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The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine—formed through the structural clustering of HPV epitopes—confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens. An effective vaccine for human papillomavirus (HPV) needs to protect from several genotypes. Here, Wang et al. provide a strategy to produce single capsomere-hybrid virus-like particle (chVLP) composed of capsomers of different genotypes and show that a nona-type chVLP induces similar levels of neutralizing antibodies as an approved HPV vaccine.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.[PUBLICATION ABSTRACT]

Infections with immunosuppressive pigeon circovirus (PiCV) pose the most severe health problem to the global pigeon breeding. The vaccination with immunogenic PiCV recombinant capsid protein (PiCV rCP) is a potential tool for disease control. Because of the high prevalence of PiCV asymptomatic infections, the subclinically infected pigeons will be vaccinated in practice. The aim of this study was to answer a question if vaccination of asymptomatic, infected with PiCV pigeons induces a similar immune response to PiCV rCP as in uninfected birds. One hundred and twenty 6-week-old carrier pigeons were divided into 4 groups (2 groups of naturally infected and uninfected with PiCV individuals). Birds from groups V and V1 were vaccinated twice with PiCV rCP mixed with an adjuvant, whereas pigeons from groups C and C1 were immunized with an adjuvant only. The expression of genes encoding IFN-γ, CD4, and CD8 T lymphocyte receptors; the number of anti-PiCV rCP IgY-secreting B cells (SBC) and anti-PiCV rCP IgY were evaluated 2, 21, 39 and 46 days post vaccination (dpv). Study results showed that the expression of CD8 and IFN-γ genes was higher in both groups of infected pigeons than in the uninfected birds, irrespective of vaccination. In the uninfected birds, the expression of these genes was insignificantly higher in the vaccinated pigeons. The anti-PiCV rCP IgY-SBC were detected on 2 and 23 dpv and seroconversion was noted on 23 and 39 dpv in V and V1 groups, respectively. In the light of the results obtained, it could be concluded that pigeon circovirus recombinant capsid protein elicits the immune response in both naturally infected and uninfected pigeons, but its rate varies depending on PiCV infectious status. The infection with PiCV masks the potential cellular immune response to the vaccination with PiCV rCP and leads to the suppression of humoral immunity.

A viral protein delivers a toxin from an insect's gut to its body cavity, providing a new strategy for controlling sap-sucking agricultural pests. The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum , without virion assembly. We show that when four aphid pest species— A. pisum , Rhopalosiphum padi , Aphis glycines and Myzus persicae —feed on a recombinant coat protein–toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein–insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

Porcine circovirus type 2 (PCV2) causes many diseases in weaned piglets, leading to serious economic losses to the pig industry. This study investigated the immune response following oral administration of Lactobacillus casei ATCC393 (L. casei 393) expressing PCV2 capsid protein (Cap) fusion with the Escherichia coli heat-labile toxin B subunit (LTB) in mice. Recombinant L. casei strains were constructed using plasmids pPG611.1 and pPG612.1. The expression and localization of proteins from recombinant pPG611.1-Cap-LTB (pPG-1-Cap-LTB)/L. casei 393 and pPG612.1-Cap-LTB (pPG-2-Cap-LTB)/L. casei 393 were detected. All recombinant strains were found to be immunogenic by oral administration in mice and developed mucosal and systemic immune responses against PCV2. The titers of specific antibodies in mice administered pPG-2-Cap-LTB/L. casei 393 were higher than those in mice administered pPG-1-Cap-LTB/L. casei 393 in serum and the mucosal samples. The mucosal immune response was not only limited to the gastrointestinal tract but was also generated in other mucosal parts. Thus, the application of recombinant L. casei could aid in vaccine development for PCV2.

