Application of nervous necrosis virus capsid protein-based antigen-presenting particles for vaccine development.

Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses.

Assessing immune factors in maternal milk and paired infant plasma antibody binding to human rhinoviruses.

Introduction: Before they can produce their own antibodies, newborns are protected from infections by transplacental transfer of maternal IgG antibodies and after birth through breast milk IgA antibodies. Rhinovirus (RV) infections are extremely common in early childhood, and while RV infections often result in only mild upper respiratory illnesses, they can also cause severe lower respiratory illnesses such as bronchiolitis and pneumonia. Methods: We used high-density peptide arrays to profile infant and maternal antibody reactivity to capsid and full proteome sequences of three human RVs - A16, B52, and C11. Results: Numerous plasma IgG and breast milk IgA RV epitopes were identified that localized to regions of the RV capsid surface and interior, and also to several non-structural proteins. While most epitopes were bound by both IgG and IgA, there were several instances where isotype-specific and RV-specific binding were observed. We also profiled 62 unique RV-C protein loop sequences characteristic of this species' capsid VP1 protein. Discussion: Many of the RV-C loop sequences were highly bound by IgG from one-year-old infants, indicating recent or ongoing active infections, or alternatively, a level of cross-reactivity among homologous RV-C sites.

CER818: A Highly Specific and Sensitive HPV L1 High-Risk Serological Lateral Flow Rapid Test for Early Detection of Cervical Cancer and Its Precursor Lesions.

Objective: The objective of the study is to validate a new human papillomavirus (HPV) L1 high-risk specific serological assay in a case-control study. Methods: Serum samples of 138 patients (cervical intraepithelial neoplasia (CIN) 1, 2, and 3 and cervical cancer), 21 vaccinees, and 246 female controls were tested for the presence of HPV L1 high-risk specific antibodies. Results: HPV L1 high-risk antibodies were detected in 100% of the CIN1 and 2, 86.6% of the CIN3 and 82.4% of the cervical cancer cases, 100% of the vaccinees, and 3.9% of the female controls. Area under the curve (AUC) was calculated with 0.91 for controls versus CIN2+, 0.923 for controls versus CIN1+, and 0.968 for controls versus CIN1/2. Conclusion: The HPV L1 high-risk specific serological lateral flow rapid test shows promising data in the field of early detection of HPV high-risk induced cervical cancer and its precursor lesions. This easy-to-use, robust, and affordable approach could offer a chance to reach women in low- or middle-income countries (LMICs) that could not be reached by HPV molecular testing-based cervical cancer screening programs.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Plant-Produced Chimeric Hepatitis E Virus-like Particles as Carriers for Antigen Presentation.

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.

Immunogenicity of a Rotavirus VP8* Multivalent Subunit Vaccine in Mice.

Rotavirus remains a significant public health threat, especially in low-income countries, where it is the leading cause of severe acute childhood gastroenteritis, contributing to over 128,500 deaths annually. Although the introduction of the Rotarix and RotaTeq vaccines in 2006 marked a milestone in reducing mortality rates, approximately 83,158 preventable deaths persisted, showing ongoing challenges in vaccine accessibility and effectiveness. To address these issues, a novel subcutaneous vaccine formulation targeting multiple rotavirus genotypes has been developed. This vaccine consists of nine VP8* proteins from nine distinct rotavirus genotypes and sub-genotypes (P[4], P[6], P[8]LI, P[8]LIII, P[8]LIV, P[9], P[11], P[14], and P[25]) expressed in E. coli. Two groups of mice were immunized either with a single immunogen, the VP8* from the rotavirus Wa strain (P[8]LI), or with the nonavalent formulation. Preliminary results from mouse immunization studies showed promising outcomes, eliciting antibody responses against six of the nine immunogens. Notably, significantly higher antibody titers against VP8* P[8]LI were observed in the group immunized with the nonavalent vaccine compared to mice specifically immunized against this genotype alone. Overall, the development of parenteral vaccines targeting multiple rotavirus genotypes represents a promising strategy in mitigating the global burden of rotavirus-related morbidity and mortality, offering new avenues for disease prevention and control.

Chimeric Viruses Enable Study of Antibody Responses to Human Rotaviruses in Mice.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human-rotavirus-specific immunity to the outer capsid, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

Prevalence of HPV16 L1 protein in oral biopsies: A diagnostic study from Ecuador.

This study was designed to investigate the expression of HPV16 L1-protein in biopsies of oral mucosa samples. The expression of HPV16 L1 protein was investigated in biopsies taken from oral mucosa from patients who required pathological diagnosis of oral lesions. Seventy-two samples were incubated with anti-L1 protein monoclonal antibodies and protein detection was revealed with diaminobenzidine. Expression of L1 protein was performed by a pathologist blinded for tissue diagnosis under light microscopy. Most of the lesions of oral mucosa were present in lining mucosa (75 %) and the most frequent lesion were mucocele (n = 17, 23.6 %), epithelial hyperplasia (n = 6, 8.33 %), fibroma (n = 5, 6.9 %) and inflammatory hyperplasia (n = 5, 6.9 %). L1 protein expression was observed only in five (6.9 %) samples (two squamous cell carcinomas, two epithelial hyperplasia, and one gingival hyperplasia). We concluded that L1 expression in oral biopsies presented a low frequency in oral mucosal biopsies samples.

