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.

Plant-derived VLP: a worthy platform to produce vaccine against SARS-CoV-2.

After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.

Vaccines based on replication incompetent Ad26 viral vectors: Standardized template with key considerations for a risk/benefit assessment.

Replication-incompetent adenoviral vectors have been under investigation as a platform to carry a variety of transgenes, and express them as a basis for vaccine development. A replication-incompetent adenoviral vector based on human adenovirus type 26 (Ad26) has been evaluated in several clinical trials. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and features of recombinant viral vector vaccines. This paper reviews features of the Ad26 vectors, including tabulation of safety and risk assessment characteristics of Ad26-based vaccines. In the Ad26 vector, deletion of E1 gene rendering the vector replication incompetent is combined with additional genetic engineering for vaccine manufacturability and transgene expression optimization. These vaccines can be manufactured in mammalian cell lines at scale providing an effective, flexible system for high-yield manufacturing. Ad26 vector vaccines have favorable thermostability profiles, compatible with vaccine supply chains. Safety data are compiled in the Ad26 vaccine safety database version 4.0, with unblinded data from 23 ongoing and completed clinical studies for 3912 participants in five different Ad26-based vaccine programs. Overall, Ad26-based vaccines have been well tolerated, with no significant safety issues identified. Evaluation of Ad26-based vaccines is continuing, with >114,000 participants vaccinated as of 4th September 2020. Extensive evaluation of immunogenicity in humans shows strong, durable humoral and cellular immune responses. Clinical trials have not revealed impact of pre-existing immunity to Ad26 on vaccine immunogenicity, even in the presence of Ad26 neutralizing antibody titers or Ad26-targeting T cell responses at baseline. The first Ad26-based vaccine, against Ebola virus, received marketing authorization from EC on 1st July 2020, as part of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen. New developments based on Ad26 vectors are underway, including a COVID-19 vaccine, which is currently in phase 3 of clinical evaluation.

E. coli expression and immunological assessment of expressed recombinant Newcastle disease virus hemagglutinin-neuraminidase protein in chickens.

Every year, the poultry industry experiences significant economic losses due to epidemics of Newcastle disease virus (NDV). Developing new vaccines by identifying and using the immunogenic hemagglutinin-neuraminidase (HN) protein can protect the poultry industry. In the present study, the full-length HN protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells, purified via affinity chromatography and detected via western blot analysis using His-specific antibodies. The purified HN protein was further evaluated in chickens to study the immune response against NDV. The successful production of HN-specific IgY proved the activity of the purified HN protein. IgY was present in the serum of immunized chickens. However, the immune response was higher in chickens immunized with purified HN protein along with complete and incomplete adjuvants than in chickens immunized with only the HN protein. Keywords: protein; Newcastle disease virus; poultry; infectious diseases; vaccines.

The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (RNA and DNA) vaccines.

Nucleic acid (DNA and RNA) vaccines are among the most advanced vaccines for COVID-19 under development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of nucleic acid vaccines. This will facilitate the assessment by key stakeholders of potential safety issues and understanding of overall benefit-risk. The structured assessment provided by the template can also help improve communication and public acceptance of licensed nucleic acid vaccines.

Noninvasive vaccination against infectious diseases.

The development of a successful vaccine, which should elicit a combination of humoral and cellular responses to control or prevent infections, is the first step in protecting against infectious diseases. A vaccine may protect against bacterial, fungal, parasitic, or viral infections in animal models, but to be effective in humans there are some issues that should be considered, such as the adjuvant, the route of vaccination, and the antigen-carrier system. While almost all licensed vaccines are injected such that inoculation is by far the most commonly used method, injection has several potential disadvantages, including pain, cross contamination, needlestick injury, under- or overdosing, and increased cost. It is also problematic for patients from rural areas of developing countries, who must travel to a hospital for vaccine administration. Noninvasive immunizations, including oral, intranasal, and transcutaneous administration of vaccines, can reduce or eliminate pain, reduce the cost of vaccinations, and increase their safety. Several preclinical and clinical studies as well as experience with licensed vaccines have demonstrated that noninvasive vaccine immunization activates cellular and humoral immunity, which protect against pathogen infections. Here we review the development of noninvasive immunization with vaccines based on live attenuated virus, recombinant adenovirus, inactivated virus, viral subunits, virus-like particles, DNA, RNA, and antigen expression in rice in preclinical and clinical studies. We predict that noninvasive vaccine administration will be more widely applied in the clinic in the near future.

