Generation Of Vaccines Research Articles

2024 White Paper on Recent Issues in Bioanalysis: Three Way-Cross Validation; Urine Clinical Analysis; Automated Methods; Regulatory Queries on Plasma Protein Binding; Automated Biospecimen Management; ELN Migration; Ultra-Sensitivity Mass Spectrometry (Part 1A - Recommendations on Advanced Strategies for Mass Spectrometry Assays, Chromatography, Sample Preparation and BMV/Regulated Bioanalysis Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV).

The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 1) covers in Part 1A the Recommendations on Mass Spectrometry Assays and Regulated Bioanalysis/BMV and in Part 1B the Regulatory Inputs on these topics. Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 3 and 4 (2025), respectively.

2024 White paper on recent issues in bioanalysis: Impact of LDT in US and IVDR in EU; AI/ML for High Parameter Flow Cytometry; The rise of Olink Technology; CDx for AAV Gene Therapies; Integrative Bioanalysis by Multiple Platforms; Super Sensitive ADA/NAb LBA (PART 2A - Recommendations on Advanced Strategies for Biomarkers, IVD/CDx Assays (BAV), Cell Based Assays (CBA), and Ligand-Binding

The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers in the Part 2A the recommendations on Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays and in Part 2B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 17 of Bioanalysis, issues 5 and 3 (2025), respectively.

2024 White Paper on Recent Issues in Bioanalysis: Evolution of Immunogenicity Assessment beyond ADA/NAb; Regulated Genomic/NGS Assays; Hypersensitivity Reactions; Minimum Noise Reduction; False Positive Range; Modernized Vaccine Approaches; NAb/TAb Correlation (PART 3A - Recommendations on Advanced Strategies for Molecular Assays and Immunogenicity of Gene Therapy, Cell Therapy, Vaccine; Biotherapeutics Immunogenicity Assessment & Clinical Relevance PART 3B - Regulatory Agencies' Input

The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agencies experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 3) covers in the Part 3A the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 4 and 5 (2025), respectively.

Enhancing DNA Vaccine Delivery Through Stearyl-Modified Cell-Penetrating Peptides: Improved Antigen Expression and Immune Response In Vitro and In Vivo.

Inefficient cellular uptake is a significant limitation to the efficacy of DNA vaccines. In this study, we introduce S-Cr9T, a stearyl-modified cell-penetrating peptide (CPP) designed to enhance DNA vaccine delivery by forming stable complexes with plasmid DNA, thereby protecting it from degradation and promoting efficient intracellular uptake. In vitro studies showed that S-Cr9T significantly improved plasmid stability and transfection efficiency, with optimal performance at an N/P ratio of 0.25. High-content imaging revealed that the S-Cr9T-plasmid complex stably adhered to the cell membrane, leading to enhanced plasmid uptake and transfection. In vivo, S-Cr9T significantly increased antigen expression and triggered a robust immune response, including a threefold increase in IFN-γ secretion and several hundred-fold increases in antibody levels compared to control groups. These findings underscore the potential of S-Cr9T to enhance DNA vaccine efficacy, offering a promising platform for advanced gene therapy and vaccination strategies.

Automated Microfluidic Systems Facilitating the Scalable and Reliable Production of Lipid Nanoparticles for Gene Delivery

AbstractLipid nanoparticles have become a promising agent for biocompatible, effective, stable, and clinically approved delivery system that protect the nucleic acid from degradation while allowing cellular uptake and release of nucleic acid. Herein, we report an advanced microfluidic device and system for synthesis of uniform shape and size of mRNA encapsulated LNPs. The device consists of five repeated donut ring structures with two inlets and one outlet microfluidic channels which are assigned for organic phase solution, aqueous phase solution, and LNP recovery, respectively. It also has a minimum microfluidic channel width of 100 μm and can produce LNPs with approximately diameter from 70 to 190 nm. These LNPs can be produced less than 2 min after loading of reagents in the instrument. To verify the practical applicability of the as-developed instrument, mRNA expressing enhanced green fluorescence protein (EGFP) is encapsulated in LNPs. The gene expression of mRNA encapsulated LNPs confirmed the mechanical and chemical stability of the LNPs and its capability of gene delivery. Based on the results, the microfluidic device and system can provide a simple, fast, reliable, easy-to-use, cost-effective, and reproducible way to be applicable in LNP-based gene therapies and vaccines for treating various diseases.

