Herpes zoster (HZ), caused by reactivation of varicella-zoster virus (VZV), imposes a substantial health burden, particularly in immunocompromised individuals. Although current HZ vaccines provide effective protection, the live-attenuated vaccine shows age-dependent and waning efficacy, whereas the recombinant zoster vaccine relies on a complex adjuvant system that may present manufacturing and supply challenges. mRNA vaccines offer a promising alternative for HZ prevention, but their efficacy depends on optimized lipid nanoparticle (LNPs) that protect mRNA and support efficient intracellular delivery. Here, we formulated LNPs for an mRNA encoding VZV glycoprotein E by partially or completely replacing cholesterol with two cholesterol-related sterols, the plant-derived sterol β-sitosterol and the immunomodulatory metabolite dendrogenin A (DDA). We found that high-level DDA substitution (≥50%) impaired both antigen-specific antibody responses and T-cell immunity. By contrast, complete β-sitosterol replacement (100% SS-LNPs) enhanced EGFP reporter signals in vitro and in vivo and produced numerically higher antigen-specific T-cell responses than cholesterol-based LNPs (Chol-LNPs), with significantly increased CD4+ T-cell polyfunctionality. Transcriptomic analysis further showed that the immune signatures induced by 100% SS-LNPs largely overlapped with those induced by Chol-LNPs, without evidence of a distinct inflammatory transcriptional program. Together, these findings identify β-sitosterol substitution as a promising formulation strategy for VZV mRNA-LNP development.

Virus transport in groundwater is a key pathway contributing to waterborne disease outbreaks and poses significant risks to public health. However, the mechanisms governing virus transport and attenuation remain poorly understood. Here, using the human enteric virus Coxsackievirus (CV-A16), we coupled experiments with a dual-marker approach (TCID50 and RT-qPCR) and a stepwise modeling strategy to quantify virus inactivation, interfacial inactivation, and adsorption during virus transport and attenuation in heterogeneous sediments. Static batch experiments revealed markedly slower CV-A16 inactivation in the aqueous phase, requiring 106-191 days to achieve a 90% loss of infectivity, compared with about 12 days in saturated sediments. Column experiments indicated that kinetic adsorption-desorption was the dominant attenuation mechanism, while surface-associated interfacial inactivation represented an additional important pathway that increased with virus adsorption, reflecting a coupled adsorption-inactivation process. Further analysis showed that inactivation rate constants ranged from 0.002 to 0.004 min-1 from coarse sand to silt, suggesting enhanced virus-solid interface reactivity in finer sediments. Furthermore, bimodal particle-size distributions from laser particle sizing and TEM observations of virus clusters indicate the formation of aggregates during transport, while zeta potential variations suggest that this process most likely occurs during the early to intermediate stages of transport. By evaluating the interplay between physical retention and biological inactivation, this study provides a mechanistic framework to improve the prediction of virus transport and the accuracy of pathogen-related health risk assessments in subsurface environments.

Introduction:Vaccination remains one of the key tools to prevent influenza pandemic. The influenza vaccine induces durable cross-subtype protection through T-cell immunity, demonstrating significant future potential. DNA vaccines are effective in sustaining the expression of antigens, which can trigger T-cell immune responses. Calcium phosphate nanoparticles can also induce T-cell immune responses by assisting in the activation of DC cells by antigens. Methods:This study developed silica-coated calcium phosphate nanoparticles (226 nm) encapsulating influenza hemagglutinin plasmids (pHAF/pHAG) via polyethyleneimine adsorption. Further analysis of its bioactivity was conducted through experiments. Results:The nanoparticles demonstrated excellent stability (PDI<0.3 for 7 days), efficient pDNA encapsulation (confirmed by UV), and sustained release (93.14% ± 4.12% at 72 h). DC2.4 cells uptake assays revealed significant antigen-presenting cell internalization (p<0.0001). BALB/c mice were immunized subcutaneously using a prime-boost-boost regimen at two-week intervals. Splenocyte analysis revealed sustained elevation of CD4+ and CD8+ T cell proportions (p<0.05) at 12 weeks post-immunization, suggesting nanoparticle-induced durable T cell immunity. Post-immunization challenge with heterologous H3N2 revealed striking protection: SCPs/pHAF conferred 100% survival, while SCPs/pHAG achieved 66% survival. Notably, SCPs/pDNA immunization significantly reduced lung viral titers versus controls (p<0.05), demonstrating robust cross-subtype protection against lethal infection. Discussion:This study establishes a significant conceptual framework for advancing the development of DNA-based influenza vaccines with sustained protective efficacy.

