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An effective vaccine against hepatitis C virus (HCV) should elicit both humoral and cellular immune responses. Previously, we characterized a bivalent vaccine candidate against hepatitis B (HBV) and HCV using chimeric HBV-HCV virus-like particles (VLP), in which the highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of HBV small surface antigen (sHBsAg). While sHBsAg_412–425 elicited cross-neutralizing antibodies, it did not trigger a T-cell response against HCV. Thus, this study aimed to develop a vaccine candidate engaging both arms of adaptive immune response, potentially offering stronger protection against HCV. We evaluated the immunogenicity of minicircle (MC) DNA vaccines encoding sHBsAg_412–425 and HCV nonstructural (NS) proteins in BALB/c mice. Co-administration of sHBsAg_412–425 and NS induced a potent T-cell response, especially against NS3 and high titers of antibodies specific to HCV E2. Additionally, these antibodies recognized native HCV envelope glycoprotein heterodimers (E1E2) across multiple HCV genotypes and showed binding profiles to E1E2 alanine mutants comparable to the broadly neutralizing AP33 antibody. Overall, the findings demonstrate that MC DNA vaccine incorporating both sHBsAg_412–425 and HCV NS protein sequences induces robust, T-cell and AP33-like antibody responses, highlighting its potential as pan-genotypic prophylactic vaccine against HCV. [ABSTRACT FROM AUTHOR]

In the recent history of the SARS-CoV-2 outbreak, vaccines have been a crucial public health tool, playing a significant role in effectively preventing infections. However, improving the efficacy while minimizing side effects remains a major challenge. In recent years, there has been growing interest in nanoparticle-based delivery systems aimed at improving antigen delivery efficiency and immunogenicity. Among these, self-assembled nanoparticles with varying sizes, shapes, and surface properties have garnered considerable attention. This paper reviews the latest advancements in the design and development of SARS-CoV-2 vaccines utilizing self-assembled materials, highlighting their advantages in delivering viral immunogens. In addition, we briefly discuss strategies for designing a broad-spectrum universal vaccine, which provides insights and ideas for dealing with possible future infectious sarbecoviruses. [ABSTRACT FROM AUTHOR]

In recent years, visible light positioning (VLP) techniques have been gaining popularity in research. Among them, the scheme of using a camera as a receiver provides a low-cost, high-precision positioning capability and easy integration with existing multimedia devices and robots. However, the pose changes of the receiver can lead to image distortion and light displacement, significantly increasing positioning errors. Addressing these errors is crucial for enhancing the accuracy of VLP. Most current solutions rely on gyroscopes or Inertial Measurement Units (IMUs) for error optimization, but these approaches often add complexity and cost to the system. To overcome these limitations, we propose a 3D positioning algorithm based on an attention mechanism convolutional neural network (CNN) aimed at reducing the errors caused by angles. We designed experiments and comparisons within a rotation angle range of ±15 degrees. The results demonstrate that the average error for 2D positioning is within 5.74 cm and the height error is within 3.92 cm, and the average error for 3D positioning is within 6.8 cm. Among the four groups of experiments for 3D positioning, compared to the traditional algorithm, the improvements were 7.931 cm, 15.569 cm, 6.004 cm, and 16.506 cm. The experiments indicate that it can be applied to high-precision visible light positioning for single-light imaging. [ABSTRACT FROM AUTHOR]

Abstract Cervical cancer is the fourth most prevalent cancer among women globally, with Thai women ranking it as the third most common. At present, a prophylactic vaccine, containing virus-like particles (VLPs) of HPV L1 capsid protein, is widely recognized as one of the major prevention strategies for cervical cancer. Unfortunately, due to a low cross-protection among subtypes, protection against each HPV subtype requires vaccination with VLPs of that specific subtype. This study aimed to optimize the cultivation medium and conditions to maximize the volumetric yield of HPV52 L1 protein produced by the recombinant H. polymorpha HPV52. The results revealed that supplementation of a complex organic nitrogen source HySoy (at 10 g/L) into SYN6 medium resulted in a significantly higher specific HPV52 L1 yield and the maximum specific and volumetric HPV52 L1 protein yield were obtained at 30℃. In this study, the volumetric yield of HPV52 L1 could be increased from 50.9 mg/L in flask-scale cultivation to 2728 mg/L in High Cell Density Cultivation or HCDC (53-folds) with a significant improvement in terms of productivity, from 0.85 mg/L.h in flask-scale cultivation to 22.7 mg/L.h in HCDC (27-folds).