Analysis of the heterologous expression, localization, and cellular response to the Zika virus E protein in vitro.

Zika virus (ZIKV) infection has been associated with damage to neural stem cells in microcephaly in newborns. The virus possesses specific tropism for glioma stem cells mediated by the ZIKV E protein. This infection causes endoplasmic reticulum stress and activation of the unfolded protein response (UPR). However, the cellular response to the expression of the ZIKV E protein alone is unknown. Therefore, in this study, we determined the effect of the expression of the ZIKV E protein on cellular responses and its subcellular localization in HEK-293T cells, due to their use as a biotechnological tool for cellular and lentiviral therapy. We observed that the ZIKV E protein is synthesized in the cytoplasm and inserted into the endoplasmic reticulum (ER), without causing activation of the UPR or cell death, and it is finally transported and located in the cell membrane. Moreover, the expression of the ZIKV E protein does not induce UPR or apoptosis in glioma cells. These results help us to better understand the characteristics of this protein and its possible use as a biotechnological tool for the creation of different gene therapy strategies, vaccines, and synthetic vectors with tropism for neural and glioma stem cells.

Transcriptome-based analysis of the molecular mechanism of recombinant protein expression in Periplaneta americana cells.

The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is widely used for the generation of a variety of gene products, including proteins, vaccines, and gene therapy vectors; however, it has some limitations, including a constrained host range and low protein yields. In a previous study, we established the RIRI-PA1 cell line, which was derived from Periplaneta americana. This cell line is susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection, which results in a higher yield production of recombinant protein within a short post-infection period of 24-48 h compared to the commonly used engineered cell line Sf21. To elucidate the basis for this phenomenon, we used RNA sequencing and transcriptome analysis of RIRI-PA1 and Sf21 cells infected with AcMNPV-GFP at 24, 72, and 168 h post-infection. Differentially expressed genes (DEGs) were identified in both cell lines. GO, eggNOG, and KEGG annotation analyses were used to identify DEGs and select candidate genes that could regulate recombinant protein expression. The results indicated a significant link between ribosomal pathway regulation and recombinant protein expression. After 24 h of AcMNPV-GFP infection, relatively high levels of protein were produced in RIRI-PA1 cells compared to Sf21 cells, which exhibited lesser enrichment of ribosomal protein-related DEGs (7 : 12). Moreover, a correlation was observed in the gene expression patterns between AcMNPV-GFP infection and recombinant protein synthesis, including genes associated with the ribosome, Toll and Imd signaling, and the cytochrome P450 pathway. Overall, our findings suggested that the ribosomal pathway might be more involved in regulation of protein expression during the early stages of RIRI-PA1 infection. The mechanisms underlying this process could have potential future applications in engineering cell modifications to reduce production time for recombinant proteins and to promote the use of IC-BEVS.

Attenuated Getah virus confers protection against multiple arthritogenic alphaviruses.

Alphaviruses are important arthropod-transmitted pathogens of humans and livestock. Getah virus (GETV) is an arthritogenic alphavirus that causes disease in horses and piglets; it also poses a potential threat to humans. A live attenuated vaccine candidate named GETV-3ΔS2-CM1, harbouring a deletion in nonstructural protein 3 and substitutions in the capsid protein, is genetically stable and exhibits robust immunogenicity. It was shown to confer passive protection to piglets born to immunized sows. In mice, a single dose of GETV-3ΔS2-CM1 protected against infection with different strains of GETV, Semliki Forest virus, Ross River virus, o'nyong'nyong virus, chikungunya virus, and Barmah Forest virus. Chimaeras based on the GETV-3ΔS2-CM1 backbone maintained both the attenuated phenotype and high immunogenicity. The safety, efficacy, and ability to induce protection against multiple alphaviruses highlights the potential of GETV-3ΔS2-CM1 and chimaeras using this backbone as promising vaccine candidates. By contributing simultaneously to the wellbeing of animals and humans, our universal next generation vaccine strategy helps to achieve "One Health" goals.