Commercial live attenuated influenza vaccines (LAIVs) usually contain a high proportion of defective interfering particles (DIPs). Given that LAIVs are not sufficiently protective worldwide, the potential to enhance their efficacy by reducing the proportion of DIPs remains largely unknown. In this study, a prepared H3N2 cold-adapted LAIV with a low proportion of DIPs exhibits delayed yet improved replication in the upper respiratory tract of mice. The low DIPs LAIV induces an increase in goblet cells, microfold cells, and neutrophils, along with enhanced antigen presentation by dendritic cells. Compared to the commercially sourced high DIPs LAIV, the low DIPs LAIV elicits enhanced mucosal and humoral immune responses, facilitates cross-neutralization in mice, and provides complete protection against lethal challenges with H3N2, H1N1 or H1N1pdm09 strains. This study offers insights into optimizing commercial LAIVs and replicative RNA virus-based vaccines by controlling DIPs.

IntroductionReactivation of the varicella-zoster virus (VZV) results in herpes zoster (HZ), which can lead to complications such as postherpetic neuralgia. The commercially available HZ subunit adjuvanted vaccine, Shingrix®, offers significant protection against HZ in older adults. However, the adjuvant system of this vaccine has limitations that necessitate the development of alternative adjuvant systems.MethodsIn this study, we established a novel adjuvant system, BK-02, composed of both the Toll-like receptor 9 (TLR9) agonist BK-02C (CpG2006) and a squalene-based oil-in-water emulsion, BK-02M (MF59), using ELISA, ELISpot, and flow cytometry analyses. ResultsOur results showed that when combined with glycoprotein E (gE), the active ingredient of a recombinant HZ vaccine, the BK-02 adjuvant system elicited significantly higher gE-specific IFN-γ+ T-cell responses (486 SFU/10⁶ cells, 121-fold increase vs gE alone) and IgG antibody titers (Lg titers 5.2 vs 3.4 for gE alone). The optimal dose (5 μg gE + 30 μg BK-02C + 1× BK-02M) for inducing gE protein-specific cellular immunity was determined in mice. This corresponded to a clinical dose of “50 μg gE + 300/500 μg BK-02C + 0.5 mL BK-02M.” Additionally, pilot-scale samples of the recombinant HZ vaccine demonstrated enhanced gE-specific CD4+ and CD8+ T-cell immune responses, compared to Shingrix®. Moreover, the gE/BK-02 adjuvant system induced a Th1-regulated mixed immune response, enabling robust cellular and humoral immunity. DiscussionThese findings indicated that the BK-02 adjuvant system is a promising adjuvant candidate for the current HZ subunit vaccines.

IntroductionUnderstanding the key factors that enable bacterial pathogens to adapt to new hosts is crucial, as host-microbe interactions not only influence host health but also drive bacterial genome diversification, thereby enhancing pathogen survival in various ecological niches.MethodsWe conducted a comparative genomic analysis of 4,366 high-quality bacterial genomes isolated from various hosts and environments. Bioinformatics databases and machine learning approaches were used to identify genomic differences in functional categories, virulence factors, and antibiotic resistance genes across different ecological niches.ResultsSignificant variability in bacterial adaptive strategies was observed. Human-associated bacteria, particularly from the phylum Pseudomonadota, exhibited higher detection rates of carbohydrate-active enzyme genes and virulence factors related to immune modulation and adhesion, indicating co-evolution with the human host. In contrast, bacteria from environmental sources, particularly those from the phyla Bacillota and Actinomycetota, showed greater enrichment in genes related to metabolism and transcriptional regulation, highlighting their high adaptability to diverse environments. Bacteria from clinical settings had higher detection rates of antibiotic resistance genes, particularly those related to fluoroquinolone resistance. Animal hosts were identified as important reservoirs of resistance genes. Key host-specific bacterial genes, such as hypB, were found to potentially play crucial roles in regulating metabolism and immune adaptation in human-associated bacteria.DiscussionThese findings highlight niche-specific genomic features and adaptive mechanisms of bacterial pathogens. This study provides valuable insights into the genetic basis of host-pathogen interactions and offers evidence to inform pathogen transmission control, infection management, and antibiotic stewardship.

