Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

The heat-labile enterotoxin B subunit bio-adjuvant linked to Newcastle disease virus recombinant hemagglutinin neuraminidase elicited a humoral immune response in animal model.

Newcastle disease is a highly contagious viral infection primarily affecting poultry, leading to significant economic losses worldwide due to its high morbidity and mortality rates. Given the severity of the disease and its impact on the poultry industry, there is an urgent need for a preventative approach to tackle this issue. Developing an efficient and effective vaccine is a valuable step toward reducing the burden of this virus. Consequently, investing in preventive measures, such as vaccination programs, is a top priority to mitigate the economic losses associated with Newcastle disease and protect the livelihoods of those relying on the poultry industry. Despite many vaccines against this viral disease, it still infects many wild and domestic birds worldwide. In this work, chimeric proteins, composed of the recombinant B subunit of Enterotoxigenic E. coli with one or two HN (Hemagglutinin-neuraminidase) subunits of NDV (LHN and LHN2, respectively), expressed using E.coli host. In-silico, in-vitro, and In-vivo procedures were performed to evaluate the immunogenicity of these proteins. The sera from immunized mice were analyzed using Western Blotting and ELISA. The LHN2 protein with an extra HN subunit elicited a higher antibody titer than the LHN protein (P<0.05). Both products could effectively elicit an immune response against NDV and can be considered a component of Newcastle disease vaccine candidates.

Filamentous bacteriophages, natural nanoparticles, for viral vaccine strategies.

Filamentous bacteriophages are natural nanoparticles formed by the self-assembly of structural proteins that have the capability of replication and infection. They are used as a highly efficient vaccine platform to enhance immunogenicity and effectively stimulate the innate and adaptive immune response. Compared with traditional vaccines, phage-based vaccines offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. This review summarizes recent research on phage-based vaccines in virus prevention. In addition, the expression systems of filamentous phage-based virus vaccines and their application principles are discussed. Moreover, the prospect of the prevention of emerging infectious diseases, such as coronavirus 2019 (COVID-19), is also discussed.

The Unique Glycosylation at Position 986 on the E2 Glycoprotein of Classical Swine Fever Virus Is Responsible for Viral Attenuation and Protection against Lethal Challenge.

Classical swine fever (CSF) is an economically important disease of pigs caused by classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 (986NYT988) on the E2 glycoprotein Domain IV of C-strain but not (986NYA988) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days postinoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the Erns, E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of the highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.

An Albumin-Binding Domain Peptide Confers Enhanced Immunoprotection Against Viral Myocarditis by CVB3 VP1 Vaccine.

Coxsackievirus B3 (CVB3)-induced viral myocarditis is a common clinical cardiovascular disease without effective available vaccine. In this study, we tried to potentiate the immunoprotection efficacy of our previous CVB3-specific VP1 protein vaccine by introducing a streptococcal protein G-derived, draining lymph nodes (dLNs)-targeting albumin-binding domain (ABD) peptide. We found that compared with the original VP1 vaccine, ABD-fused VP1 (ABD-VP1) vaccine gained the new ability to efficiently bind murine albumin both in vitro and in vivo, possessed a much longer serum half-life in serum and exhibited more abundance in the dLNs after immunization. Accordingly, ABD-VP1 immunization not only significantly facilitated the enrichment and maturation of dendritic cells (DCs), induced higher percentages of IFN-γ+ CD8 + cells in the dLNs, but also robustly promoted VP1-induced T cell proliferation and cytotoxic T lymphocyte (CTL) responses in the spleens. More importantly, ABD-VP1 also elicited higher percentages of protective CD44hi CD62Lhi memory T cells in dLNs and spleens. Consequently, obvious protective effect against viral myocarditis was conferred by ABD-VP1 vaccine compared to the VP1 vaccine, reflected by the less body weight loss, improved cardiac function, alleviated cardiac histomorphological changes and an increased 28-day survival rate. Our results indicated that the ABD might be a promising immune-enhancing regime for vaccine design and development.

Development of a Novel Multi-Epitope Vaccine Against Crimean-Congo Hemorrhagic Fever Virus: An Integrated Reverse Vaccinology, Vaccine Informatics and Biophysics Approach.

Crimean-Congo hemorrhagic fever (CCHF) is a highly severe and virulent viral disease of zoonotic origin, caused by a tick-born CCHF virus (CCHFV). The virus is endemic in many countries and has a mortality rate between 10% and 40%. As there is no licensed vaccine or therapeutic options available to treat CCHF, the present study was designed to focus on application of modern computational approaches to propose a multi-epitope vaccine (MEV) expressing antigenic determinants prioritized from the CCHFV genome. Integrated computational analyses revealed the presence of 9 immunodominant epitopes from Nucleoprotein (N), RNA dependent RNA polymerase (RdRp), Glycoprotein N (Gn/G2), and Glycoprotein C (Gc/G1). Together these epitopes were observed to cover 99.74% of the world populations. The epitopes demonstrated excellent binding affinity for the B- and T-cell reference set of alleles, the high antigenic potential, non-allergenic nature, excellent solubility, zero percent toxicity and interferon-gamma induction potential. The epitopes were engineered into an MEV through suitable linkers and adjuvating with an appropriate adjuvant molecule. The recombinant vaccine sequence revealed all favorable physicochemical properties allowing the ease of experimental analysis in vivo and in vitro. The vaccine 3D structure was established ab initio. Furthermore, the vaccine displayed excellent binding affinity for critical innate immune receptors: TLR2 (-14.33 kcal/mol) and TLR3 (-6.95 kcal/mol). Vaccine binding with these receptors was dynamically analyzed in terms of complex stability and interaction energetics. Finally, we speculate the vaccine sequence reported here has excellent potential to evoke protective and specific immune responses subject to evaluation of downstream experimental analysis.

Advances in vaccine delivery systems against viral infectious diseases.

Although vaccines are available for many infectious diseases, there are still unresolved infectious diseases that threaten global public health. In particular, the rapid spread of unpredictable, highly contagious viruses has recorded numerous infection cases and deaths, and has changed our lives socially or economically through social distancing and wearing masks. The pandemics of unpredictable, highly contagious viruses increase the ever-high social need for rapid vaccine development. Nanotechnologies may hold promise and expedite the development of vaccines against newly emerging infectious viruses. As potential nanoplatforms for delivering antigens to immune cells, delivery systems based on lipids, polymers, proteins, and inorganic nanomaterials have been studied. These nanoplatforms have been tested as a means to deliver vaccines not as a whole, but in the form of protein subunits or as DNA or mRNA sequences encoding the antigen proteins of viruses. This review covers the current status of nanomaterial-based delivery systems for viral antigens, with highlights on nanovaccines against recently emerging infectious viruses, such as severe acute respiratory syndrome coronavirus-2, Middle East respiratory syndrome coronavirus, and Zika virus.

Exploring the out of sight antigens of SARS-CoV-2 to design a candidate multi-epitope vaccine by utilizing immunoinformatics approaches.

SARS-CoV-2 causes a severe respiratory disease called COVID-19. Currently, global health is facing its devastating outbreak. However, there is no vaccine available against this virus up to now. In this study, a novel multi-epitope vaccine against SARS-CoV-2 was designed to provoke both innate and adaptive immune responses. The immunodominant regions of six non-structural proteins (nsp7, nsp8, nsp9, nsp10, nsp12 and nsp14) of SARS-CoV-2 were selected by multiple immunoinformatic tools to provoke T cell immune response. Also, immunodominant fragment of the functional region of SARS-CoV-2 spike (400-510 residues) protein was selected for inducing neutralizing antibodies production. The selected regions' sequences were connected to each other by furin-sensitive linker (RVRR). Moreover, the functional region of ß-defensin as a well-known agonist for the TLR-4/MD complex was added at the N-terminus of the vaccine using (EAAAK)3 linker. Also, a CD4 + T-helper epitope, PADRE, was used at the C-terminal of the vaccine by GPGPG and A(EAAAK)2A linkers to form the final vaccine construct. The physicochemical properties, allergenicity, antigenicity, functionality and population coverage of the final vaccine construct were analyzed. The final vaccine construct was an immunogenic, non-allergen and unfunctional protein which contained multiple CD8 + and CD4 + overlapping epitopes, IFN-γ inducing epitopes, linear and conformational B cell epitopes. It could form stable and significant interactions with TLR-4/MD according to molecular docking and dynamics simulations. Global population coverage of the vaccine for HLA-I and II were estimated 96.2% and 97.1%, respectively. At last, the final vaccine construct was reverse translated to design the DNA vaccine. Although the designed vaccine exhibited high efficacy in silico, further experimental validation is necessary.

The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

Genome based evolutionary lineage of SARS-CoV-2 towards the development of novel chimeric vaccine.

The present study aimed to predict a novel chimeric vaccine by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein, membrane protein, envelope protein and nucleocapsid protein were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. However, the in vivo and in vitro validation of suggested vaccine molecule might be ensured with wet lab trials using model animals for the implementation of the presented data.

A candidate multi-epitope vaccine against SARS-CoV-2.

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.

A Novel Vaccine Employing Non-Replicating Rabies Virus Expressing Chimeric SARS-CoV-2 Spike Protein Domains: Functional Inhibition of Viral/Nicotinic Acetylcholine Receptor Complexes.

