopes using a 0.45 µM concentration was carried out by using a systems biology approach. Our results suggest a panel of epitopes that could be used to immunize populations to protect against multiple diseases caused by EBV.