Current seasonal influenza vaccines offer limited protection against influenza viruses due to genetic drift. The urgent need for a universal influenza vaccine to combat highly mutated strains is evident. This study utilized the conserved HA2 subunit of hemagglutinin (HA) and a short linear epitope of HA2 (HA2-16) from the H3 influenza virus to conjugate with ferritin, resulting in the construction of recombinant immunogens termed HA2-F and HA2-16-F, respectively. In vitro characterization confirmed the self-assembly of prokaryotically expressed HA2-F and HA2-16-F into nanoparticles (NPs). To simulate natural virus infection in the vaccinated population, intranasal infection with the whole H3N2 virus was administered as a final boost. Enhanced binding activity to A/Hong Kong/4801/2014 (H3N2) and A/17/California/2009/38 (H1N1) virus was detected in the HA2-16 group induced by the A/Wisconsin/67/2005 (H3N2) virus boost (Titer >10(4)). Furthermore, higher titers of neutralizing antibodies were elicited by HA2-16-F NP (ID50: 50.4-631.0) compared to those by HA2-F NP (ID50: 20.3-178.2). These results demonstrated that the H3N2 virus boost focused the antibody response on the HA2-16 epitope. Additionally, our immunization strategy was found to reduce serum ferritin reactive antibodies. In summary, HA2-16 not only holds promise as a vaccine candidate but also exhibits significant potential for influenza vaccine production, particularly in enhancing the levels of induced stem-directed antibodies. This study contributes to the development of recombinant immunogens for improved influenza vaccine efficacy.

Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors’ infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeLa-based cell lines that stably encode the rep and cap genes from AAV serotype 2. Co-infection with wild-type (WT) adenoviruses provides the helper functions for expression of these genes, and the use of quantitative PCR (qPCR)/droplet digital PCR (ddPCR) serves as the endpoint method for the detection of infectious events. However, TCID50 assays using these HeLa-based rep cap trans-complementing cell lines have traditionally been regarded as challenging due to high variability, stability of the integrated genes, and safety concerns associated with the use of WT helper viruses. Here we developed a novel method for infectious titration of rAAV using our vector “tetracycline-enabled self-silencing adenovirus” (TESSA); we engineered it to deliver and express the AAV2 rep genes and adenoviral helper functions for rAAV genome replication, independent of the cell type. This approach allows the infectious titration of rAAV serotypes in cell lines permissive to adenovirus but without the production of adenoviral particles for improved safety, therefore benefiting GMP analytical requirements for rAAV gene therapies.

Purpose:Tick-borne encephalitis virus (TBEV) infections result in severe central nervous system diseases in humans across Asia and Europe. In China, cases of tick-borne encephalitis are primarily caused by the Far East subtype of TBEV, which exhibits a distinct disease course compared to other extensively studied subtypes. However, there is limited knowledge regarding the nucleic acid and serological diagnostic characteristics of patients infected with the TBEV in China, which is the focus of investigation in the present study. Methods:This study established a TaqMan qPCR approach to detect TBEV RNA in the serum with optimal specificity, sensitivity, and precision. Using TaqMan qPCR and ELISA assay for TBEV IgM detection, serum samples from 63 hospitalized patients bitten by ticks in Northeast China were investigated for diagnostic characteristics. Results:Twenty-five patients were positive for viral RNA; nineteen patients were positive for IgM, and nine were positive for both viral RNA and IgM. Through comparative analysis, TBEV RNA copies were negatively correlated with the virus incubation period. IgM levels were positively correlated with the clinical symptom scores of patients. The severity of clinical symptoms and the length after the tick bite could be used to predict the IgM occurrence. Furthermore, IgM levels and viral RNA copies were not correlated in double-positive patients. Conclusion:Both nucleic acid and serological detection methods exhibited distinct windows for detecting TBEV infection, with some overlap, and were associated with specific correlated factors. This study provided novel insights into the diagnosis and course of TBEV-induced tick-borne encephalitis in China.

Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Drinking groundwater contamination by pathogenic viruses represents a serious risk to worldwide public health, particularly for enteric viruses, which exhibit high prevalence and occurrence during outbreaks. Understanding how enteric viruses adsorb in groundwater is essential to protecting human health and ensuring the sustainable use of water resources. The adsorption properties of Coxsackievirus A16 (CA16), a common gastrointestinal virus that spreads through groundwater, were investigated in this work. A typical batch equilibrium approach was used to investigate CA16 adsorption and factors that influence it. In a laboratory recognized nationally as a biosafety level 2 facility, stringent research protocols were followed to guarantee compliance with experimental standards. The variables that were investigated included the size of the sediment particles, the starting concentration of the virus, temperature, pH level, and humic acid content. The findings showed that the CA16 virus was more strongly attracted to finer sediment particles and that its adsorption increased as the size of the sediment particle decreased. Furthermore, it was discovered that higher temperatures improved the CA16 virus’s ability to bind to sediment particles. The pH of the aqueous environment has a significant effect on the effectiveness of virus adsorption; higher effectiveness was seen in acidic environments. Furthermore, it was found that the presence of humic acid decreased the ability of clay to adsorb CA16, suggesting that humic acid has a detrimental influence on clay’s ability to adsorb viruses. The examination of kinetic models demonstrated that, in every scenario examined, the adsorption process of CA16 adhered to the pseudo-second-order kinetics model. Additionally, the Langmuir and Freundlich isotherm models were used to assess the equilibrium data that were collected in this investigation. The outcomes amply proved that the most accurate representation of the adsorption equilibrium was given by the Langmuir isotherm model. The study offered a solid scientific foundation for treating groundwater and creating plans to stop the spread of viruses.

Genital herpes (GH) has become one of the most common sexually transmitted diseases worldwide, and it is spreading rapidly in developing countries. Approximately 90% of GH cases are caused by HSV-2. Therapeutic HSV-2 vaccines are intended for people already infected with HSV-2 with the goal of reducing clinical recurrences and recurrent virus shedding. In our previous work, we evaluated recombinant adenovirus-based vaccines, including rAd-gD2ΔUL25, rAd-ΔUL25, and rAd-gD2, for their potency as prophylactic vaccines. In this study, we evaluated these three vaccines as therapeutic vaccines against acute and recurrent diseases in intravaginal challenged guinea pigs. Compared with the control groups, the recombinant vaccine rAd-gD2ΔUL25 induced a higher titer of the binding antibody, and rAd-gD2 + rAd-ΔUL25 induced a higher titer of the neutralizing antibody. Both rAd-gD2ΔUL25 and rAd-gD2 + rAd-ΔUL25 vaccines significantly enhanced the survival rate by 50% compared to rAd-gD2 and reduced viral replication in the genital tract and recurrent genital skin disease. Our findings provide a new perspective for HSV-2 therapeutic vaccine research and provide a new technique to curtail the increasing spread of HSV-2.

HIV-1 vaccines have been challenging to develop, partly due to the high level of genetic variation in its genome. Thus, a vaccine that can induce cross-reactive neutralization activities will be needed. Studies on the co-evolution of antibodies and viruses indicate that mimicking the natural infection is likely to induce broadly neutralizing antibodies (bnAbs). We generated the consensus Env sequence for each time point in subject CH505, who developed broad neutralization activities, and selected five critical time points before broad neutralization was detected. These consensus sequences were designed to express stable Env trimers. Priming with the transmitted/founder Env timer and sequential boosting with these consensus Env trimers from different time points induced broader and more potent neutralizing activities than the BG505 Env trimer in guinea pigs. Analysis of the neutralization profiles showed that sequential immunization of Env trimers favored nAbs with gp120/gp41 interface specificity while the BG505 Env trimer favored nAbs with V2 specificity. The unique features such as consensus sequences, stable Env trimers and the sequential immunization to mimic natural infection likely has allowed the induction of improved neutralization responses.

Efficient manufacture of recombinant adeno-associated virus (rAAV) vectors for gene therapy remains challenging. Packaging cell lines containing stable integration of the AAV rep/cap genes have been explored, however rAAV production needs to be induced using wild-type adenoviruses to promote episomal amplification of the integrated rep/cap genes by mobilizing a cis -acting replication element (CARE). The adenovirus proteins responsible are not fully defined, and using adenovirus during rAAV manufacture leads to contamination of the rAAV preparation. ‘TESSA’ is a helper adenovirus with a self-repressing Major Late Promoter (MLP). Its helper functions enable efficient rAAV manufacture when the rep and cap genes are provided in trans but is unable to support rAAV production from stable packaging cells. Using rAAV-packaging cell line HeLaRC32, we show that expression of the adenovirus L4 22/33K unit is essential for rep/cap amplification but the proteins are titrated away by binding to replicating adenovirus genomes. siRNA-knockdown of the adenovirus DNA polymerase or the use of a thermosensitive TESSA mutant decreased adenovirus genome replication whilst maintaining MLP repression, thereby recovering rep/cap amplification and efficient rAAV manufacture. Our findings have direct implications for engineering more efficient adenovirus helpers and superior rAAV packaging/producer cells.