The emergence of the novel ß-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic of coronavirus disease 2019 (COVID-19). Clinical studies have documented that potentially severe neurological symptoms are associated with SARS-CoV-2 infection, thereby suggesting direct CNS penetration by the virus. Prior studies have demonstrated that the destructive neurological effects of rabies virus (RABV) infections are mediated by CNS transport of the virus tightly bound to the nicotinic acetylcholine receptor (nAChR). By comparison, it has been hypothesized that a similar mechanism exists to explain the multiple neurological effects of SARS-CoV-2 via binding to peripheral nAChRs followed by orthograde or retrograde transport into the CNS. Genetic engineering of the RABV has been employed to generate novel vaccines consisting of non-replicating RABV particles expressing chimeric capsid proteins containing human immunodeficiency virus 1 (HIV-1), Middle East respiratory syndrome (MERS-CoV), Ebolavirus, and hepatitis C virus (HCV) sequences. Accordingly, we present a critical discussion that integrates lessons learned from prior RABV research and vaccine development into a working model of a SARS-CoV-2 vaccine that selectively targets and neutralizes CNS penetration of a tightly bound viral nAChR complex.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine.

The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

Cell culture propagation of foot-and-mouth disease virus: adaptive amino acid substitutions in structural proteins and their functional implications.

Foot-and-mouth disease is endemic in livestock in large parts of Africa and Asia, where it is an important driver of food insecurity and a major obstacle to agricultural development and the international trade in animal products. Virtually all commercially available vaccines are inactivated whole-virus vaccines produced in cell culture, but the adaptation of a field isolate of the virus to growth in culture is laborious and time-consuming. This is of particular concern for the development of vaccines to newly emerging virus lineages, where long lead times from virus isolate to vaccine can delay the implementation of effective control programs. High antigen yields in production cells are also necessary to make vaccines affordable for less developed countries in endemic areas. Therefore, a rational approach to cell culture adaptation that combines prior knowledge of common adaptive mutations and reverse genetics techniques is urgently required. This review provides an overview of amino acid exchanges in the viral capsid proteins in the context of adaptation to cell culture.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha.

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro.

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

Investigation of cell culture conditions for optimal foot-and-mouth disease virus production.

BACKGROUND: Foot-and-mouth disease is a highly contagious and economically devastating disease with endemic occurrence in many parts of the world. Vaccination is the method of choice to eradicate the disease and to limit the viral spread. The vaccine production process is based on mammalian cell culture, in which the viral yield varies in dependence of the composition of the culture media. For foot-and-mouth disease virus (FMDV), very little is known about the culture media components that are necessary to grow the virus to high titers in cell culture. RESULTS: This study examined the influence of increasing concentrations of glucose, glutamine, ammonium chloride and different cell densities on the yield of FMDV. While an excess of glucose or glutamine does not affect the viral yield, increasing cell density reduces the viral titer by a log10 step at a cell density of 3 × 106 cells/mL. This can be mitigated by performing a 100% media exchange before infection of the cells. CONCLUSIONS: The reasons for the diminished viral growth, if no complete media exchange has been performed prior to infection, remain unclear and further studies are necessary to investigate the causes more deeply. For now, the results argue for a vaccine production process with 100% media exchange to reliably obtain high viral titers.

Design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics.

Severe acute respiratory syndrome (SARS) is endemic in South China and is continuing to spread worldwide since the 2003 outbreak, affecting human population of 37 countries till present. SARS is caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV). In the present study, we have designed two multi-epitope vaccines (MEVs) composed of cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell epitopes overlap, bearing the potential to elicit cellular as well as humoral immune response. We have used truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 as molecular adjuvants at N-terminal of both the MEVs. Selected overlapping epitopes of both the MEVs were further validated for stable molecular interactions with their respective human leukocyte antigen class I and II allele binders. Moreover, CTL epitopes were further studied for their molecular interaction with transporter associated with antigen processing. Furthermore, after tertiary structure modelling, both the MEVs were validated for their stable molecular interaction with Toll-like receptors 2 and 4. Codon-optimized cDNA of both the MEVs was analysed for their potential high level of expression in the mammalian cell line (Human) needed for their further in vivo testing. Overall, the present study proposes in silico validated design of two MEVs against SARS composed of specific epitopes with the potential to cause a high level of SARS-CoV specific cellular as well as humoral immune response. Communicated by Ramaswamy H. Sarma.

Production of virus-like particles for vaccines.

Virus-like particles (VLPs) are nanostructures that resemble the structures of viruses. They are composed of one or more structural proteins that can be arranged in several layers and can also contain a lipid outer envelope. VLPs trigger a high humoral and cellular immune response due to their repetitive structures. A key factor regarding VLP safety is the lack of viral genomic material, which enhances safety during both manufacture and administration. Contemporary VLP production may take advantage of several systems, including bacterial, yeast, insect and mammalian cells. The choice of production platform depends on several factors, including cost and the need for post-translational modifications (PTMs), which can be essential in generating an optimal immune response. Some VLP-based vaccines designed to prevent several infectious diseases are already approved and on the market, with many others at the clinical trial or research stage. Interest in this technology has recently increased due to its advantages over classical vaccines. This paper reviews the state-of-the-art of VLP production systems and the newest generation of VLP-based vaccines now available.