Reverse vaccinology-based multi-epitope vaccine design against Indian group A rotavirus targeting VP7, VP4, and VP6 proteins.

Rotavirus, a primary contributor to severe cases of infantile gastroenteritis on a global scale, results in significant morbidity and mortality in the under-five population, particularly in middle to low-income countries, including India. WHO-approved live-attenuated vaccines are linked to a heightened susceptibility to intussusception and exhibit low efficacy, primarily attributed to the high genetic diversity of rotavirus, varying over time and across different geographic regions. Herein, molecular data on Indian rotavirus A (RVA) has been reviewed through phylogenetic analysis, revealing G1P[8] to be the prevalent strain of RVA in India. The conserved capsid protein sequences of VP7, VP4 and VP6 were used to examine helper T lymphocyte, cytotoxic T lymphocyte and linear B-cell epitopes. Twenty epitopes were identified after evaluation of factors such as antigenicity, non-allergenicity, non-toxicity, and stability. These epitopes were then interconnected using suitable linkers and an N-terminal beta defensin adjuvant. The in silico designed vaccine exhibited structural stability and interactions with integrins (αvß3 and αIIbß3) and toll-like receptors (TLR2 and TLR4) indicated by docking and normal mode analyses. The immune simulation profile of the designed RVA multiepitope vaccine exhibited its potential to trigger humoral as well as cell-mediated immunity, indicating that it is a promising immunogen. These computational findings indicate potential efficacy of the designed vaccine against rotavirus infection.

Exploring the nuclear proteins, viral capsid protein, and early antigen protein using immunoinformatic and molecular modeling approaches to design a vaccine candidate against Epstein Barr virus.

The available Epstein Barr virus vaccine has tirelessly harnessed the gp350 glycoprotein as its target epitope, but the result has not been preventive. Right here, we designed a global multi-epitope vaccine for EBV; with special attention to making sure all strains and preventive antigens are covered. Using a robust computational vaccine design approach, our proposed vaccine is armed with 6-16 mers linear B-cell epitopes, 4-9 mer CTL epitopes, and 8-15 mer HTL epitopes which are verified to induce interleukin 4, 10 & IFN-gamma. We employed deep computational mining coupled with expert intelligence in designing the vaccine, using human Beta defensin-3-which has been reported to induce the same TLRs as EBV-as the adjuvant. The tendency of the vaccine to cause autoimmune disorder is quenched by the assurance that the construct contains no EBNA-1 homolog. The protein vaccine construct exhibited excellent physicochemical attributes such as Aliphatic index 59.55 and GRAVY - 0.710; and a ProsaWeb Z score of - 3.04. Further computational analysis revealed the vaccine docked favorably with EBV indicted TLR 1, 2, 4 & 9 with satisfactory interaction patterns. With global coverage of 85.75% and the stable molecular dynamics result obtained for the best two interactions, we are optimistic that our nontoxic, non-allergenic multi-epitope vaccine will help to ameliorate the EBV-associated diseases-which include various malignancies, tumors, and cancers-preventively.

Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

Co-expression of VP2, NS1 and NS2-Nt proteins by an MVA viral vector induces complete protection against bluetongue virus.

Introduction: Bluetongue (BT), caused by bluetongue virus (BTV), is an important arthropod-borne livestock disease listed by the World Organization for Animal Health. Live-attenuated and inactivated vaccines have permitted to control BT but they do not simultaneously protect against the myriad of BTV serotypes. Recently, we identified the highly conserved BTV nonstructural protein NS1 and the N-terminal region of NS2 as antigens capable of conferring multiserotype protection against BTV. Methods: Here, we designed Modified Vaccinia Ankara (MVA) viral vectors that expressed BTV-4 proteins VP2 or VP7 along with NS1 and NS2-Nt as well as MVAs that expressed proteins VP2, VP7 or NS1 and NS2-Nt. Results: Immunization of IFNAR(-/-) mice with two doses of MVA-NS1-2A-NS2-Nt protected mice from BTV-4M infection by the induction of an antigen-specific T cell immune response. Despite rMVA expressing VP7 alone were not protective in the IFNAR(-/-) mouse model, inclusion of VP7 in the vaccine formulation amplified the cell-mediated response induced by NS1 and NS2-Nt. Expression of VP2 elicited protective non-cross-reactive neutralizing antibodies (nAbs) in immunized animals and improved the protection observed in the MVA-NS1-2A-NS2-Nt immunized mice when these three BTV antigens were co-expressed. Moreover, vaccines candidates co-expressing VP2 or VP7 along with NS1 and NS2-Nt provided multiserotype protection. We assessed protective efficacy of both vaccine candidates in sheep against virulent challenge with BTV-4M. Discussion: Immunization with MVA-VP7-NS1-2A-NS2-Nt partially dumped viral replication and clinical disease whereas administration of MVA-VP2-NS1-2A-NS2-Nt promoted a complete protection, preventing viraemia and the pathology produced by BTV infection.