Cold adapted live attenuated influenza vaccines can effectively prevent human disease and death caused by influenza virus. Since chicken embryos are used as the culture substrate for the large-scale production of influenza vaccines, cold adapted live attenuated influenza vaccines may be contaminated by exogenous avian viruses. Rapid and sensitive methods such as TaqMan-based quantitative PCR are needed for the detection of exogenous avian viruses during cold adapted live attenuated influenza vaccines production. In this study, a TaqMan-based quantitative PCR method was established for the detection of three common exogenous avian viruses, including fowl adenovirus type I, type Ⅲ and avian leukosis virus. Avian virus-encoding plasmids purified in high-performance liquid chromatography were essential for sensitivity analysis. The sensitivity reached 1 copy per reaction for each of the avian virus plasmids. Standard curves showed a strong linear relationship. The TaqMan-based quantitative PCR method had high specificity and no cross-reactivity with other irrelevant viruses. Furthermore, the established TaqMan-based quantitative PCR can effectively detect 0.1 TCID50 of each avian virus without or with interference from the influenza virus nucleic acid. Ultimately, this method was used to test three master seed lots of monovalent cold adapted live attenuated influenza vaccine, and the results showed that no fowl adenovirus type I, type Ⅲ or avian leukosis virus contamination, which were consistent with serological methods. The TaqMan-based quantitative PCR method for the determination of extraneous avian viruses in cold adapted live attenuated influenza vaccines met the requirement for both conventional and emergency inspection on cold adapted live attenuated influenza vaccines.

Rabies virus (RV) is an acute zoonotic infectious disease characterized by invariably lethal encephalitis of viral origin. Currently available vaccines are cumbersome to produce, required intensive immunization to induce and maintain protective immunity. DNA vaccination is an efficient strategy for rabies prophylaxis. In this study, we designed three constructs wherein the RV glycorpotein (G protein) ectodomain was cloned as mRVG∆t and expressed as a foldon trimerization domain fusion protein (tRVG∆t) with different linkers. Compared with mRVG∆t, tRVG∆t possessed more trimer-specific binding sites. Mice immunized with tRVG∆t elicited 1.4 folds higher RV neutralizing antibody responses compared to that with mRVG∆t. Antibody isotyping and cytokine ELISA results indicate that the tRVG∆t DNA vaccine could boost cellular immune responses. tRVG∆t immunization also protected 83% of mice from RV challenge 14 days post-vaccination. Thus, our version of the RV DNA vaccine can guide and assist future studies on related vaccines.

Genital herpes caused by herpes simplex virus type 2 (HSV-2) poses a global health issue. HSV-2 infection increases the risk of acquiring HIV infection. Studies have demonstrated that HSV-2 subunit vaccines have potential benefits, but require adjuvants to induce a balanced Th1/Th2 response. To develop a novel, effective vaccine, in this study, a truncated glycoprotein D (aa 1-285) of HSV-2 was formulated with an Al(OH)3 adjuvant, three squalene adjuvants, zMF59, zAS03, and zAS02, or a mucosal adjuvant, bacterium-like particles (BLPs). The immunogenicity of these subunit vaccines was evaluated in mice. After three immunizations, vaccines formulated with Al(OH)3, zMF59, zAS03, and zAS02 (intramuscularly) induced higher titers of neutralizing antibody than that formulated without adjuvant, and in particular, mice immunized with the vaccine plus zAS02 had the highest neutralizing antibody titers and tended to produce a more balanced immune reaction than others. Intranasal gD2-PA-BLPs also induced excellent IgA levels and a more balanced Th1 and Th2 responses than intranasal gD2. After challenge with a lethal dose of HSV-2, all five adjuvants exhibited a positive effect in improving the survival rate. zAS02 and gD2-PA-BLPs enhanced survival by 50% and 25%, respectively, when compared with the vaccine without adjuvant. zAS02 was the only adjuvant that resulted in complete vaginal virus clearance and genital lesion healing within eight days. These results demonstrate the potential of using zAS02 as a subunit vaccine adjuvant, and BLPs as a mucosal vaccine adjuvant.