Efficacy evaluation of a bivalent subunit vaccine against epidemic PEDV heterologous strains with low cross-protection.

Variant Porcine epidemic diarrhea virus (PEDV), which causes diarrhea and high mortality in piglets, has become a major pathogen, and co-epidemics of different subtypes of the virus have become a very thorny problem for the clinical prevention and control of PEDV. However, cross-protection between epidemic G2a and G2b subtype strains has not been observed, and there is currently no vaccine against both G2a and G2b strains. In this study, we demonstrate the low cross-protection between G2a and G2b strains with piglet immunization and challenge tests. The trimeric full-length S proteins of G2a and G2b variants were purified and a bivalent subunit vaccine against PEDV G2a/G2b-S was developed. In active and passive immune protection tests, the bivalent subunit vaccine produced high neutralizing antibody titers and S-specific immunoglobulin G (IgG) and IgA titers against both the G2a and G2b strains in piglets and sows. In the attack phase of the viruses, the clinical symptoms and microscopic lesions in the immunized groups were significantly alleviated. Importantly, the PEDV G2a/G2b-S bivalent subunit vaccine conferred effective passive immunity against PEDV G2a and G2b challenges in the form of colostrum-derived antibodies from the immunized sows. In conclusion, our data demonstrate the low cross-protection of PEDV epidemic G2a and G2b strains and show that the G2a/G2b-S bivalent subunit vaccine is protective against both G2a and G2b strains. It is therefore a candidate vaccine for PEDV prevention. IMPORTANCE: The detection rate of PEDV G2a subtype strains is currently increasing. Although commercial vaccines are available, most vaccines do not exert an ideal protective effect against these strains. Furthermore, there is no definitive research into the cross-protection between G2a and G2b strains, and no bivalent vaccine provides joint protection against both. Therefore, in this study, we investigated the cross-protection between PEDV G2a and G2b strains and designed a candidate bivalent subunit vaccine combining the trimeric S proteins of the G2a and G2b subtypes. We demonstrate that the cross-protection between strains G2a and G2b is poor and that this bivalent subunit vaccine protects piglets from viral attack by inducing both active and passive immunity. This study emphasizes the effectiveness of the PEDV G2a/G2b-S bivalent subunit vaccine and provides a feasible method for the development of efficient PEDV vaccines.

Designing a multi-epitope subunit vaccine against Orf virus using molecular docking and molecular dynamics.

Orf virus (ORFV) is an acute contact, epitheliotropic, zoonotic, and double-stranded DNA virus that causes significant economic losses in the livestock industry. The objective of this study is to design an immunoinformatics-based multi-epitope subunit vaccine against ORFV. Various immunodominant cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B-cell epitopes from the B2L, F1L, and 080 protein of ORFV were selected and linked by short connectors to construct a multi-epitope subunit vaccine. Immunogenicity was enhanced by adding an adjuvant ß-defensin to the N-terminal of the vaccine using the EAAAK linker. The vaccine exhibited a significant degree of antigenicity and solubility, without allergenicity or toxicity. The 3D formation of the vaccine was subsequently anticipated, improved, and verified. The optimized model exhibited a lower Z-score of -4.33, indicating higher quality. Molecular docking results demonstrated that the vaccine strongly binds to TLR2 and TLR4. Molecular dynamics results indicated that the docked vaccine-TLR complexes were stable. Immune simulation analyses further confirmed that the vaccine can induce a marked increase in IgG and IgM antibody titers, and elevated levels of IFN-γ and IL-2. Finally, the optimized DNA sequence of the vaccine was cloned into the vector pET28a (+) for high expression in the E.coli expression system. Overall, the designed multi-epitope subunit vaccine is highly stable and can induce robust humoral and cellular immunity, making it a promising vaccine candidate against ORFV.

Synergistic immune augmentation enabled by covalently conjugating TLR4 and NOD2 agonists.