Abstract An effective vaccine against hepatitis C virus (HCV) should elicit both humoral and cellular immune responses. Previously, we characterized a bivalent vaccine candidate against hepatitis B (HBV) and HCV using chimeric HBV-HCV virus-like particles (VLP), in which the highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of HBV small surface antigen (sHBsAg). While sHBsAg_412–425 elicited cross-neutralizing antibodies, it did not trigger a T-cell response against HCV. Thus, this study aimed to develop a vaccine candidate engaging both arms of adaptive immune response, potentially offering stronger protection against HCV. We evaluated the immunogenicity of minicircle (MC) DNA vaccines encoding sHBsAg_412–425 and HCV nonstructural (NS) proteins in BALB/c mice. Co-administration of sHBsAg_412–425 and NS induced a potent T-cell response, especially against NS3 and high titers of antibodies specific to HCV E2. Additionally, these antibodies recognized native HCV envelope glycoprotein heterodimers (E1E2) across multiple HCV genotypes and showed binding profiles to E1E2 alanine mutants comparable to the broadly neutralizing AP33 antibody. Overall, the findings demonstrate that MC DNA vaccine incorporating both sHBsAg_412–425 and HCV NS protein sequences induces robust, T-cell and AP33-like antibody responses, highlighting its potential as pan-genotypic prophylactic vaccine against HCV.

Circular RNAs (circRNAs) are a class of single-stranded RNAs with a closed loop lacking 5ʹ and 3ʹ ends. These circRNAs are translatable and, therefore, have a potential in developing vaccine. CircRNA vaccines have been shown to be more stable, safe, easy to manufacture and scale-up production when compared to mRNA vaccines. However, these vaccines also suffer from several drawbacks such as low circularization efficiency for longer RNA precursor and usage of lipid nano particles (LNPs) in their delivery. LNPs have been shown to require large amounts of RNA due to their indirect delivery from endosome to cytosol. Besides, individual components of LNPs provide reactogenicity. Usage of virus like particles (VLPs) can improve the increased production and targeted delivery of circRNA vaccines and show no reactogenicity. Moreover, VLPs has also been used to produce vaccines against several diseases such as hepatitis C virus (HCV) etc. In this article, we will discuss about the methods used to enhance synthesis or circularization efficiency of circRNA. Moreover, we will also discuss about the significance of VLPs as the delivery vehicle for circRNA and their possible usage as the dual vaccine.

Canan Bayraktar,1,* Alisan Kayabolen,1,* Arda Odabas,1 Aysegul Durgun,1 Ipek Kok,1 Kenan Sevinc,1 Aroon Supramaniam,2 Adi Idris,2,3 Tugba Bagci-Onder1 1Koç University Research Center for Translational Medicine (KUTTAM), Koç University, Istanbul, Turkey; 2Menzies Health Institute Queensland, School of Medical Science Griffith University, Gold Coast Campus, Brisbane, QLD, Australia; 3Centre for Immunology and Infection Control, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia*These authors contributed equally to this workCorrespondence: Tugba Bagci-Onder; Alisan Kayabolen, Email tuonder@ku.edu.tr; akayabol@mit.eduPurpose: Over the past three years, extensive research has been dedicated to understanding and combating COVID-19. Targeting the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor has emerged as a promising therapeutic strategy against SARS-CoV-2. This study aimed to develop ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus.Methods: Virus-like particles were generated through the utilization of a packaging plasmid in conjunction with a plasmid containing the ACE2 envelope sequence. Subsequently, ACE2-VLPs and ACE2-EVs were purified via ultracentrifugation. The quantification of VLPs was validated through multiple methods, including Nanosight 3000, TEM imaging, and Western blot analysis. Various packaging systems were explored to optimize the ACE2-VLP configuration for enhanced neutralization capabilities. The evaluation of neutralization effectiveness was conducted using pseudoviruses bearing different spike protein variants. Furthermore, the study assessed the neutralization potential against the Omicron BA.1 variant in Vero E6 cells.Results: ACE2-VLPs showed a high neutralization capacity even at low doses and demonstrated superior efficacy in in vitro pseudoviral assays compared to extracellular vesicles carrying ACE2. ACE2-VLPs remained stable under various environmental temperatures and effectively blocked all tested variants of concern in vitro. Notably, they exhibited significant neutralization against Omicron BA.1 variant in Vero E6 cells. Given their superior efficacy compared to extracellular vesicles and proven success against live virus, ACE2-VLPs stand out as crucial candidates for treating SARS-CoV-2 infections.Conclusion: This novel therapeutic approach of coating VLPs with receptor particles provides a proof-of-concept for designing effective neutralization strategies for other viral diseases in the future.Keywords: ACE2, virus like particles, SARS-CoV-2, neutralization, VLP, neutralization, escape mutations, spike protein