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50-60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates. * N-terminal truncations promote the soluble expression of HPV L1 proteins in E. coli and their self-assembly of T = 7 icosahedral particle in vitro * An HPV 9-valent vaccine candidate was formulated with E. coli-derived HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 VLPs, and conferred comparable immunogenicity with Gardasil 9

•Immunization with VLPs displaying HPV L2 peptides elicits long-lasting, protective antibody responses in mice.•High titer antibody induction is predominantly due to the multivalent display of the L2 peptide, not adjuvants.•Encapsidated RNA within the VLPs skews the IgG response from isotypes associated with a Th2-type to a Th1-type response.•Alum effectively adjuvants bacteriophage VLPs displaying L2. Antibodies targeting epitopes within the amino terminus of the minor capsid protein L2 of human papillomavirus (HPV) are broadly neutralizing against diverse HPV isolates. We have constructed bacteriophage virus-like particle (VLP)-based vaccines that display short L2 peptides and elicit high-titer and broadly protective antibody responses. Here, we further characterize two additional features of these VLP-based vaccines; the longevity of protection and the role of endogenous and exogenous adjuvants on the magnitude and characteristics of the antibody response. We show that vaccinated mice have long-lived antibody responses against L2, persisting over 18 months after vaccination. Vaccinated mice were strongly protected against infection by diverse HPV pseudoviruses over a year after immunization. We also show that exogenous and endogenous adjuvants (LPS and encapsidated single-stranded RNA) have minor effects on antibody titers. Immunization with VLPs containing encapsidated ssRNA predominantly shifts the response to a Th1, rather than a Th2-like response. Importantly, immunization with L2-VLPs (without endogenous and exogenous adjuvants) in the presence of alum hydroxide elicited a robust antibody response.

Background Nanocarriers targeting the blood-brain barrier (BBB) are promising drug delivery systems to enhance the penetration of therapeutic molecules into the brain. Immunotherapy, particularly monoclonal antibodies designed to bind amyloid-beta peptides have become a promising strategy for Alzheimer’s disease, but ensuring efficacy and safety is challenging and crucial for these therapies. Our aim was to develop an innovative nanocarriers conjugated with PepH3, a cationic peptide derived from Dengue virus type-2 capsid protein that crosses the BBB and acts as a shuttle peptide for the encapsulated single domain antibody (sdAb) recognizing Aβ oligomers. Results PepH3 peptide enhanced the uptake of the nanoparticles (NPs) into brain endothelial cells, and transcytosis of sdAb, as a potential therapeutic molecule, across both rat and human BBB culture models. The cargo uptake was a temperature dependent active process that was reduced by metabolic and endocytosis inhibitors. The cellular uptake of the cationic PepH3-tagged NPs decreased when the negative surface charge of brain endothelial cells became more positive after treatments with a cationic lipid or with neuraminidase by digesting the glycocalyx. The NPs colocalized mostly with endoplasmic reticulum and Golgi apparatus and not with lysosomes, indicating the cargo may avoid cellular degradation. Conclusions Our results support that combination of NPs with a potential brain shuttle peptide such as PepH3 peptide can improve the delivery of antibody fragments across the BBB.

Original reports of adeno-associated virus (AAV) vector-mediated gene transfer to the muscle resulted in high-level β-galactosidase (β-gal) expression and the promise of a viral vector that was largely nonimmunogenic. Subsequent attempts to utilize these vectors for genetic vaccination, however, demonstrated that it was possible to activate cellular and humoral immunity to AAV-encoded antigens. These findings fueled years of investigation into factors impacting the immunogenicity of recombinant AAV-mediated gene delivery, including route of administration, dose, host species, capsid serotype, and transgene product. In cases where AAV vectors could avoid transgene-directed immunity, it became clear that mechanisms of tolerance were at work, varying between ignorance, anergy/deletion, or active suppression. Here, we follow the field of AAV gene therapy from inception, as investigators have worked to understand the delicate balance between AAV-mediated tolerance and the activation of immunity. This review discusses our current appreciation of AAV vector immunology, with a specific focus on the transgene-specific T cell response.

Background Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer’s patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis. Results We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer’s patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis. Conclusions Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.