Enhancing the efficacy of subunit vaccines relies significantly on the utilization of potent adjuvants, particularly those capable of triggering multiple immune pathways. To achieve synergistic immune augmentation by Toll-like receptor 4 agonist (TLR4a) and nucleotide-binding oligomerization-domain-containing protein 2 agonist (NOD2a), in this work, we conjugated RC529 (TLR4a) and MDP (NOD2a) to give RC529-MDP, and evaluated its adjuvanticity for OVA antigen. Compared to the unconjugated RC529+MDP, RC529-MDP remarkably enhanced innate immune responses with 6.8-fold increase in IL-6 cytokine, and promoted the maturation of antigen-presenting cells (APCs), possibly because of the conjugation of multiple agonists ensuring their delivery to the same cell and activation of various signaling pathways within that cell. Furthermore, RC529-MDP improved OVA-specific antibody response, T cells response and the memory T cells ratio relative to the unconjugated mixture. Therefore, covalently conjugating TLR4 agonist and NOD2 agonist was an effective strategy to enhance immune responses, providing the potential to design and develop more effective vaccines.

Enhanced immune response with baculovirus-expressed BoHV-1 glycoprotein D in vaccine development.

Bovine herpesvirus 1 (BoHV-1), a significant pathogen in the alpha-herpesvirus subfamily, primarily infects cattle and causes the upper respiratory disease known as infectious bovine rhinotracheitis (IBR). In silico studies evaluated the BoHV-1 D protein to be non-allergenic, non-toxic, and highly antigenic, highlighting its potential as an antigen for vaccine development. Therefore, this study aimed to evaluate the efficacy of a subunit vaccine using the ectodomain of glycoprotein D (gD34-380) as an antigen. The truncated gD was successfully cloned and expressed in both Escherichia coli (E. coli, termed EgD) and baculovirus (termed BgD) systems, with expected molecular weights of 65 kDa and 50 kDa, respectively. For the vaccine formulation, the gD proteins were used either alone or in combination with in-house inactivated BoHV-1. Vaccination of mice and bovines showed that baculovirus-expressed gD34-380 accelerated the antibody response. Moreover, the BgD-vaccinated group also showed significantly higher neutralizing antibody levels against BoHV-1 than the control group (p<0.0001). In conclusion, our study found that BgD from BoHV-1 can increase the immune response and enhance vaccine efficacy.

A Magnetically Driven Biodegradable Microsphere with Mass Production Capability for Subunit Vaccine Delivery and Enhanced Immunotherapy.

Subunit vaccines have emerged as a promising strategy in immunotherapy for combating viral infections and cancer. Nevertheless, the clinical application of subunit vaccines is hindered by limitations in antigen delivery efficiency, characterized by rapid clearance and inadequate cellular uptake. Here, a novel subunit vaccine delivery system utilizing ovalbumin@magnetic nanoparticles (OVA@MNPs) encapsulated within biodegradable gelatin methacryloyl (GelMA) microspheres was proposed to enhance the efficacy of antigen delivery. OVA@MNPs-loaded GelMA microspheres, denoted as OMGMs, can be navigated through magnetic fields to deliver subunit vaccines into the lymphatic system efficiently. Moreover, the biodegradable OMGMs enabled the sustained release of subunit vaccines, concentrating OVA around lymph nodes and enhancing the efficacy of induced immune response. OMGMs were produced through a microfluidic droplet generation technique, enabling mass production. In murine models, OMGMs successfully accumulated antigens in lymph nodes abundant in antigen-presenting cells, leading to enhanced cellular and humoral immunity and pronounced antitumor effects with a single booster immunization. In conclusion, these findings highlight the promise of OMGMs as a practical subunit vaccination approach, thus addressing the limitations associated with antigen delivery efficiency and paving the way for advanced immunotherapeutic strategies.

Enhancing protective immunity against bacterial infection via coating nano-Rehmannia glutinosa polysaccharide with outer membrane vesicles.

With the coming of the post-antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non-replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live-attenuated vaccines. Here, we developed a novel nano-vaccine by coating OMVs onto PEGylated nano-Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL-OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL-OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial-specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T-cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV-coated nano-carriers in antimicrobial immunotherapy.