Abstract Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake. • Antibody Fc can be surface-displayed on virions and virus-like particles (VLPs). • Fc-displaying viruses/VLPs undergo enhanced uptake by antigen-presenting cells. • Imaging cytometry depicts enhanced Fc-displayed virus internalization. • Loading antigen-compatible design can harness immunization regimens. [ABSTRACT FROM AUTHOR]

Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of the ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). For VLP production, rVVs expressing Env, Gag-Pol (HIV-1/SIV), and the cowpox virus hr gene, which overcomes the restriction of vaccinia virus replication in CHO cells, were used. The CHO Lec1 engineered cell line lacking GlcNAc-TI was used for generating VLPs with Env proteins containing a cytoplasmic (CT) domain affecting the surface subunit (SU) conformation. This has created the opportunity to modulate the glycan composition, and refine the conditions for their production, and optimize approaches to overcoming HIV-1 resistance associated with abundant glycosylation. [ABSTRACT FROM AUTHOR]

A vaccine protecting against malaria caused by Plasmodium falciparum is urgently needed. The blood-stage invasion complex PCRCR consists of the five malarial proteins PfPTRAMP, PfCSS, PfRipr, PfCyRPA, and PfRH5. As each subcomponent represents an essential and highly conserved antigen, PCRCR is considered a promising vaccine target. Furthermore, antibodies targeting the complex can block red blood cell invasion by the malaria parasite. However, extremely high titers of neutralizing antibodies are needed for this invasion-blocking effect, and a vaccine based on soluble PfRH5 protein has proven insufficient in inducing a protective response in a clinical trial. Here, we present the results of two approaches to increase the neutralizing antibody titers: (A) immunofocusing and (B) increasing the immunogenicity of the antigen via multivalent display on capsid virus-like particles (cVLPs). The immunofocusing strategies included vaccinating with peptides capable of binding the invasion-blocking anti-PfCyRPA monoclonal antibody CyP1.9, as well as removing non-neutralizing epitopes of PfCyRPA through truncation. Vaccination with PfCyRPA coupled to the AP205 cVLP induced nearly two-fold higher IgG responses compared to vaccinating with soluble PfCyRPA protein. Immunofocusing using a linear peptide greatly increased the neutralizing capacity of the anti-PfCyRPA antibodies. However, significantly lower total anti-PfCyRPA titers were achieved using this strategy. Our results underline the potential of a cVLP-based malaria vaccine including full-length PfCyRPA, which could be combined with other leading malaria vaccine antigens presented on cVLPs. [ABSTRACT FROM AUTHOR]

Herpes zoster (HZ), also known as shingles, remains a significant global health issue and most commonly seen in elderly individuals with an early exposure history to varicella-zoster virus (VZV). Currently, the licensed vaccine Shingrix, which comprises a recombinant VZV glycoprotein E (gE) formulated with a potent adjuvant AS01B, is the most effective shingles vaccine on the market. However, undesired reactogenicity and increasing global demand causing vaccine shortage, prompting the development of novel shingles vaccines. Here, we developed novel vaccine candidates utilising multiple nanoparticle (NP) platforms to display the recombinant gE antigen, formulated in an MF59-biosimilar adjuvant. In naïve mice, all tested NP vaccines induced higher humoral and cellular immune responses than Shingrix, among which, the gEM candidate induced the highest cellular response. In live attenuated VZV (VZV LAV)-primed mouse and rhesus macaque models, the gEM candidate elicited superior cellmediated immunity (CMI) over Shingrix. Collectively, we demonstrated that NP technology remains a suitable tool for developing shingles vaccine, and the reported gEM construct is a highly promising candidate in the next-generation shingles vaccine development. [ABSTRACT FROM AUTHOR]

Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells. [ABSTRACT FROM AUTHOR]

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells. [ABSTRACT FROM AUTHOR]

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression. [ABSTRACT FROM AUTHOR]