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.

Assessment of a novel recombinant vesicular stomatitis virus with triple mutations in its matrix protein as a vaccine for pigs.

Vesicular stomatitis virus (VSV) causes a serious vesicular disease responsible for economic losses in the livestock industry. Currently, there are no suitable vaccines to prevent VSV infection. Although the structural matrix (M) protein of VSV has been shown to be a virulence factor in rodent models, its role in the pathogenicity of VSV infection in livestock species is unknown. We hypothesized that VSV with mutations in the M protein represents a novel live attenuated vaccine candidate. To test this, we introduced mutations into VSV M protein using reverse genetics and assessed their attenuation both in vitro and in pigs, an important natural host of VSV. A recombinant VSV with a triple amino acid mutation in M protein (VSVMT) demonstrated a significantly reduced ability to inhibit the type I interferon (IFN) signaling pathway and to shutoff host gene expression compared to WT-VSV and a mutant virus with a single amino acid deletion (VSVΔM51). Inoculation of pigs with VSVMT induced no apparent vesicular lesions but stimulated virus-neutralizing antibodies and animals were protected against virulent VSV challenge infection. These data demonstrate that the M protein is an important virulence factor for VSV in swine and VSVMT represents a novel vaccine candidate for VSV infections in pigs.

Live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment.

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called "chimeric virus vaccines"). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for marketing (to date in Australia, Thailand, Malaysia, and the Philippines) is a vaccine against the flavivirus, Japanese encephalitis (JE), which employs a licensed vaccine (yellow fever 17D) as a vector. In this vaccine, two envelope proteins (prM-E) of YF 17D virus were exchanged for the corresponding genes of JE virus, with additional attenuating mutations incorporated into the JE gene inserts. Similar vaccines have been constructed by inserting prM-E genes of dengue and West Nile into YF 17D virus and are in late stage clinical studies. The dengue vaccine is, however, more complex in that it requires a mixture of four live vectors each expressing one of the four dengue serotypes. This vaccine has been evaluated in multiple clinical trials. No significant safety concerns have been found. The Phase 3 trials met their endpoints in terms of overall reduction of confirmed dengue fever, and, most importantly a significant reduction in severe dengue and hospitalization due to dengue. However, based on results that have been published so far, efficacy in preventing serotype 2 infection is less than that for the other three serotypes. In the development of these chimeric vaccines, an important series of comparative studies of safety and efficacy were made using the parental YF 17D vaccine virus as a benchmark. In this paper, we use a standardized template describing the key characteristics of the novel flavivirus vaccine vectors, in comparison to the parental YF 17D vaccine. The template facilitates scientific discourse among key stakeholders by increasing the transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.

Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs.

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 10(5) TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted.

Plant-based vaccines: novel and low-cost possible route for Mediterranean innovative vaccination strategies.

A plant bioreactor has enormous capability as a system that supports many biological activities, that is, production of plant bodies, virus-like particles (VLPs), and vaccines. Foreign gene expression is an efficient mechanism for getting protein vaccines against different human viral and nonviral diseases. Plants make it easy to deal with safe, inexpensive, and provide trouble-free storage. The broad spectrum of safe gene promoters is being used to avoid risk assessments. Engineered virus-based vectors have no side effect. The process can be manipulated as follows: (a) retrieve and select gene encoding, use an antigenic protein from GenBank and/or from a viral-genome sequence, (b) design and construct hybrid-virus vectors (viral vector with a gene of interest) eventually flanked by plant-specific genetic regulatory elements for constitutive expression for obtaining chimeric virus, (c) gene transformation and/or transfection, for transient expression, into a plant-host model, that is, tobacco, to get protocols processed positively, and then moving into edible host plants, (d) confirmation of protein expression by bioassay, PCR-associated tests (RT-PCR), Northern and Western blotting analysis, and serological assay (ELISA), (e) expression for adjuvant recombinant protein seeking better antigenicity, (f) extraction and purification of expressed protein for identification and dosing, (g) antigenicity capability evaluated using parental or oral delivery in animal models (mice and/or rabbit immunization), and (h) growing of construct-treated edible crops in protective green houses. Some successful cases of heterologous gene-expressed protein, as edible vaccine, are being discussed, that is, hepatitis C virus (HCV). R9 mimotope, also named hypervariable region 1 (HVR1), was derived from the HVR1 of HCV. It was used as a potential neutralizing epitope of HCV. The mimotope was expressed using cucumber mosaic virus coat protein (CP), alfalfa mosaic virus CP P3/RNA3, and tobacco mosaic virus (TMV) CP-tobacco mild green mosaic virus (TMGMV) CP as expression vectors into tobacco plants. Expressed recombinant protein has not only been confirmed as a therapeutic but also as a diagnostic tool. Herpes simplex virus 2 (HSV-2), HSV-2 gD, and HSV-2 VP16 subunits were transfected into tobacco plants, using TMV CP-TMGMV CP expression vectors.

Assessment of the potential relationship between egg quality and infectious bronchitis virus infection in Australian layer flocks.

OBJECTIVE: This investigation aimed to determine if there was a relationship between the production of eggs with poor internal quality, as measured by poor Haugh units, by Australian layer flocks and the detection of infectious bronchitis virus (IBV) in the hens. Other risk factors including flock size, flock type, flock age, chicken breed and vaccination frequency were also assessed. METHODS: The study group comprised 17 flocks from 14 farms. Data relating to the factors investigated were requested on a regular basis. The Haugh unit data were used to grade eggs as good or poor based on the age and flock at the time of data collection. Cloacal swabs were collected from 20 chickens in each flock approximately every 6 weeks. RESULTS: IBV was detected from a majority of the flocks and in 68% of cases the IBV strain detected was an A-vaccine-related field strain. Three variant strains were detected. Detection of IBV in a flock, the farm type and flock size were identified as potential risk factors for the production of eggs with poor Haugh units. CONCLUSION: IBV is prevalent in Australian layer flocks, but infection was primarily subclinical. The results complement previous reports indicating that there are many potential risk factors for the production of eggs with poor Haugh units.

Lumpy skin disease: preliminary vaccine efficacy assessment and overview on outbreak impact in dairy cattle at Debre Zeit, central Ethiopia.

This study was conducted in and around Debre Zeit town to assess the field efficacy of LSD vaccine in use and overview associated disease impact. The study comprised cross-sectional and retrospective study design which employed active disease follow-up, semi-structured questionnaire survey and molecular techniques. The finding revealed that the Kenyan sheep pox vaccine strain used for the control of LSD did not confer expected protection. From the total of 476 animals observed, 22.9% and 2.31% cattle were found sick and dead due to LSD, respectively. Breed specific morbidity rate was 22.5% in Holstein Friesian-zebu cross and 25.9% in local zebu breed. The disease was observed to be more serious in young animals and also in females. A trend of seasonality was also observed in its occurrence. The study finding urges the need for investigation of vaccine failure including vaccine matching and alternative vaccine development.

Environmental risk assessment of clinical trials involving modified vaccinia virus Ankara (MVA)-based vectors.

The modified vaccinia virus Ankara (MVA) strain, which has been developed as a vaccine against smallpox, is since the nineties widely tested in clinical trials as recombinant vector for vaccination or gene therapy applications. Although MVA is renowned for its safety, several biosafety aspects need to be considered when performing the risk assessment of a recombinant MVA (rMVA). This paper presents the biosafety issues and the main lessons learned from the evaluation of the clinical trials with rMVA performed in Belgium. Factors such as the specific characteristics of the rMVA, the inserted foreign sequences/transgene, its ability for reconversion, recombination and dissemination in the population and the environment are the main points of attention. Measures to prevent or manage identified risks are also discussed.

