Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Fluorescent-protein co-expression to select CHO cells expressing high quantities of vaccine antigens.

Cell line development for production of vaccine antigens or therapeutic proteins typically involves transfection, selection, and enrichment for high-expressing cells. Enrichment methods include minipool enrichment, antibody-based enrichment, and enrichment based on co-expressed fluorescent biosensor proteins. However, these methods have limitations regarding labor and cost intensity, the generation of antibodies and assurance of their viral safety, and potential expression-interference or signal-saturation of the co-expressed fluorescent protein. To improve the method of fluorescent-protein co-expression, expression constructs were created that constitutively express a model vaccine antigen together with one of three fluorescent proteins having translation initiation controlled by a wildtype or mutant internal ribosome entry site (IRES), for a total of six constructs. The constructs were transfected into Chinese hamster ovary cells (CHO) cells, enriched for high fluorescence, cultured, and tested in a mini bioreactor to identify the most promising construct. The fluorescent protein, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a mutant IRES performed best and was further tested with three additional vaccine antigens. Across the four vaccine antigens, the FUCCI fluorescent protein yielded productivity enhancements, without the need for generating an antibody and assuring its viral safety. Furthermore, FUCCI protein was present in negligible quantities in the cell supernatant, indicating a low risk for contaminating drug substances or vaccine antigen.

Genome editing and its role in vaccine, diagnosis, and therapeutic advancement.

Genome editing involves precise modification of specific nucleotides in the genome using nucleases like CRISPR/Cas, ZFN, or TALEN, leading to increased efficiency of homologous recombination (HR) for gene editing, and it can result in gene disruption events via non-homologous end joining (NHEJ) or homology-driven repair (HDR). Genome editing, particularly CRISPR-Cas9, revolutionizes vaccine development by enabling precise modifications of pathogen genomes, leading to enhanced vaccine efficacy and safety. It allows for tailored antigen optimization, improved vector design, and deeper insights into host genes' impact on vaccine responses, ultimately enhancing vaccine development and manufacturing processes. This review highlights different types of genome editing methods, their associated risks, approaches to overcome the shortcomings, and the diverse roles of genome editing.

Conditional splicing system for tight control of viral overlapping genes.

Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

Advances of Recombinant Adenoviral Vectors in Preclinical and Clinical Applications.

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.

Engineered EVs with pathogen proteins: promising vaccine alternatives to LNP-mRNA vaccines.

Extracellular vesicles (EVs) are tiny, lipid membrane-bound structures that are released by most cells. They play a vital role in facilitating intercellular communication by delivering bioactive cargoes to recipient cells and triggering cellular as well as biological responses. EVs have enormous potential for therapeutic applications as native or engineered exosomes. Native EVs are naturally released by cells without undergoing any modifications to either the exosomes or the cells that secrete them. In contrast, engineered EVs have been deliberately modified post-secretion or through genetic engineering of the secreting cells to alter their composition. Here we propose that engineered EVs displaying pathogen proteins could serve as promising alternatives to lipid nanoparticle (LNP)-mRNA vaccines. By leveraging their unique characteristics, these engineered EVs have the potential to overcome certain limitations associated with LNP-mRNA vaccines.

Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses.

IMPORTANCE: messenger RNA (mRNA) vaccines are a key technology in combating existing and emerging infectious diseases. However, the inherent instability of mRNA and the nonspecificity of lipid nanoparticle-encapsulated (LNP) delivery systems result in the need for cold storage and a relatively short-duration immune response to mRNA vaccines. Herein, we develop a novel vaccine in the form of circRNAs encapsulated in LNPs, and the circular structure of the circRNAs enhances their stability. Lyophilization is considered the most effective method for the long-term preservation of RNA vaccines. However, this process may result in irreversible damage to the nanoparticles, particularly the potential disruption of targeting modifications on LNPs. During the selection of lymph node-targeting ligands, we found that LNPs modified with mannose maintained their physical properties almost unchanged after lyophilization. Additionally, the targeting specificity and immunogenicity remained unaffected. In contrast, even with the addition of cryoprotectants such as sucrose, the physical properties of LNPs were impaired, leading to an obvious decrease in immunogenicity. This may be attributed to the protective role of mannose on the surface of LNPs during lyophilization. Freshly prepared and lyophilized mLNP-circRNA vaccines elicited comparable immune responses in both the rabies virus model and the SARS-CoV-2 model. Our data demonstrated that mLNP-circRNA vaccines elicit robust immune responses while improving stability after lyophilization, with no compromise in tissue targeting specificity. Therefore, mannose-modified LNP-circRNA vaccines represent a promising vaccine design strategy.

RNA vaccines: A transformational advance.

Vaccines have stemmed many infectious diseases, but when SARS-CoV-2 emerged, traditional vaccine development would not have been fast enough. This year's Nobel Prize in Physiology or Medicine recognizes work that enabled the rapid development of mRNA vaccines, which halted the COVID-19 pandemic. The feat was a product of basic biological insights coupled with technological innovations, which have transformed vaccine design.