In this paper, we consider a visible light positioning (VLP) system, where an array of photo diodes combined with apertures is used as a directional receiver and a set of inexpensive and energy-efficient light-emitting diodes (LEDs) is used as transmitters. The paper focuses on the optimisation of the layout of the transmitter, i.e., the number and placement of the LEDs, to meet the wanted position estimation accuracy levels. To this end, we evaluate the Cramer–Rao bound (CRB), which is a lower bound on the mean-squared error (MSE) of the position estimate, to analyse the influence of the LEDs' placement. In contrast to other works, where only the location of the LEDs was considered and/or the optimisation was carried out through simulations, in this work, the optimisation is carried out analytically and considers all the parameters involved in the VLP system as well as the illumination. Based on our results, we formulate simple rules of thumb with which we can determine the spacing between LEDs and the minimum number of LEDs, as well as their position on the ceiling, while also taking into account the requirements for the illumination. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a unique asexual blood-stage malaria vaccine candidate because of its high conservation and essential biological function of binding to basigin on the erythrocyte surface. Recent studies by Barrett et al. , Wang et al. , and King et al. , have brought RH5-based vaccine development a step forward based on a rational antigen design strategy. [ABSTRACT FROM AUTHOR]

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system. • 1:2 dilution of the culture one day after infection is most resource-efficient. • Infection strategy suitable for large scale. • Reducing seed virus by a factor of 20 in comparison to reference process. • Sf 9 HCD culture for low-footprint cell proliferation reduces volume by > 60%. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes. [ABSTRACT FROM AUTHOR]

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP–cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research. [ABSTRACT FROM AUTHOR]

A number of endogenous genes in the human genome encode retroviral gag-like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen (PNMA) family members encode a gag-like capsid domain, but their ability to assemble as capsids and traffic between cells remains mostly uncharacterized. Here, we systematically investigate human PNMA proteins and find that a number of PNMAs are secreted by human cells. We determine that PNMA2 forms icosahedral capsids efficiently but does not naturally encapsidate nucleic acids. We resolve the cryoelectron microscopy (cryo-EM) structure of PNMA2 and leverage the structure to design engineered PNMA2 (ePNMA2) particles with RNA packaging abilities. Recombinantly purified ePNMA2 proteins package mRNA molecules into icosahedral capsids and can function as delivery vehicles in mammalian cell lines, demonstrating the potential for engineered endogenous capsids as a nucleic acid therapy delivery modality. [ABSTRACT FROM AUTHOR]

The rapid growth in the prevalence of infectious diseases requires timely action from drug developers. In recent years, the COVID-19 pandemic has demonstrated the unpreparedness of the population for such emergencies. The introduction of modern methods of Design of Experiments (DoE) is required to accelerate the process of drug development and bring a drug to market. The main objective of this study was to develop an ion-triggered in situ system for intranasal delivery of VLP using a Quality by Design approach. Based on a literature review and initial studies, the key QTPP, CQA, CPP, and CMA were identified to develop a novel delivery system for virus-like particles. As a result of the studies on the quality attributes of the developed delivery system, an ion-triggered in situ gel meeting all the specified parameters was obtained using the Quality by Design method. [ABSTRACT FROM AUTHOR]

The development of HIV prophylactic vaccines is facing an impasse, since all phase IIb/III clinical trials were halted in 2023 without demonstrating efficacy. Thus, the field is in need of developing novel immunogens and vaccination strategies that induce broadly neutralising antibodies together with potent Fc-dependent effector functions, as well as protective cross-reactive CD4+ and CD8+ T cell responses. Nucleic acid vaccines, particularly mRNA vaccines, have been one of the major groundbreaking advances in the current decade. Nucleic acid vaccines may help recalibrate the HIV vaccine field towards the use of delivery systems that allow the proper expression of immunogens as a sole antigen (i.e., membrane-bound trimeric envelope glycoproteins) or even to be displayed in a multiantigen platform that will be synthesised by the host. In this review, we will summarise how the multiple HIV vaccine strategies pursued in the last 40 years of HIV research have driven current vaccine development, which are the most relevant immunogens identified so far to induce balanced adaptive immune responses, and how they can benefit from the acceptance of nucleic acid vaccines in the market by reducing the limitations of previous delivery systems. The incorporation of nucleic acid vaccines into the current heterogeneous repertoire of vaccine platforms may represent an invaluable opportunity to reignite the fight against HIV. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2–220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low. [ABSTRACT FROM AUTHOR]

ABSTRACT The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays.IMPORTANCEThe study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