Recombinant VLP vaccines synthesized in plant expression systems: current updates and prospects

The development and creation of a new generation vaccines based on recombinant proteins that assemble into virus-like particles (VLPs), as well as recombinant proteins in the form of nanoparticles are promising directions in modern biotechnology. Due to their large size (20–200 nm) and a multiplicity of viral antigenic determinants on the surface, VLPs can stimulate strong humoral and cellular immune responses. The review considered the main types of VLPs, as well as the features and disadvantages of the main expression systems used for their biosynthesis. The main focus was on plant expression systems that ensure the biosynthesis of a target recombinant protein from a DNA matrix integrated into the nuclear or chloroplast genomes of a plant (stable expression) or from a matrix for temporary production of the target product (transient expression). Various approaches for increasing the yield of VLP-forming recombinant proteins, including fusion with a transit peptide that directed the protein into the chloroplast, were discussed. The possibility of accumulation of recombinant proteins expressed in plants and intended for creation of VLP-vaccines in another type of nanoparticles, protein bodies, using specific signal sequences was also considered.

Coxiella burnetii Nine Mile phase I primary infection derived protective immunity against C. burnetii reinfection in mice depends on both B and T cells, but T cells play a critical role.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes acute and chronic Q fever in humans. Acute Q fever is usually a flu-like, self-limiting or treatable illness, but some infections can turn into a severe and sometimes fatal chronic disease. There is currently no FDA-approved vaccine available for the prevention of human Q fever in the US, development of a safe and effective vaccine for the prevention of human Q fever remains an important goal for public health. However, there is a fundamental gap in knowledge regarding the mechanism of protective immunity against C. burnetii infection. To understand the mechanism of C. burnetii infection induced protective immunity, we examined if C. burnetii Nine Mile phase I (NMI) infection induces protection against C. burnetii reinfection in mice. Our results indicate that NMI-infected mice conferred significant protection against C. burnetii reinfection. We also found that NMI infection derived protection did not depend on the routes of infection and antibodies are required for NMI infection derived protection. In addition, NMI infection elicited a comparable level of protection in Wild type, CD4+ T cell deficient, and CD8+ T cell deficient mice, partial protection in B cell deficient mice but no protection in T cell deficient mice. These results suggest that both B cells and T cells are required for NMI-infection derived protection, but T cells may play a critical role. Therefore, the new generation vaccine for the prevention of human Q fever should be focused on boosting both humoral and T cell immune responses.

Revolutionizing Veterinary Health with Viral Vector-Based Vaccines.

Vaccines signify one of the economical and reasonable means to prevent and eradicate the important infectious diseases. Conventional vaccines like live attenuated and inactivated vaccines comprise of whole pathogen either in attenuated or killed form. While, new generation vaccines have been designed to elicit immune response by genetically modifying only the nucleic acid portion of that pathogen. These new generation therapeutics include mRNA vaccines, DNA plasmid vaccines, chimeric vaccines and recombinant viral vector-based vaccines. Nucleic acid based vaccines use genetic material itself thus, they are highly stable and potent in nature to induce long-lasting immune response. Amongst these novel vaccine platforms, viral vector-based vaccines is one such emerging field which has proven to be extremely effective and potent. Nowadays, veterinary medicine has also accepted this innovative vectored vaccine platform to develop an effective control strategy against certain important viral diseases of animals. Viral vector-based vaccine uses various DNA and RNA viruses of human or animal origin to carry an immunogenic transgene of target pathogen. These vaccines enhance both humoral and cell mediated immune response without use of any accessory immune-stimulants. Till today, several viruses have been modified to be characterized as vaccine vectors. Currently, large number of research programs are going on to develop vectored vaccines and novel viral vector for veterinary use. In the present review, different kinds of viral vectored vaccines having veterinary importance have been discussed.

Baculoviruses in Integrated Pest Management of Fall Armyworm, (Spodoptera frugiperda) (Lepidoptera: Noctuidae): Structure, Classification and Application

Baculoviruses are crucial biological agents in integrated pest management (IPM), particularly for controlling lepidopteran pests in agriculture. These are highly specific, eco-friendly viruses characterized by rod-shaped nucleocapsids containing a protein-encased genome. The Baculoviridae family comprises four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus, each targeting specific insect orders. Nucleo polyhedron virus (NPVs) and granuloviruses (GVs) are extensively used in pest management due to their high virulence and specificity, ensuring safety for non-target organisms. Ascoviruses are dsDNA viruses that infect Spodoptera frugiperda larvae, transmitted primarily by parasitoids. Despite high virulence, their use in pest control is limited due to ineffective oral transmission. Combining ascoviruses with other pathogens, such as Bacillus thuringiensis kurstaki (Btk), may enhance their pest control potential. The infection process begins when insect larvae ingest viral occlusion bodies, leading to systemic infection and host death. Despite their narrow host range and slower action compared to chemical insecticides, baculoviruses are invaluable in sustainable agriculture. Their application has expanded beyond pest control to include gene therapy and vaccine production. This review examines the structure, classification, infection process, and current status of baculoviruses in controlling the fall armyworm, Spodoptera frugiperda, a significant agricultural pest. By exploring their potential as a cornerstone in biological pest management, we highlight the importance of baculoviruses in developing sustainable and environmentally friendly pest control strategies. The integration of baculoviruses into IPM programs offers a promising approach to reduce chemical pesticide use, preserve biodiversity, and enhance crop protection, ultimately contributing to more sustainable agricultural practices and food security.

Safety pharmacology of human endogenous retrovirus-enveloped baculoviral DNA vaccines against SARS-CoV-2 in Sprague-Dawley rats and beagle dogs

The coronavirus disease 2019 (COVID-19) emerged as a major global health crisis, posing significant health, economic, and social challenges. Vaccine development has been a crucial response to the severe-acute-respiratory-syndrome-related coronavirus-2 pandemic owing to the critical role of immunization in controlling infectious diseases, leading to the expedited development of several effective vaccines. Although mRNA platform-based COVID-19 vaccines authorized under emergency-use authorization have been administered globally, concerns regarding the vaccines have increased owing to the occurrence of various side effects. The present study aimed to evaluate the safety of a non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) vaccine encoding SARS-CoV-2 antigens. Owing to the limited number of existing safety pharmacology studies on AcHERV as a viral vector vaccine, we conducted neurobehavior (Modified Irwin’s Test), body temperature, and respiratory function studies in rats and cardiovascular system studies in male beagle dogs, which were administered the AcHERV-COVID-19 vaccine using telemetry. The safety assessment revealed no significant toxicological alterations. However, in rats, both sexes administered with the AcHERV-COVID-19 vaccine exhibited a temporary increase in body temperature, which normalized or showed signs of recovery. In conclusion, AcHERV-COVID-19 demonstrates a sufficient safety profile that supports its potential evaluation in future clinical trials.

Safety and Efficacy of Antiviral Drugs and Vaccines in Pregnant Women: Insights from Physiologically Based Pharmacokinetic Modeling and Integration of Viral Infection Dynamics.

Addressing the complexities of managing viral infections during pregnancy is essential for informed medical decision-making. This comprehensive review delves into the management of key viral infections impacting pregnant women, namely Human Immunodeficiency Virus (HIV), Hepatitis B Virus/Hepatitis C Virus (HBV/HCV), Influenza, Cytomegalovirus (CMV), and SARS-CoV-2 (COVID-19). We evaluate the safety and efficacy profiles of antiviral treatments for each infection, while also exploring innovative avenues such as gene vaccines and their potential in mitigating viral threats during pregnancy. Additionally, the review examines strategies to overcome challenges, encompassing prophylactic and therapeutic vaccine research, regulatory considerations, and safety protocols. Utilizing advanced methodologies, including PBPK modeling, machine learning, artificial intelligence, and causal inference, we can amplify our comprehension and decision-making capabilities in this intricate domain. This narrative review aims to shed light on diverse approaches and ongoing advancements, this review aims to foster progress in antiviral therapy for pregnant women, improving maternal and fetal health outcomes.

Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses.

Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.

The BCG vaccine, advantages, and disadvantages of introducing new generation vaccines against Mycobacterium tuberculosis.

Tuberculosis (TB) is consistently ranked among the deadliest diseases worldwide, causing millions of deaths annually. Mycobacterium tuberculosis is the causative agent for this infection. Different antibiotics and vaccines have been discussed as potential treatments and prevention. Currently, there is only one licensed vaccine against TB, Bacillus Calmette-Guérin (BCG). Despite its protective efficacy against TB in children, BCG has failed to protect adults against pulmonary TB, lacks therapeutic value, and can cause complications in immunocompromised individuals. In this review, BCG, the most widely administered vaccine, is discussed, and the newest vaccines available in medicine are discussed. Based on the restrictions that prevent optimal BCG efficacy and the vaccines that are now being tested in various clinical studies, some criteria need to be considered in designing future vaccines.

The Immune System-A Double-Edged Sword for Adenovirus-Based Therapies.

Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine, and oncology applications, Ad-based platforms offer advantages, including ease of genetic manipulation, scale of production, and well-established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that must be overcome for adenovirus-based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects on the downstream impact of an adenovirus-based therapeutic. This review focuses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, in the context of innate and adaptive immune responses. We summarise different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.