Anti-tick vaccine candidate subolesin is important for blood feeding and innate immune gene expression in soft ticks.

Subolesin is a conserved molecule in both hard and soft ticks and is considered as an effective candidate molecule for the development of anti-tick vaccine. Previous studies have reported the role of subolesin in blood feeding, reproduction, development, and gene expression in hard ticks. However, studies addressing the role of subolesin in soft ticks are limited. In this study, we report that subolesin is not only important in soft tick Ornithodoros turicata americanus blood feeding but also in the regulation of innate immune gene expression in these ticks. We identified and characterized several putative innate immune genes including Toll, Lysozyme precursor (Lp), fibrinogen-domain containing protein (FDP), cystatin and ML-domain containing protein (MLD) in O. turicata americanus ticks. Quantitative real-time polymerase chain reaction analysis revealed the expression of these genes in both O. turicata americanus salivary glands and midgut and in all developmental stages of these soft ticks. Significantly increased expression of fdp was noted in salivary glands and midgut upon O. turicata americanus blood feeding. Furthermore, RNAi-mediated knockdown of O. turicata americanus subolesin expression affected blood feeding and innate immune gene expression in these ticks. Significant downregulation of toll, lp, fdp, cystatin, and mld transcripts was evident in sub-dsRNA-treated ticks when compared to the levels noted in mock-dsRNA-treated control. Collectively, our study not only reports identification and characterization of various innate immune genes in O. turicata americanus ticks but also provides evidence on the role of subolesin in blood feeding and innate immune gene expression in these medically important ticks.

Regulatory perspective for quality evaluation of lipid nanoparticle-based mRNA vaccines in China.

In recent years, urgent unmet medical needs due to the COVID-19 pandemic have accelerated the application of mRNA technology in vaccine development, leading to some of the first approvals of mRNA vaccines in human history by regulatory agencies around the world. For market authorization, comprehensive chemistry, manufacturing and control (CMC) information is required to assure the safety and quality consistency of mRNA vaccines. Evaluating mRNA vaccines for new virus variants poses a challenge for regulators, given the rapid optimization and development based on prior platform knowledge to accelerate the development process, which is traditionally limited for biological products. Here we summarize the current regulatory considerations of CMC evaluation on mRNA vaccines based on the scientific knowledge available, which will be updated with the advance of mRNA biology and pharmaceutical science.

Innovative approaches for the control of ticks and tick-borne diseases.

Ticks and tick-borne diseases constitute a major threat for human and animal health worldwide. Vaccines for the control of tick infestations and transmitted pathogens still represents a challenge for science and health. Vaccines have evolved with antigens derived from inactivated pathogens to recombinant proteins and vaccinomics approaches. Recently, vaccines for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown the efficacy of new antigen delivery platforms. However, until now only two vaccines based on recombinant Bm86/Bm95 antigens have been registered and commercialized for the control of cattle-tick infestations. Nevertheless, recently new technologies and approaches are under consideration for vaccine development for the control of ticks and tick-borne pathogens. Genetic manipulation of tick commensal bacteria converted enemies into friends. Frankenbacteriosis was used to control tick pathogen infection. Based on these results, the way forward is to develop new paratransgenic interventions and vaccine delivery platforms for the control of tick-borne diseases.

Plant glycoengineering for designing next-generation vaccines and therapeutic proteins.

Protein glycosylation has a huge impact on biological processes in all domains of life. The type of glycan present on a recombinant glycoprotein depends on protein intrinsic features and the glycosylation repertoire of the cell type used for expression. Glycoengineering approaches are used to eliminate unwanted glycan modifications and to facilitate the coordinated expression of glycosylation enzymes or whole metabolic pathways to furnish glycans with distinct modifications. The formation of tailored glycans enables structure-function studies and optimization of therapeutic proteins used in different applications. While recombinant proteins or proteins from natural sources can be in vitro glycoengineered using glycosyltransferases or chemoenzymatic synthesis, many approaches use genetic engineering involving the elimination of endogenous genes and introduction of heterologous genes to cell-based production systems. Plant glycoengineering enables the in planta production of recombinant glycoproteins with human or animal-type glycans that resemble natural glycosylation or contain novel glycan structures. This review summarizes key achievements in glycoengineering of plants and highlights current developments aiming to make plants more suitable for the production of a diverse range of recombinant glycoproteins for innovative therapies.

Reverse Vaccinology for Influenza A Virus: From Genome Sequencing to Vaccine Design.

Reverse vaccinology (RV) consists in the identification of potentially protective antigens expressed by any organism starting from genomic information and derived from in silico analysis, with the aim of promoting the discovery of new candidate vaccines against different types of pathogens. This approach makes use of bioinformatics techniques to screen the whole genomic sequence of a specific pathogen for the identification of the epitopes that could elicit the best immune response. The use of in silico techniques allows to reduce dramatically both the time and cost required for the identification of a potential vaccine, also facilitating the laborious process of selection of those antigens that, with a traditional approach, would be completely impossible to detect or culture. RV methodologies have been successfully applied for the identification of new vaccines against serogroup B meningococcus (MenB), Bacillus anthracis, Streptococcus pneumonia, Staphylococcus aureus, Chlamydia pneumoniae, Porphyromonas gingivalis, Edwardsiella tarda, and Mycobacterium tuberculosis. As a case of study, we will go in depth into the application of RV techniques on Influenza A virus.