Studies on the human virome based on the application of metagenomic approaches involve overcoming a series of challenges and limitations inherent not only to the biological features of viruses, but also to methodological pitfalls which different approaches have tried to minimize. These approaches fall into two main categories: bulk-metagenomes and virus-like particle (VLP) enrichment. In order to address issues associated with commonly used experimental procedures to assess the degree of reliability, representativeness, and reproducibility, we designed a comparative analysis applied to three experimental protocols, one based on bulk-metagenomes and two based on VLP enrichment. These protocols were applied to stool samples from 10 adult participants, including two replicas per protocol and subject. We evaluated the performances of the three methods, not only through the analysis of the resulting composition, abundance, and diversity of the virome via taxonomical classification and type of molecule (DNA versus RNA, single stranded vs. double stranded), but also according to how the a priori identical replicas differed from each other according to the extraction methods used. Our results highlight the strengths and weaknesses of each approach, offering valuable insights and tailored recommendations for drawing reliable conclusions based on specific research goals. [ABSTRACT FROM AUTHOR]

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE–Rev module to enhance the packaging of Cas9 nuclease into VLPs. [ABSTRACT FROM AUTHOR]

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported, but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compound in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV depletion from the extracellular medium restored transfection efficiency in conditions that suffer from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UDP-glucose ceramide glucosyltransferase (UGCG) enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12 × 106 cells/mL. These results suggest that the interaction between polyplexes and extracellular and intracellular vesicles plays a crucial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.

In recent years, visible light positioning (VLP) techniques have been gaining popularity in research. Among them, the scheme of using a camera as a receiver provides a low-cost, high-precision positioning capability and easy integration with existing multimedia devices and robots. However, the pose changes of the receiver can lead to image distortion and light displacement, significantly increasing positioning errors. Addressing these errors is crucial for enhancing the accuracy of VLP. Most current solutions rely on gyroscopes or Inertial Measurement Units (IMUs) for error optimization, but these approaches often add complexity and cost to the system. To overcome these limitations, we propose a 3D positioning algorithm based on an attention mechanism convolutional neural network (CNN) aimed at reducing the errors caused by angles. We designed experiments and comparisons within a rotation angle range of ±15 degrees. The results demonstrate that the average error for 2D positioning is within 5.74 cm and the height error is within 3.92 cm, and the average error for 3D positioning is within 6.8 cm. Among the four groups of experiments for 3D positioning, compared to the traditional algorithm, the improvements were 7.931 cm, 15.569 cm, 6.004 cm, and 16.506 cm. The experiments indicate that it can be applied to high-precision visible light positioning for single-light imaging.

A vaccine protecting against malaria caused by Plasmodium falciparum is urgently needed. The blood-stage invasion complex PCRCR consists of the five malarial proteins PfPTRAMP, PfCSS, PfRipr, PfCyRPA, and PfRH5. As each subcomponent represents an essential and highly conserved antigen, PCRCR is considered a promising vaccine target. Furthermore, antibodies targeting the complex can block red blood cell invasion by the malaria parasite. However, extremely high titers of neutralizing antibodies are needed for this invasion-blocking effect, and a vaccine based on soluble PfRH5 protein has proven insufficient in inducing a protective response in a clinical trial. Here, we present the results of two approaches to increase the neutralizing antibody titers: (A) immunofocusing and (B) increasing the immunogenicity of the antigen via multivalent display on capsid virus-like particles (cVLPs). The immunofocusing strategies included vaccinating with peptides capable of binding the invasion-blocking anti-PfCyRPA monoclonal antibody CyP1.9, as well as removing non-neutralizing epitopes of PfCyRPA through truncation. Vaccination with PfCyRPA coupled to the AP205 cVLP induced nearly two-fold higher IgG responses compared to vaccinating with soluble PfCyRPA protein. Immunofocusing using a linear peptide greatly increased the neutralizing capacity of the anti-PfCyRPA antibodies. However, significantly lower total anti-PfCyRPA titers were achieved using this strategy. Our results underline the potential of a cVLP-based malaria vaccine including full-length PfCyRPA, which could be combined with other leading malaria vaccine antigens presented on cVLPs.

Introduction: The Human papillomaviruses (HPV) main capsid protein L1 is naturally capable to self-assemble as virus-like particles (VLPs). There are different recombinant protein expression systems such as bacteria, yeast, insect, plant, and mammalian cells for generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced HPV-L1 protein by BL21/pET32a expression system and VLP production was confirmed.Material & Method: The recombinant plasmid pET32/L1 was transformed into Escherichia. coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and sequence analysis. The expression of HPV16-L1 fusion protein in E. coli BL21was identified by SDS-PAGE and Western blotting. VLPs were evaluated using electron microscopy.Result: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by Western blotting. VLPs were confirmed using electron microscopy.Conclusion: In this study, we established a high-efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production.

Triggering intrusions of phreatic eruptions are often observed as seismic and ground deformation signals on a time scale of minutes. The current understanding of hydrothermal intrusions still needs improvement to obtain insight into the eruption scale from the observables. We examine local geophysical data from the precursory hydrothermal intrusion of the 2018 phreatic eruption of Kusatsu‐Shirane volcano. To achieve an integrated intrusion model, we divide analyzing time window into the onset, middle, and climax. Focusing on the transient response of tilt data for the sudden pressurization, we estimate a vertical tensile opening (1.7 × 103 m3/s in 40 s) at 1.1 km depth for the intrusion onset. Pressurization can represent the start of vapourization. Very long period (VLP, 0.033–0.1 Hz) seismic signals are adopted to constrain the middle and climax phases. We obtained two sequential semi‐horizontal tensile crack oscillation sources with peak volume changes of 3.6 × 104–1.9 × 105 m3 at 0.3–0.6 km depths. The second VLP source acted as a final trigger of the eruption to cause depressurization in the shallow portion of the intruded region, which is constrained as having reached 0.1 km depth by surface deformation. Simultaneously, we find another depressurization originated from depth in the climax due to a decrease in the hydrothermal intrusion rate. Through comparison with the 2014 Ontake phreatic eruption, the total inflation volume may correlate with eruption scales. Intruded hydrothermal fluid and local structure characteristics also may have to be considered to evaluate the eruptions scales from inferred signal source intensity. Plain Language Summary: Phreatic eruptions, emitting non‐fresh magma fragments, are driven by underground water and gas mixtures intruding into the subsurface. Local geophysical observations often detect intrusions as seismic and ground deformation signals. Those observation data potentially provide insight into eruption and hazard scale evaluations. However, such an interpretation is still challenging for the current understanding. This paper examines seismic and ground deformation data associated with the precursory intrusion of a phreatic eruption at Kusatsu‐Shirane volcano in Japan, revealing a detailed hydrothermal intrusion process. Using ground deformation data, we have found that an aqueous intrusion started at 1.1 km depth. This starting process may represent the vapourization onset of underground water and gas mixtures. By analyzing seismic signals having a longer dominant period (>10 s), we revealed the intrusion path from the starting region to beneath the 2018 eruption vent and the final triggering for the eruption. Through comparisons with other phreatic eruptions, we find that the total deformation volume may correlate with the eruption intensity scales such as the mass emitted volcanic ash and the maximum eruption cloud height. However, there is still room for considering hydrothermal fluid characteristics and local structures to evaluate the eruption scale properly from observed data. Key Points: A sudden hydrothermal intrusion onset is constrained by adopting transient tiltmeter responsesVery long period band seismic signals reflect the hydrothermal intrusion behaviors during the middle and climax phasesTilt and seismic records show precursory depressurization in shallow depth as the final eruption trigger [ABSTRACT FROM AUTHOR]

Studies into the viral fraction of complex microbial communities, like in the mammalian gut, have recently garnered much interest. Yet there is still no standardized protocol for extracting viruses from such samples, and the protocols that exist employ procedures that skew the viral community of the sample one way or another. The first step of the extraction pipeline often consists of the basic filtering of macromolecules and bacteria, yet even this affects the viruses in a strain-specific manner. In this study, we investigate a protocol for viral extraction based on ultrafiltration and how the choice of ultrafilter might influence the extracted viral community. Clinical samples (feces, vaginal swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4, c2, Φ6, Φ29, Φx174, and Φ2972), filtered, and quantified using spot and plaque assays to estimate the loss in recovery. The enveloped Φ6 phage is especially severely affected by the choice of filter, but also tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that the pore size of ultrafilters may affect the recovery of phages in a strain- and sample-dependent manner, suggesting the need for greater thought when selecting filters for virus extraction. [ABSTRACT FROM AUTHOR]