The presence of broadly neutralizing anti-SARS-CoV-2 RBD antibodies elicited by primary series and booster dose of COVID-19 vaccine.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design.

Active immunization with inhibin DNA vaccine promotes spermatogenesis and testicular development in rats

ABSTRACT The objective of the present study was to investigate the potential role of immunization against INH on testicular development, serum reproductive hormone (FSH, LH and T) concentrations, testicular biochemical indexes (GGT, ALP, ACP, LDH, α-neutral glucosidase and carnitine) and testicular cell apoptosis in rats. Forty healthy male rats were randomly divided into four groups (n = 10 per group), and respectively immunized with 0.5 mL of C500/pVAX-asd-IS-L (108 CFU/ mL), C500/pVAX-asd-IS-M (109 CFU/ mL), C500/pVAX-asd-IS-H (1010 CFU/ mL) or C500 (1010 CFU/ mL) (thrice with an interval of 2 weeks). P/N values increased after vaccination and differed (p < 0.05) at 1 week and sharply increased at 2 weeks following the booster vaccination (p < 0.01); P/N values indicated that the immune response was induced and production of anti-INH antibodies in rats; Immunization of INH vaccines enhanced serum concentrations of T and FSH, while reduced LH concentrations (p < 0.05); The testis weight, volume, testis coefficient, epididymal weight, sperm count in the epididymis, and the sperm deformity results indicated that INH gene vaccine immunization could promote testicular development and enhance sperm production rate in rats. After immunization of INH gene vaccines, GGT activity was markedly increased, but ALP, LDH and α-neutral glucosidase activities were observably reduced (p < 0.05). Furthermore, flow cytometry results demonstrated that the apoptosis rate of the testicular cells was extremely increased in the C500/pVAX-asd-IS-M group (p < 0.01). These findings illustrate a functional cross-link between FSH, T, LH and spermatogenic markers via inhibin to maintain spermatogenesis. INH immunization may have a positive effect on spermatogenesis and testicular development in male rats.

Grafting chromatographic monoliths with charged linear polymers for highly productive and selective protein or plasmid DNA purification

Increased requirements for plasmid DNA (pDNA) in gene therapy and vaccination efforts bring the need for efficient large-scale production processes with high purity and productivity. Chromatographic purification of pDNA is often a bottleneck due to low dynamic binding capacities (DBC), pDNA recovery and/or low selectivity of columns. Ion exchangers (IEX) with charged groups on surface extenders (polyions formed by a grafting reaction and subsequents modification) have attracted attention due to their greatly enhanced productivity compared with traditional IEX. The ultimate goal of our investigation was to increase the DBC for pDNA on a monolith stationary phase by grafting the surface with linear polymethacrylate brushes (grafted chains), while retaining high pDNA recovery and chromatographic selectivity between pDNA and RNA impurities. The inner walls of the Convective Interaction Media® monolith channels were successfully coated with polyglycidyl methacrylate linear chains of varying densities and lengths by employing a controlled radical polymerization reaction. Glycidyl groups throughout the length of the formed grafted chains were converted to cation- (CEX) or anion-exchanging (AEX) moieties. Preliminary chromatographic evaluation of novel grafted CEX columns for different model proteins exhibited 10-times higher DBC with high elution recovery (99 %). Similar DBC improvement was proven for 7.3 kilobase pair pDNA, where the DBC10 increased from 1.8 mg pDNA per mL of non-grafted chromatographic support up to 15.2 mg per the most densely distributed and the longest grafted chains, modified with weak AEX groups. However, the DBC increase was partially at the expense of pDNA elution recovery, which presumably decreased due to entanglement of pDNA molecules inside the dense grafted layer. To confirm the hypothesis, we have prepared a series of columns differing in length and density of grafted chains and tested them for pDNA capture. A satisfactory compromise between high pDNA DBC and elution recovery was found with relatively long and low density grafted chains. The optimal grafted AEX column with 1 mL bed volume was evaluated for pDNA capture from neutralized E. coli lysate. With a capacity of 13.5 mg pDNA per mL support, ≥95 % elution recovery, and complete RNA clearance, the pDNA was successfully purified at loading flow rate of at least 15 column volumes per min. According to our knowledge and literature search, these process characteristics would enable one of the highest pDNA isolation productivities to be achieved with currently tested IEX.