Phase I Clinical Study of the Subunit Betulin-Adjuvanted Tetravalent Candidate Influenza Vaccine TetraFluBet.

OBJECTIVES: This study aimed to determine the safety, tolerability and immunogenicity of TetraFluBet, an inactivated subunit influenza vaccine that contains a corpuscular immuno-adjuvant derived from natural betulin. METHODS: We conducted a prospective, randomized, open-labeled, single-center, phase I trial. The study was conducted in two stages: 5 volunteers in stage I and 25 volunteers in stage II. Eligible participants received one single dose (20 µg/0.5 mL) of TetraFluBet intramuscularly. Participants were followed for adverse events and reactogenicity. Seroconversion rate, seroprotection level, geometric mean titers (GMTs) of virus-neutralizing antibodies, IFN-γ induction and cell-mediated immunity were assessed. RESULTS: A total of 30 participants were enrolled. No vaccine-related serious adverse events were observed. The proportions of study participants with 4-fold seroconversions assessed by the HI assay (with 95% CIs) were 80.0% (62.7; 90.5), 70.0% (52.1; 83.3), 63.3% (45.5; 78.1) and 73.3% (55.6; 85.8) for influenza virus subtypes A (H1N1), A (H3N2), B1 and B2, respectively. Seroprotection levels (with 95% CIs) were 83.3% (66.4; 92.7), 83.3% (66.4; 92.7), 73.3% (55.6; 85.8) and 66.7% (48.8; 80.8), respectively. The fold increases in the GMTs of virus-neutralizing antibodies for subtype H1N1 was 6.50, for subtype H3N2 was 3.03, for subtype B1 was 3.56 and for subtype B2 was 6.07. The population of cytotoxic T-cells increased significantly in the post-vaccination period, indicating a strong CD3+CD8+ response. CONCLUSIONS: TetraFluBet tetravalent inactivated subunit vaccine with adjuvant demonstrated pronounced immunogenic properties, leading to the formation of both specific humoral and cellular immunity at a 20 µg dose.

Effects of Rotavirus NSP4 on the Immune Response and Protection of Rotavirus-Norovirus Recombinant Subunit Vaccines in Different Immune Pathways.

Diarrheal disease continues to be a major cause of global morbidity and mortality among children under 5 years of age. To address the current issues associated with oral attenuated rotavirus vaccines, the study of parenteral rotavirus vaccines has promising prospects. In our previous study, we reported that rotavirus nonstructural protein 4 (NSP4) did not increase the IgG antibody titer of co-immune antigen but did have a protective effect against diarrhea via the intramuscular injection method. Here, we explored whether NSP4 can exert adjuvant effects on mucosal immune pathways. In this study, we immunized mice via muscle and nasal routes, gavaged them with the rotavirus Wa strain or the rotavirus SA11 strain, and then tested the protective effects of immune sera against both viruses. The results revealed that the serum-specific VP8* IgG antibody titers of the mice immunized via the nasal route were much lower than those of the mice immunized by intramuscular injection, and the specific IgA antibodies were almost undetectable in the bronchoalveolar lavage fluid (BALF). NSP4 did not increase the titer of specific VP8* antibodies in either immune pathway. Therefore, in the two vaccines (PP-NSP4-VP8* and PP-VP8*+NSP4) used in this study, NSP4 was unable to perform its potential adjuvant role through the mucosal immune pathway. Instead, NSP4 was used as a co-immunized antigen to stimulate the mice to produce specific binding antibodies that play a protective role against diarrhea.

Effectively Evaluating a Novel Consensus Subunit Vaccine Candidate to Prevent the H9N2 Avian Influenza Virus.

The enormous effects of avian influenza on poultry production and the possible health risks to humans have drawn much attention to this disease. The H9N2 subtype of avian influenza virus is widely prevalent among poultry, posing a direct threat to humans through infection or by contributing internal genes to various zoonotic strains of avian influenza. Despite the widespread use of H9N2 subtype vaccines, outbreaks of the virus persist due to the rapid antigenic drift and shifts in the influenza virus. As a result, it is critical to develop a broader spectrum of H9N2 subtype avian influenza vaccines and evaluate their effectiveness. In this study, a recombinant baculovirus expressing the broad-spectrum HA protein was obtained via bioinformatics analysis and a baculovirus expression system (BES). This recombinant hemagglutinin (HA) protein displayed cross-reactivity to positive sera against several subbranch H9 subtype AIVs. An adjuvant and purified HA protein were then used to create an rHA vaccine candidate. Evaluation of the vaccine demonstrated that subcutaneous immunization of the neck with the rHA vaccine candidate stimulated a robust immune response, providing complete clinical protection against various H9N2 virus challenges. Additionally, virus shedding was more effectively inhibited by rHA than by the commercial vaccine. Thus, our findings illustrate the efficacy of the rHA vaccine candidate in shielding chickens against the H9N2 virus challenge, underscoring its potential as an alternative to conventional vaccines.

Cost-Effectiveness Analysis of Herpes Zoster Vaccination in a Chinese Population: Recombinant Subunit Vaccine versus Live Attenuated Vaccine.

BACKGROUND: Currently, the recombinant subunit vaccine and live attenuated vaccine in the prevention of herpes zoster are approved for marketing in China. This study aims to evaluate the cost-effectiveness of the recombinant subunit vaccine and the live attenuated vaccine in the Chinese population. METHODS: A decision tree-Markov analysis model was utilized to estimate expected costs and quality-adjusted life years (QALYs), comparing the lifetime cost-effectiveness of vaccination with the recombinant subunit vaccine (London, United Kingdom, Shingrix, GSK) to that of the live attenuated vaccine (Changchun, China, Ganwei, Changchun Bcht) in the Chinese population, with the primary outcome measure being the incremental cost-effectiveness ratio (ICER). RESULTS: In the base-case analysis, the ICERs for the recombinant subunit vaccine ranged by age from USD 3428 to USD 5743 per QALY, while the ICERs for the live attenuated vaccine ranged from USD 4017 to USD 18,254 per QALY, compared with no vaccination. Among all age groups, the category of 60 to 69 years was the optimal age for vaccination. The results were most sensitive to changes in herpes zoster incidence, vaccine efficacy, and discount rate. Even with a two-dose compliance rate of 20% for the recombinant subunit vaccine, vaccination remained cost-effective. ZVL would need to reduce costs by at least 12.2% compared to RZV to have a cost-effectiveness advantage. CONCLUSIONS: The recombinant subunit vaccine and the live attenuated vaccine were both cost-effective in the Chinese population, but, relatively, the recombinant subunit vaccine had a greater advantage in disease prevention and cost-effectiveness in all age groups above 50 years.

A Rationally Designed H5 Hemagglutinin Subunit Vaccine Provides Broad-Spectrum Protection against Various H5Nx Highly Pathogenic Avian Influenza Viruses in Chickens.

The evolution of the H5 highly pathogenic avian influenza (HPAI) viruses has led to the emergence of distinct groups with genetically similar clusters of hemagglutinin (HA) sequences. In this study, a consensus H5 HA sequence was cloned into the baculovirus expression system. The HA protein was expressed in baculovirus-infected insect cells and utilized as the antigen for the production of an oil emulsion-based H5 avian influenza vaccine (rBacH5Con5Mut). Twenty-one-day-old SPF chickens were immunized with this vaccine and then challenged at 21 days post-vaccination with clade 2.3.2.1, clade 2.3.4.4, and clade 7.2 of H5 HPAI viruses. The sera of vaccinated chickens exhibited high hemagglutination inhibition (HI) titers against the rBacH5 vaccine antigen, while lower HI titers were observed against the different challenge virus H5 hemagglutinins. Furthermore, the rBacH5Con5Mut vaccine provided 100% protection from mortality and clinical signs. Virus isolation results showed that oropharyngeal and cloacal shedding was prevented in 100% of the vaccinated chickens when challenged with clade 2.3.2.1 and clade 2.3.4.4 H5 viruses. When the rBacH5Con5Mut vaccine candidate was administrated at one day of age, 100% protection was demonstrated against the challenge of clade 2.3.4.4 virus at three weeks of age, indicating the potential of this vaccine for hatchery vaccination. Overall, A single immunization of rBacH5Con5Mut vaccine candidate with a consensus HA antigen can protect chickens against different clades of H5 HPAI viruses throughout the rearing period of broiler chickens without a boost, thus fulfilling the criteria for an efficacious broad-spectrum H5 avian influenza vaccine.

An immunoinformatics approach for design and validation of multi-subunit vaccine against Plasmodium falciparum from essential hypothetical proteins.

Malaria, caused by Plasmodium falciparum, remains a pressing global health concern. Advancements in combating this parasite involve the development of a protein vaccine. This study employs immunoinformatics to identify potential vaccine candidates within the repertoire of 218 P. falciparum exported essential proteins identified through saturaturation mutagenesis study. Our screening approach narrows down to 65 Plasmodium-exported proteins with uncharacterized functions while exhibiting non-mutability in CDS (coding sequences). The transmembrane helix, antigenicity, allergenicity of the shortlisted proteins was assessed through diverse prediction algorithm, culminating in the identification of five promising vaccination contenders, based on probability scores. We discerned B-cell, helper T-lymphocyte, and cytotoxic T-lymphocyte epitopes. Two proteins with the most favorable epitope were harnessed to construct a multi-subunit vaccine, through judicious linker integration. Employing the I-TASSER software, three-dimensional models of the constituent proteins was obtained and was validated using diverse tools like ProSA, VERIFY3D, and ERRAT. The modelled proteins underwent Molecular Dynamics (MD) simulation in a solvent environment to evaluate the stability of the multi-subunit vaccine. Furthermore, we conducted molecular docking through the ClusPro web server to elucidate potential interactions with Toll-like receptors (TLR2 and TLR4). Docking scores revealed a pronounced affinity of the multi-subunit vaccine for TLR2. Significantly, 100 ns MD simulation of the protein-receptor complex unveiled a persistent hydrogen bond linkage between the ARG63 residue of the sub-unit vaccine and the GLU32 residue of the TLR2 receptor. These findings collectively advocate the potential efficacy of the first multi-subunit vaccine from the potential hypothetical proteins of P. falciparum. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01696-w.

Cationic pH-sensitive liposome-based subunit tuberculosis vaccine induces protection in mice challenged with Mycobacterium tuberculosis.

Tuberculosis (TB) has been and still is a global emergency for centuries. Prevention of disease through vaccination would have a major impact on disease prevalence, but the only available current vaccine, BCG, has insufficient impact. In this article, a novel subunit vaccine against TB was developed, using the Ag85B-ESAT6-Rv2034 fusion antigen, two adjuvants - CpG and MPLA, and a cationic pH-sensitive liposome as a delivery system, representing a new TB vaccine delivery strategy not previously reported for TB. In vitro in human dendritic cells (DCs), the adjuvanted formulation induced a significant increase in the production of (innate) cytokines and chemokines compared to the liposome without additional adjuvants. In vivo, the new vaccine administrated subcutaneously significantly reduced Mycobacterium tuberculosis (Mtb) bacterial load in the lungs and spleens of mice, significantly outperforming results from mice vaccinated with the antigen mixed with adjuvants without liposomes. In-depth analysis underpinned the vaccine's effectiveness in terms of its capacity to induce polyfunctional CD4+ and CD8+ T-cell responses, both considered essential for controlling Mtb infection. Also noteworthy was the differential abundance of various CD69+ B-cell subpopulations, which included IL17-A-producing B-cells. The vaccine stimulated robust antigen-specific antibody titers, further extending its potential as a novel protective agent against TB.