Porcine circovirus (PCV) 2d is a common genotype in South Korea, and the cross-protective ability of PCV2a-based vaccines has been reported recently. In this study, a PCV2d vaccine candidate was selected, and its protective efficacy against the PCV2d isolate was evaluated. From 2016 to 2020, 234 PCV2d isolates were phylogenetically analyzed using open reading frame 2 (ORF2) sequences and classified into four subgroups: PCV2d-1, PCV2d-2, PCV2d-3, and PCV2d-4. Except for PCV2d-4, which consisted of ungrouped isolates, the three subgroups showed distinct differences at amino acid positions 53 and 169 in the ORF2. The detection rates of PCV2d-1, PCV2d-2, and PCV2d-3 were 36.5, 37.4, and 3.7%, respectively, and representative isolates were selected from each subgroup (QIA244, QIA126, and QIA169, respectively). In the neutralization assay, QIA244 showed the lowest neutralization efficiency among the three PCV2a-based vaccines, whereas the virus-like particles of QIA244 (rQIA244) provided broader protection against the three genotypes than did those of QIA126 and rQIA169. To further evaluate rQIA244 in pigs, the experimental groups were divided into rQIA244-vaccine (2dVac), commercial PCV2a-vaccine (2aVac), and no-vaccination (noVac) groups. The 2dVac effectively reduced the copy number of PCV2d in blood and tissues, as well as in tissue lesions, compared to the effect of 2aVac. Collectively, 2dVac provided by QIA244 ORF2 successfully demonstrated protective efficacy against the currently prevalent PCV2d in vitro neutralization and in vivo assays. [ABSTRACT FROM AUTHOR]

In this paper, we consider a visible light positioning (VLP) system, where an array of photo diodes combined with apertures is used as a directional receiver and a set of inexpensive and energy-efficient light-emitting diodes (LEDs) is used as transmitters. The paper focuses on the optimisation of the layout of the transmitter, i.e., the number and placement of the LEDs, to meet the wanted position estimation accuracy levels. To this end, we evaluate the Cramer–Rao bound (CRB), which is a lower bound on the mean-squared error (MSE) of the position estimate, to analyse the influence of the LEDs’ placement. In contrast to other works, where only the location of the LEDs was considered and/or the optimisation was carried out through simulations, in this work, the optimisation is carried out analytically and considers all the parameters involved in the VLP system as well as the illumination. Based on our results, we formulate simple rules of thumb with which we can determine the spacing between LEDs and the minimum number of LEDs, as well as their position on the ceiling, while also taking into account the requirements for the illumination.

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2–220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.

The development of HIV prophylactic vaccines is facing an impasse, since all phase IIb/III clinical trials were halted in 2023 without demonstrating efficacy. Thus, the field is in need of developing novel immunogens and vaccination strategies that induce broadly neutralising antibodies together with potent Fc-dependent effector functions, as well as protective cross-reactive CD4+ and CD8+ T cell responses. Nucleic acid vaccines, particularly mRNA vaccines, have been one of the major groundbreaking advances in the current decade. Nucleic acid vaccines may help recalibrate the HIV vaccine field towards the use of delivery systems that allow the proper expression of immunogens as a sole antigen (i.e., membrane-bound trimeric envelope glycoproteins) or even to be displayed in a multiantigen platform that will be synthesised by the host. In this review, we will summarise how the multiple HIV vaccine strategies pursued in the last 40 years of HIV research have driven current vaccine development, which are the most relevant immunogens identified so far to induce balanced adaptive immune responses, and how they can benefit from the acceptance of nucleic acid vaccines in the market by reducing the limitations of previous delivery systems. The incorporation of nucleic acid vaccines into the current heterogeneous repertoire of vaccine platforms may represent an invaluable opportunity to reignite the fight against HIV.

Background & Aims: Rat hepatitis E virus (Rocahepevirus ratti; HEV-C1) is an emerging cause of hepatitis E that is divergent from conventional human-infecting HEV variants (Paslahepevirus balayani; HEV-A). Validated serological assays for HEV-C1 are lacking. We aimed to develop a parallel enzymatic immunoassay (EIA) system that identifies individuals with HEV-C1 exposure. We also aimed to conduct the first HEV-C1 seroprevalence study in humans using this validated EIA system. Methods: Expressed HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) peptides were characterised. Blood samples were simultaneously tested in HEV-A4 p239 and HEV-C1 p241 IgG EIAs. An optical density (OD) cut-off-based interpretation algorithm for identifying samples seropositive for HEV-A or HEV-C1 was validated using RT-PCR-positive infection sera. This algorithm was used to measure HEV-C1 seroprevalence in 599 solid organ transplant recipients and 599 age-matched immunocompetent individuals. Results: Both peptides formed virus-like particles. When run in HEV-A4 p239 and HEV-C1 p241 EIAs, HEV-A and HEV-C1 RT-PCR-positive samples formed distinct clusters with minimal overlap in a two-dimensional plot of optical density values. The final EIA interpretation algorithm showed high agreement with RT-PCR results (Cohen’s κ = 0.959) and was able to differentiate HEV-A and HEV-C1 infection sera with an accuracy of 94.2% (95% CI: 85.8–98.4%). HEV-C1 IgG seroprevalence was 7/599 (1.2%) among solid organ transplant recipients and 4/599 (0.7%) among immunocompetent individuals. Five of 11 (45.5%) of these patients had history of transient hepatitis of unknown cause. Conclusions: HEV-C1 exposure was identified in 11/1198 (0.92%) individuals in Hong Kong indicating endemic exposure. This is the first estimate of HEV-C1 seroprevalence in humans. The parallel IgG EIA algorithm is a valuable tool for investigating epidemiology and risk factors for HEV-C1 infection. Impact and Implications: Rat hepatitis E virus has recently been discovered to infect humans, but antibody tests for this infection are lacking, making it difficult to gauge how common this infection is. We developed an antibody test algorithm that can identify individuals with past rat hepatitis E virus exposure. We used this algorithm to estimate rat hepatitis E exposure rates in humans in Hong Kong and found that approximately 1% of all tested people had been exposed to this virus previously.