[Fusion expression of Asia I type FMDV neutralizing epitope with heavy chain constant region of sheep IgG and the assessment of its immunogenicity].

VP1 is a major antigenic protein of foot-and-mouth disease virus(FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. We designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136-160aa and 198-211aa of a strain of type Asia I FMDV, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Inoculation with 100 microg FsIgG induced strong neutralizing antibody response in guinea pigs, and FshIgG inoculated guinea pigs were also protected against 200 ID50 FMDV challenge. Our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection.

Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease.

Beak and feather disease virus (BFDV) is a significant pathogen of wild Australasian and African psittacine birds. We assessed the immunogenicity of recombinant BFDV capsid (recBFDVcap) to protect against the development of psittacine beak and feather disease (PBFD). Long-billed corellas (Cacatua tenuirostris) (n=13) received (by injection) 1 ml vaccine containing 10 microg recBFDVcap on day 0 and 0.4 ml vaccine containing 66.8 microg recBFDVcap on day 11. All vaccinated corellas and five non-vaccinated control corellas were given 0.4 ml BFDV suspension [titre=log(2) 12 haemagglutination units (HAU) 50 microl(-1)] intramuscularly and 0.1 ml orally 16 days after booster vaccination. Blood was collected during the vaccination period and blood and feathers were collected after BFDV administration. Testing of blood samples included BFDV DNA detection by PCR and quantitative PCR (qPCR) as well as antibody detection by haemagglutination inhibition (HI) and on feather samples, BFDV DNA and antigen was detected by haemagglutination (HA) and qPCR. Four of 97 blood samples collected from vaccinated birds after virus challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR-detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry. Non-vaccinated control corellas developed transient feather lesions and had PCR, HI and HA test results consistent with PBFD. They were BFDV PCR-positive for up to 41 days post-challenge and qPCR demonstrated reduced virus replication in vaccinated birds compared with non-vaccinated control birds.

Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: advantages for immunization strategies and vaccine implementation.

Rabbit hemorrhagic disease virus (RHDV) VP60 capsid protein was recently expressed at approximately 1.5 gL(-1) associated with the disruption pellet of Pichia pastoris, thus requiring an additional process of extraction by solubilization. Consequently, the expression of a soluble variant of VP60 was undertaken in order to attain an easier approach for vaccine production. The VP60 gene was cloned without secretion signal under the transcriptional control of the AOX1 yeast promoter. The antigen obtained was intracellular and soluble at approximately 480 mg L(-1). Its characterization by size-exclusion HPLC, ultracentrifugation, and electron microscopy, showed the presence of high molecular weight structures similar in mass, size and buoyant density to native RHDV. The antigenic profile was similar to that from authentic virions as determined with monoclonal antibodies directed against RHDV conformational epitopes. These analyses, conducted on VP60 obtained insoluble in P. pastoris revealed the formation of protein aggregates rather than the presence of ordered multimeric structures. An immunization trial was conducted in which the soluble VP60 was employed by subcutaneous (s.c.) injection either purified by a single chromatographic step or contained within raw disruption supernatant, emulsified in Montanide 888. The insoluble variant was administered as a yeast extract powder by oral and s.c. routes. The earliest IgG response, titers and persistence of antibodies were studied by competition ELISA and hemagglutination inhibition (HI) assays. All rabbits immunized with the yeast-derived antigens developed a strong RHDV-specific response (including the "RHDVa" subtype) that lasted over one year after the primary immunization. Early HI titers up to 1/40 960 were generated. The immune response was similar to that induced by VP60 from Sf9 cells and superior to the response elicited with inactivated RHDV. Overall it was found that the soluble VP60 multimers, safely and easily produced in P. pastoris, are a valuable candidate for the rational implementation of a low-cost, scalable subunit vaccine against RHDV.

Assessment of bovine herpesvirus 4 based vector in chicken.

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.