Production and evaluation of three kinds of vaccines against largemouth bass virus, and DNA vaccines show great application prospects.

Largemouth bass virus (LMBV) infections has resulted in high mortality and economic losses to the global largemouth bass industry and has seriously restricted the healthy development of the bass aquaculture industry. There are currently no antiviral therapies available for the control of this disease. In this study, we developed three types of vaccine against LMBV; whole virus inactivated vaccine (I), a subunit vaccine composed of the major viral capsid protein MCP (S) as well as an MCP DNA vaccine(D), These were employed using differing immunization and booster strategies spaced 2 weeks apart as follows: II, SS, DD and DS. We found that all vaccine groups induced humoral and cellular immune responses and protected largemouth bass from a lethal LMBV challenge to varying degrees and DD produced the best overall effect. Specifically, the levels of specific IgM in serum in all immunized groups were elevated and significantly higher than those in the control group. Moreover, the expression of humoral immunity (CD4 and IgM) and cellular immunity (MHCI-α) as well as cytokines (IL-1ß) was increased, and the activity of immunity-related enzymes ACP, AKP, LZM, and T-SOD in the serum was significantly enhanced. In addition, the relative percent survival of fish following an LMBV lethal challenge 4 weeks after the initial immunizations were high for each group: DD(89.5 %),DS(63.2 %),SS(50 %) and II (44.7 %). These results indicated that the MCP DNA vaccine is the most suitable and promising vaccine candidate for the effective control of LMBV disease.

Exploiting membrane vesicles derived from avian pathogenic Escherichia coli as a cross-protective subunit vaccine candidate against avian colibacillosis.

Avian pathogenic Escherichia coli (APEC) is a notable pathogen that frequently leads to avian colibacillosis, posing a substantial risk to both the poultry industry and public health. The commercial vaccines against avian colibacillosis are primarily inactivated vaccines, but their effectiveness is limited to specific serotypes. Recent advances have highlighted bacterial membrane vesicles (MV) as a promising candidate in vaccine research. How to produce bacterial MVs vaccines on a large scale is a significant challenge for the industrialization of MVs. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in lipopolysaccharides (LPS). Here, we evaluated the immunoprotective efficacy of MVs derived from the LPS low-expressed APEC strain FY26ΔmsbB, which was an APEC mutant strain with a deletion of the msbB gene. The nitrogen cavitation technique was employed to extract APEC MVs, with results indicating a significant increase in MVs yield compared to that obtained under natural culture. The immunization effectiveness was assessed, revealing that FY26ΔmsbB MVs elicited an antibody response of laying hens and facilitated bacterial clearance. Protective efficacy studies demonstrated that immunization with FY26ΔmsbB MVs conferred the immune protection in chickens challenged with the wild-type APEC strain FY26. Notably, LPS low-carried MVs recovered from the mutant FY26ΔmsbB also displayed cross-protective capabilities, and effectively safeguarding against infections caused by O1, O7, O45, O78, and O101 serotypes virulent APEC strains. These findings suggest that MVs generated from the LPS low-expressed APEC strain FY26ΔmsbB represent a novel and empirically validated subunit vaccine for the prevention and control of infections by various APEC serotypes.

Recombinant Subunit Vaccine Candidate against the Bovine Viral Diarrhea Virus.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.

Vaccination with OprB porin, and its epitopes offers protection against A. baumannii infections in mice.