A novel virus‐like particle (VLP)‐based multivalent recombinant human papillomavirus (HPV) vaccine was developed and evaluated in human, including 14 HPV‐type specific VLP antigens (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The pseudovirus‐based neutralizing assay (PBNA) method is widely used for immunogenicity assessment of HPV vaccine in clinical trials. However, as many as 14 antigen‐specific antibody levels need be determined, PBNA is, for many reasons, challenging and time‐consuming. In this study, we developed a Luminex immunological assay (LIA) and a competitive Luminex immunological assay (cLIA). These methods increase the throughput, reproducibility and precision, as well as reduce the complexity. All assay parameters showed good characteristics in the validation of both methods, benefiting from highly purified and structurally correct VLPs, high specific antibodies, standard VLP‐microspheres and PE‐mAbs conjugating process, adequate assay development and stable system. Validation data support the use of both methods for immunogenicity assessment in clinical trials. LIA showed higher sensitivity than cLIA, and due to limited epitopes of mAb, cLIA detected lower antibody responses, and therefore, fewer antibodies. This work not only supports clinical trials of 14‐valent HPV vaccines more efficiently and reliably, but also provides a set of validation strategies and usable standards for general vaccine immunogenicity testing. [ABSTRACT FROM AUTHOR]

This study focused on developing an influenza vaccine delivered in polymeric nanoparticles (NPs) using dissolving microneedles. We first formulated an influenza extracellular matrix protein 2 virus-like particle (M2e VLP)-loaded with poly(lactic-co-glycolic) acid (PLGA) nanoparticles, yielding M2e5x VLP PLGA NPs. The vaccine particles were characterized for their physical properties and in vitro immunogenicity. Next, the M2e5x VLP PLGA NPs, along with the adjuvant Alhydrogel® and monophosphoryl lipid A® (MPL-A®) PLGA NPs, were loaded into fast-dissolving microneedles. The vaccine microneedle patches were then evaluated in vivo in a murine model. The results from this study demonstrated that the vaccine nanoparticles effectively stimulated antigen-presenting cells in vitro resulting in enhanced autophagy, nitric oxide, and antigen presentation. In mice, the vaccine elicited M2e-specific antibodies in both serum and lung supernatants (post-challenge) and induced significant expression of CD4+ and CD8+ populations in the lymph nodes and spleens of immunized mice. Hence, this study demonstrated that polymeric particulates for antigen and adjuvant encapsulation, delivered using fast-dissolving microneedles, significantly enhanced the immunogenicity of a conserved influenza antigen. [ABSTRACT FROM AUTHOR]

Background: The human papillomavirus (HPV) main capsid protein L1 is naturally capable of self-assembly as virus-like particles (VLPs). There are different recombinant protein expression systems, such as bacteria, yeast, insect, plant, and mammalian cells, for the generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced the HPV16-L1 protein by BL21/pET32a expression system, and VLP production was confirmed. Methods: The recombinant plasmid pET32/L1 was transformed into Escherichia coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and nested polymerase chain reaction (PCR). The expression of HPV16-L1 fusion protein in Escherichia coli BL21 was identified by SDS-PAGE and western blotting. Electron microscopy was used to evaluate VLP formation. Results: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60 kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by western blotting. The VLPs were confirmed using electron microscopy. Conclusion: In this study, we established an efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production. [ABSTRACT FROM AUTHOR]

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses. [ABSTRACT FROM AUTHOR]