Oral immunization against foot-and-mouth disease virus using recombinant Saccharomyces cerevisiae with the improved expression of the codon-optimized VP1 fusion protein.

VP1, a major immunogenic protein of foot-and-mouth disease virus (FMDV), facilitates viral attachment and entry into host cells. VP1 possesses critical epitope sequences responsible for inducing neutralizing antibodies but its expression using Saccharomyces cerevisiae has been hampered despite evidence that the presence of VP1 does not negatively impact the yeast's biology. In this study, we fused proteins to enhance VP1 expression using S. cerevisiae. Among short P1 chimeras containing VP1 including VP3-VP1 and VP2-VP1, VP3-VP1 fusion proteins showed higher expression levels than VP2-VP1. We subsequently designed new fusion proteins, of which 20 amino acids of N-terminal VP3 fused with VP1-Co1 (referred to 20aaVP3-VP1-Co1) showed the highest expression level. Lowering the culture temperature from 30 °C to 20 °C further enhanced fusion protein production. The highest expression level of 20aaVP3-VP1-Co1 was estimated to be 7.7 mg/L, which is comparable to other heterologous proteins produced using our S. cerevisiae expression system. Oral administration of the cell expressing 20aaVP3-VP1-Co1 induced VP1-specific IgG and IgA responses in mice. The S. cerevisiae-expressed 20aaVP3-VP1-Co1 fusion protein induced a significant immune response to the FMDV structural epitope protein, which opens the possibility of an oral FMDV vaccine.

Design of multi-epitope vaccine against porcine rotavirus using computational biology and molecular dynamics simulation approaches.

Porcine Rotavirus (PoRV) is a significant pathogen affecting swine-rearing regions globally, presenting a substantial threat to the economic development of the livestock sector. At present, no specific pharmaceuticals are available for this disease, and treatment options remain exceedingly limited. This study seeks to design a multi-epitope peptide vaccine for PoRV employing bioinformatics approaches to robustly activate T-cell and B-cell immune responses. Two antigenic proteins, VP7 and VP8*, were selected from PoRV, and potential immunogenic T-cell and B-cell epitopes were predicted using immunoinformatic tools. These epitopes were further screened according to non-toxicity, antigenicity, non-allergenicity, and immunogenicity criteria. The selected epitopes were linked with linkers to form a novel multi-epitope vaccine construct, with the PADRE sequence (AKFVAAWTLKAAA) and RS09 peptide attached at the N-terminus of the designed peptide chain to enhance the vaccine's antigenicity. Protein-protein docking of the vaccine constructs with toll-like receptors (TLR3 and TLR4) was conducted using computational methods, with the lowest energy docking results selected as the optimal predictive model. Subsequently, molecular dynamics (MD) simulation methods were employed to assess the stability of the protein vaccine constructs and TLR3 and TLR4 receptors. The results indicated that the vaccine-TLR3 and vaccine-TLR4 docking models remained stable throughout the simulation period. Additionally, the C-IMMSIM tool was utilized to determine the immunogenic triggering capability of the vaccine protein, demonstrating that the constructed vaccine protein could induce both cell-mediated and humoral immune responses, thereby playing a role in eliciting host immune responses. In conclusion, this study successfully constructed a multi-epitope vaccine against PoRV and validated the stability and efficacy of the vaccine through computational analysis. However, as the study is purely computational, experimental evaluation is required to validate the safety and immunogenicity of the newly constructed vaccine protein.

A nanoparticle vaccine based on the VP121-26 and VP2 structural proteins of Senecavirus A induces robust protective immune responses.

Senecavirus A (SVA) is a causative agent that can cause vesicular disease in swine, which causes a great threat to the swine husbandry in the world. Therefore, it is necessary to develop a vaccine that can effectively prevent the spread of SVA. In this study, we developed a 24-polymeric nano-scaffold using ß-annulus peptide from tomato bushy effect virus (TBSV) by coupling this antigen to SVA B cell epitope VP121-26 and VP2 proteins via linkers, respectively. The SVA-based nanoparticle protein of the VP1(B)-ß-VP2 was expressed and purified by low-cost prokaryotic system to prepare a SVA nanoparticle vaccine. The immunological protective effect of SVA nanoparticle vaccine was evaluated in mouse and swine models, respectively. The results suggested that both mice and swine could induce high levels SVA neutralizing antibodies and IgG antibodies after two doses immunization. In addition, the swine challenge protection experiment showed that the protection rate of immune SVA nanoparticle vaccine and SVA inactivated vaccine both were 80 %, while the negative control had no protection effect. It demonstrated that SVA nanoparticle vaccine effectively prevented SVA infection in swine. In summary, the preparation of SVA vaccine by using ß-annulus peptide is a promising candidate vaccine for prevent SVA transmission, and provides a new idea for the development of novel SVA vaccines.

Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages.

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

Generation of single-round infectious rotavirus with a mutation in the intermediate capsid protein VP6.

Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.