A. baumannii is a deadly antimicrobial resistance pathogen that acquires drug resistance through different mechanisms. Therefore, it is necessary to investigate all its virulence factors and design effective vaccines against it. For this purpose, OprB, an outer membrane porin, was investigated in this study, and its secondary and tertiary structures, physicochemical properties, and B-T epitopes were determined. The vaccine potential of this protein and its linear, non-continuous, and chimeric epitopes were also in-vivo analyzed. Based on the results, two surface epitopes and one non-continuous epitope were identified. Surface contiguous epitopes were produced recombinantly and non-continuous epitope sequences were synthesized and then produced. The chimeric epitope was also produced via the SOE-PCR technique. Active and passive immunization of mice with the whole OprB protein, non-continuous epitope, contiguous epitopes, two epitopes in chimeric form, as well as the mixture of two purified epitopes showed that the survival level and total IgG titer of the mice compared to non-vaccinated mice or mice that were vaccinated with an internal fragment increased significantly. The bacterial load in the immunized mice's lung, liver, kidney, and spleen was much lower than in the control groups, and the TNF-α, IFN-γ, and IL-6 cytokines levels were also lower in these groups and were similar to the naive mice. On the other hand, subunit vaccines showed acceptable safety and due to their minimal cross-activity, their use is much safer.

Conjugation of TLR7 and TLR7/8 agonists onto weak protein antigen via versatile oxime ligation for enhanced vaccine efficacy.

Protein-based subunit vaccines are weakly immunogenic, and developing self-adjuvanting vaccines with adjuvant conjugated to antigen is a promising approach for generating optimal immune responses. Here, we report a novel adjuvant-protein conjugate vaccine based on versatile oxime ligation technique. Firstly, the adjuvant properties of a series of TLR7 and TLR7/8 small molecule agonists in self-adjuvanting vaccines were systematically compared by coupling them to proteins in consistent ratio via p-carboxybenzaldehyde (p-CBA) for the first time. All conjugate vaccines induced cytokine secretion in murine and human macrophages in vitro, and promoted specific antibody production in vivo. Notably, a conjugate containing imidazoquinoline TLR7/8 agonist (TLR7/8a1) showed the greatest enhancement in Th1/2 balanced antibody response. To minimize the interference with the protein antigenic integrity, we further developed a systematic glycoconjugation strategy to conjugate this TLR7/8a1 onto the glycan chains of SARS-CoV-2 S1 glycoprotein via oxime ligation, in which S1 containing different numbers of aldehyde groups were obtained by differential periodate oxidation. The resulting TLR7/8a1-S1 conjugate triggered a potent humoral and cellular immunity in vivo. Together these data demonstrate the promise of these TLR7 and TLR7/8 agonists as effective built-in adjuvants, and the versatile oxime ligation strategy might broaden potential applications in designing different conjugate vaccines.

Revolutionizing Nipah virus vaccinology: insights into subunit vaccine development strategies and immunological advances.

The Nipah virus (NiV), a zoonotic virus in the Henipavirus genus of the Paramyxoviridae family, emerged in Malaysia in 1998 and later spread globally. Diseased patients may have a 40- 70% chance of fatality depending on the severity and early medication. The recent outbreak of NiV was reported in Kerala (India) by a new strain of MCL-19-H-1134 isolate. Currently, no vaccines are available, highlighting the critical need for a conclusive remedy. Our study aims to develop a subunit vaccine against the NiV by analyzing its proteome. NiV genome and proteome sequences were obtained from the NCBI database. A phylogenetic tree was constructed based on genome alignment. T-cell, helper T-cell, and B-cell epitopes were predicted from the protein sequences using NetCTL-1.2, NetMHCIIPan-4.1, and IEDB servers, respectively. High-affinity epitopes for human receptors were selected to construct a multi-epitope vaccine (MEV). These epitopes' antigenicity, toxicity, and allergenicity were evaluated using VaxiJen, AllergenFP-v.1.0, and AllergenFP algorithms. Molecular interactions with specific receptors were analyzed using PyRx and ClusPro. Amino acid interactions were visualized and analyzed using PyMOL and LigPlot. Immuno-simulation was conducted using C-ImmSim to assess the immune response elicited by the MEV. Finally, the vaccine cDNA was inserted into the pET28a(+) expression vector using SnapGene tool for in silico cloning in an E. coli host. The potential for an imminent outbreak cannot be overlooked. A subunit vaccine is more cost-effective and time-efficient. With additional in vitro and in vivo validation, this vaccine could become a superior preventive measure against NiV disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00246-9.

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.