COVID-19 vaccine design using reverse and structural vaccinology, ontology-based literature mining and machine learning.

Rational vaccine design, especially vaccine antigen identification and optimization, is critical to successful and efficient vaccine development against various infectious diseases including coronavirus disease 2019 (COVID-19). In general, computational vaccine design includes three major stages: (i) identification and annotation of experimentally verified gold standard protective antigens through literature mining, (ii) rational vaccine design using reverse vaccinology (RV) and structural vaccinology (SV) and (iii) post-licensure vaccine success and adverse event surveillance and its usage for vaccine design. Protegen is a database of experimentally verified protective antigens, which can be used as gold standard data for rational vaccine design. RV predicts protective antigen targets primarily from genome sequence analysis. SV refines antigens through structural engineering. Recently, RV and SV approaches, with the support of various machine learning methods, have been applied to COVID-19 vaccine design. The analysis of post-licensure vaccine adverse event report data also provides valuable results in terms of vaccine safety and how vaccines should be used or paused. Ontology standardizes and incorporates heterogeneous data and knowledge in a human- and computer-interpretable manner, further supporting machine learning and vaccine design. Future directions on rational vaccine design are discussed.

Variations in antibody repertoires correlate with vaccine responses.

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold the key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated data set, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study reveals that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

Quantifying the effectiveness of betaherpesvirus-vectored transmissible vaccines.

Transmissible vaccines have the potential to revolutionize how zoonotic pathogens are controlled within wildlife reservoirs. A key challenge that must be overcome is identifying viral vectors that can rapidly spread immunity through a reservoir population. Because they are broadly distributed taxonomically, species specific, and stable to genetic manipulation, betaherpesviruses are leading candidates for use as transmissible vaccine vectors. Here we evaluate the likely effectiveness of betaherpesvirus-vectored transmissible vaccines by developing and parameterizing a mathematical model using data from captive and free-living mouse populations infected with murine cytomegalovirus (MCMV). Simulations of our parameterized model demonstrate rapid and effective control for a range of pathogens, with pathogen elimination frequently occurring within a year of vaccine introduction. Our results also suggest, however, that the effectiveness of transmissible vaccines may vary across reservoir populations and with respect to the specific vector strain used to construct the vaccine.

Impact of Molecular Modification on the Efficiency of Recombinant Baculovirus Vector Invasion to Mammalian Cells and Its Immunogenicity in Mice.

The baculovirus display system (BDS), an excellent eukaryotic surface display technology that offers the advantages of safety, efficiency, and economy, is widely used in biomedicine. A previous study using rBacmid-Δgp64-ires-gp64 expressed in low copy numbers of the gp64 gene achieved high-efficiency expression and co-display of three fluorescent proteins (GFP, YFP, and mCherry). However, low expression of GP64 in recombinant baculoviruses also reduces the efficiency of recombinant baculovirus transduction into mammalian cells. In addition, the baculovirus promoter has no expression activity in mammalian cells and thus cannot meet the application requirements of baculoviral vectors for the BDS. Based on previous research, this study first determined the expression activity of promoters in insect Spodoptera frugiperda 9 cells and mammalian cells and successfully screened the very early promoter pie1 to mediate the co-expression of multiple genes. Second, utilizing the envelope display effect of the INVASIN and VSVG proteins, the efficiency of transduction of recombinant baculovirus particles into non-host cells was significantly improved. Finally, based on the above improvement, a recombinant baculovirus vector displaying four antigen proteins with high efficiency was constructed. Compared with traditional BDSs, the rBacmid-Δgp64 system exhibited increased display efficiency of the target protein by approximately 3-fold and induced an approximately 4-fold increase in the titer of serum antibodies to target antigens in Bal B/c mice. This study systematically explored the application of a new multi-gene co-display technology applicable to multi-vaccine research, and the results provide a foundation for the development of novel BDS technologies.

Inhalable mRNA vaccines for respiratory diseases: a roadmap.

Global implementation of messenger RNA (mRNA) vaccines represents an enormous advance with far-reaching implications for respiratory disease treatment. mRNA vaccines offer exceptional efficacy and versatile capacity to be adapted to new viruses and variants; however, critical questions remain regarding immune persistence and formulation stability. This represents a significant opportunity for developing next-generation, inhaled mRNA vaccines with the ability to drive long-lasting, tissue-specific memory responses needed for rapid recall and immediate local protection. Advances in pulmonary delivery technologies offer potential to overcome translational challenges including design of aerosol-stable and lung-stable formulations, navigation of pulmonary biological barriers, and a lack of predictive models and measurement techniques. We highlight recent advances in each of these challenge areas to illuminate the path to translation.

Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae.

Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships.