Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research.

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.

Investigation of a subunit protein vaccine for HFRS based on a consensus sequence between envelope glycoproteins of HTNV and SEOV.

Due to the global resurgence of hemorrhagic fever with renal syndrome (HFRS), more attention is being focused on this dangerous illness. In China and Korea, the only vaccines available are the virus-inactivated vaccine against Hantaan virus (HTNV) or Seoul virus (SEOV), but their efficacy and safety are inadequate. Therefore, it is important to develop new vaccines that are safer and more efficient to neutralize and regulate areas with a high prevalence of HFRS. We employed bioinformatics methods to design a recombinant protein vaccine based on conserved regions of protein consensus sequences in HTNV and SEOV membranes. The S2 Drosophila expression system was utilized to enhance protein expression, solubility and immunogenicity. After the Gn and Gc proteins of HTNV and SEOV were successfully expressed, mice were immunized, and the humoral immunity, cellular immunity, and in vivo protection of the HFRS universal subunit vaccine were systematically evaluated in mouse models. These results indicated that the HFRS subunit vaccine generated elevated levels of binding and neutralizing antibodies, particularly IgG1, compared to that of the traditional inactivated HFRS vaccine. Additionally, the spleen cells of immunized mice secreted IFN-r and IL-4 cytokines effectively. Moreover, the HTNV-Gc protein vaccine successfully protected suckling mice from HTNV infection and stimulated GC responses. In this research, a new scientific approach is investigated to develop a universal HFRS subunit protein vaccine that is capable of producing effective humoral and cellular immunity in mice. The results suggest that this vaccine could be a promising candidate for preventing HFRS in humans.

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses.

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8+ T cell responses. Moreover, E2-E1-LNP induced CD8+ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.

A single nonsynonymous mutation on ZIKV E protein-coding sequences leads to markedly increased neurovirulence in vivo.

Zika virus (ZIKV) can infect a wide range of tissues including the developmental brain of human fetus. Whether specific viral genetic variants are linked to neuropathology is incompletely understood. To address this, we have intracranially serially passaged a clinical ZIKV isolate (SW01) in neonatal mice and discovered variants that exhibit markedly increased virulence and neurotropism. Deep sequencing analysis combining with molecular virology studies revealed that a single 67D (Aspartic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism in vivo. Notably, virus clones with D67N mutation had higher viral production and caused more severe cytopathic effect (CPE) in human neural astrocytes U251 â€‹cells in vitro, indicating its potential neurological toxicity to human brain. These findings revealed that a single mutation D67N on ZIKV envelope may lead to severe neuro lesion that may help to explain the neurovirulence of ZIKV and suggest monitoring the occurrence of this mutation during nature infection may be important.

A high-dose inoculum size results in persistent viral infection and arthritis in mice infected with chikungunya virus.

Chikungunya virus (CHIKV) is an emerging mosquito-transmitted alphavirus that leads to acute fever and chronic debilitating polyarthralgia. To date, the mechanism underlying chronic recurrent arthralgia is unknown. In the present study, newborn wild-type C57BL/6 mice were infected with CHIKV, and the virological and pathological features of CHIKV infection were analyzed over a period of 50 days. Acute viral infection was readily established by footpad inoculation of CHIKV at doses ranging from 10 plaque forming unit (PFU) to 106 PFU, during which inoculation dose-dependent viral RNA and skeletal muscle damage were detected in the foot tissues. However, persistent CHIKV was observed only when the mice were infected with a high dose of 106 PFU of CHIKV, in which low copy numbers (103-104) of viral positive strand RNA were continuously detectable in the feet from 29 to 50 dpi, along with a low level and progressive reduction in virus-specific CD8+ T cell responses. In contrast, viral negative strand RNA was detected at 50 dpi but not at 29 dpi and was accompanied by significant local skeletal muscle damage at 50 dpi when mild synovial hyperplasia appeared in the foot joints, although the damage was briefly repaired at 29 dpi. These results demonstrated that a high viral inoculation dose leads to viral persistence and progression to chronic tissue damage after recovery from acute infection. Taken together, these results provide a useful tool for elucidating the pathogenesis of persistent CHIKV infection and viral relapse-associated chronic arthritis.

A Combined Adjuvant TF-Al Consisting of TFPR1 and Aluminum Hydroxide Augments Strong Humoral and Cellular Immune Responses in Both C57BL/6 and BALB/c Mice.

TFPR1 is a novel adjuvant for protein and peptide antigens, which has been demonstrated in BALB/c mice in our previous studies; however, its adjuvanticity in mice with different genetic backgrounds remains unknown, and its adjuvanticity needs to be improved to fit the requirements for various vaccines. In this study, we first compared the adjuvanticity of TFPR1 in two commonly used inbred mouse strains, BALB/c and C57BL/6 mice, in vitro and in vivo, and demonstrated that TFPR1 activated TLR2 to exert its immune activity in vivo. Next, to prove the feasibility of TFPR1 acting as a major component of combined adjuvants, we prepared a combined adjuvant, TF-Al, by formulating TFPR1 and alum at a certain ratio and compared its adjuvanticity with that of TFPR1 and alum alone using OVA and recombinant HBsAg as model antigens in both BALB/c and C57BL/6 mice. Results showed that TFPR1 acts as an effective vaccine adjuvant in both BALB/c mice and C57BL/6 mice, and further demonstrated the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TF-Al, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future.

Nlrc3 Knockout Mice Showed Renal Pathological Changes After HTNV Infection.

Hantaan virus (HTNV) infects humans and causes hemorrhagic fever with renal syndrome (HFRS). The development of well-characterized animal models of HFRS could accelerate the testing of vaccine candidates and therapeutic agents and provide a useful tool for studying the pathogenesis of HFRS. Because NLRC3 has multiple immunoregulatory roles, we investigated the susceptibility of Nlrc3-/- mice to HTNV infection in order to establish a new model of HFRS. Nlrc3-/- mice developed weight loss, renal hemorrhage, and tubule dilation after HTNV infection, recapitulating many clinical symptoms of human HFRS. Moreover, infected Nlrc3-/- mice showed higher viral loads in serum, spleen, and kidney than wild type C57BL/6 (WT) mice, and some of them manifested more hematological disorders and significant pathological changes within multiple organs than WT mice. Our results identify that HTNV infected Nlrc3-/- mice can develop clinical symptoms and pathological changes resembling patients with HFRS, suggesting a new model for studying the pathogenesis and testing of candidate vaccines and therapeutics.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

The immunologic dominance of an epitope within a rationally designed poly-epitope vaccine is influenced by multiple factors.

INTRODUCTION: CD4+ T cells are essential for inducing optimal CD8+ T cell and antibody-producing B cell responses and maintaining their long-term immunological memory. Therefore, CD4+ T cells are a critical component in HIV vaccine development. Due to enormous viral gene variation and significant human host genetic diversity, HIV vaccines may need to be custom-made for different countries. METHODS: Previously, we designed a CD4+ T cell vaccine based on Chinese HIV isolates and HLA-DR alleles using bioinformatics tools and predicted that 20 epitopes could cover 98.1% of the Chinese population. In vivo testing of the poly-epitope antigen in mice only activated specific T cells for some epitopes. To elucidate the mechanism of the observed differential immunogenicity, we examined poly-epitope antigen processing and presentation using in vitro and in vivo analytical methods. RESULTS: Enzymatic digestion indicated that all 20 epitopes comprising the poly-epitope antigen could be liberated, but MHC II binding assays showed that neither binding affinity nor dissociation rate was associated with the magnitude of T cell immune responses elicited by each peptide epitope in vaccinated mice. Mass spectrometry analysis of MHC II-bound peptides suggested that the abundance of endogenously processed peptides bound to MHC II molecules was significantly associated with the relative immunodominance of these epitopes. CONCLUSION: These results provide a new rationale for improving the design and testing of poly-epitope vaccines for HIV and other diseases.

Vaccines and Therapeutics Against Hantaviruses.

Hantaviruses (HVs) are rodent-transmitted viruses that can cause hantavirus cardiopulmonary syndrome (HCPS) in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. Together, these viruses have annually caused approximately 200,000 human infections worldwide in recent years, with a case fatality rate of 5-15% for HFRS and up to 40% for HCPS. There is currently no effective treatment available for either HFRS or HCPS. Only whole virus inactivated vaccines against HTNV or SEOV are licensed for use in the Republic of Korea and China, but the protective efficacies of these vaccines are uncertain. To a large extent, the immune correlates of protection against hantavirus are not known. In this review, we summarized the epidemiology, virology, and pathogenesis of four HFRS-causing viruses, HTNV, SEOV, PUUV, and DOBV, and two HCPS-causing viruses, ANDV and SNV, and then discussed the existing knowledge on vaccines and therapeutics against these diseases. We think that this information will shed light on the rational development of new vaccines and treatments.

A novel polyepitope vaccine elicited HIV peptide specific CD4+ T cell responses in HLA-A2/DRB1 transgenic mice.

Human immunodeficiency (HIV) infection is a leading global health problem that causes approximately one million deaths each year. Although antiretroviral therapy can slow down the disease progression and improve the quality of life of infected individuals, it cannot eradicate the virus. A successful vaccine is one of the most cost-effective alternatives to control the incidence and mortality of HIV infection. CD4+ T cells play a key role in orchestrating other forms of human immune responses, therefore, an HIV vaccine that includes a component capable of eliciting CD4+ T cell responses is highly desirable. To this end, we have previously designed a polypeptide vaccine comprised of multiple CD4+ T cell epitopes. In the current study, we tested the immunogenicity of this vaccine in mouse models by using IFN-γELISPOT and intracellular cytokine staining assays. We found that several epitopes in this vaccine elicited CD4+ T cell immune responses in both congenic mice and human HLA-A2/DRB1 transgenic mice. These new epitopes may be further tested for their ability to augment immune responses elicited by other forms of HIV vaccines.

Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli.

To design an epitope-based vaccine for Human immunodeficiency virus (HIV), we previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen DR types. To test the immunogenicity of this vaccine in vivo, a corresponding antigen needs to be prepared. To this end, we constructed a recombinant plasmid containing DNA encoding the epitopes and GPGPG spacers and a 6-His tag for verification of protein expression and ease of purification, and then transformed Escherichia coli cells with the plasmid. After IPTG induction, the recombinant protein was expressed in the form of mainly inclusion bodies. To stabilize the structure of denatured inclusion bodies for efficient purification and renaturation in vitro, we transferred the dissolved inclusion bodies from 7 mol/L guanidine hydrochloride to 8 mol/L urea. Under denaturing conditions, the vaccine protein was purified by a 3-step process including ion-exchange chromatography and affinity column, and then renatured by stepwise dialysis. Together, the above described procedures generated 43 mg of vaccine protein per litre of fermentation medium, and the final product reached approximately 95% purity. The purified protein was capable of eliciting antigen-specific T-cell responses in immunized mice.

Designing peptide-based HIV vaccine for Chinese.

CD4+ T cells are central to the induction and maintenance of CD8+ T cell and antibody-producing B cell responses, and the latter are essential for the protection against disease in subjects with HIV infection. How to elicit HIV-specific CD4+ T cell responses in a given population using vaccines is one of the major areas of current HIV vaccine research. To design vaccine that targets specifically Chinese, we assembled a database that is comprised of sequences from 821 Chinese HIV isolates and 46 human leukocyte antigen (HLA) DR alleles identified in Chinese population. We then predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and also Chinese HLA-DR types. We suggest that testing this vaccine experimentally will facilitate the development of a CD4+ T cell vaccine especially catered for Chinese.