Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Characterization of cellular immune response in hamsters immunized with recombinant vaccines against leptospirosis based on LipL32:LemA:LigAni chimeric protein.

In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.

Endotoxin-free gram-negative bacterium as a system for production and secretion of recombinant proteins.

Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines. KEY POINTS: • Novel molecular tools for protein expression in non-model bacteria. • Bacteria with GSL instead of LPS as a potential vector for protein delivery.

Assessment of the Immunoprotective Efficacy of Recombinant 14-3-3 Protein and Dense Granule Protein 10 (GRA10) as Candidate Antigens for Rabbit Vaccines against Eimeria intestinalis.

Eimeria intestinalis infects rabbits, causing severe intestinal coccidiosis. Prolonged anticoccidial drug use might lead to coccidia resistance and drug residues in food. Thus, vaccines are required to control rabbit coccidiosis. In this study, recombinant E. intestinalis 14-3-3 and GRA10 proteins (rEi-14-3-3 and rEi-GRA10) were obtained via prokaryotic expression and used as recombinant subunit vaccines. Fifty 30-day-old rabbits were randomly grouped as follows: PBS-uninfected group, PBS-infected group, Trx-His-S control group, and rEi-14-3-3 and rEi-GRA10 immunized groups. The rabbits were subcutaneously immunized twice at 2-week intervals, challenged with 7 × 104 sporulated oocysts, and sacrificed 14 days later. The protective effects were assessed via clinical signs, relative weight gain, oocyst reduction, mean intestinal lesion score, ACI (anticoccidial index), cytokine, and specific antibody levels in sera. The rEi-14-3-3 and rEi-GRA10 groups had higher relative weight gain rates of 81.94% and 73.61% (p < 0.05), and higher oocyst reduction rates of 86.13% and 84.87% (p < 0.05), respectively. The two immunized groups had fewer intestinal lesions (p < 0.05) and higher IgG levels (p < 0.05). Higher levels of IL-2, IL-4, and IFN-γ cytokines in the rEi-14-3-3 group (p < 0.05) and a higher level of IFN-γ in the rEi-GRA10 group (p < 0.05) were observed. The ACI values of the rEi-14-3-3 and rEi-GRA10 groups were 168.24 and 159.91, with good and moderate protective effects, respectively. Both rEi-14-3-3 and rEi-GRA10 induced humoral immunity in the rabbits. In addition, rEi-14-3-3 induced Th1- and Th2-type immune responses. Both recombinant proteins were protective against E. intestinalis infection in rabbits, with rEi-14-3-3 showing a better protective effect.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita.

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1ß, and TLR-22 were significantly up-regulated at early time points (3 h-72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

Protective immunity following vaccination with a recombinant multiple-epitope protein of bovine herpesvirus type I in a rabbit model.

Bovine herpesvirus type 1 (BoHV-1) causes considerable economic losses to the cow industry. Vaccination remains an effective strategy to control the diseases associated with BoHV-1. However, live vaccines present safety concerns, especially in pregnant cows; thus, nonreplicating vaccines have been developed to control the disease. The envelope glycoproteins of BoHV-1 induce a protective immune response. In this work, selected epitopes on glycoproteins gD, gC, and gB were constructed in triplicate with linker peptides. Vaccination of rabbits demonstrated that P2-gD/gC/gB with AAYAAY induced higher specific antibodies than that with GGGGS linker. P2-gD/gC/gB with AAYAAY linker was fused with bovine interleukin-6 (BoIL-6) or rabbit IL-6 (RaIL-6) and bacterially expressed. Rabbits were intramuscularly immunized with 100 µg of P2-gD/gC/gB-BoIL-6, P2-gD/gC/gB-RaIL-6, P2-gD/gC/gB, P2-gD/gC/gB plus BoIL-6, P2-(gD-a)3-BoIL-6, or P2-(gD-a)3 emulsified with ISA 206 adjuvant thrice at 3-week intervals. P2-gD/gC/gB-BoIL-6 generated a higher titer of BoHV-1-specific antibodies, neutralizing antibodies, interferon (IFN)-γ, and IL-4 compared with P2-gD/gC/gB plus BoIL-6, P2-gD/gC/gB-RaIL-6, or other formulation. P2-gD/gC/gB-BoIL-6 triggered similar levels of antibodies and significantly higher titer of IFN-γ and IL-4 compared with inactivated bovine viral diarrhea (BVD)-infectious bovine rhinotracheitis (IBR) vaccine. Rabbits vaccinated with P2-gD/gC/gB-BoIL-6 dramatically reduced viral shedding and tissue lesions in lungs and trachea after viral challenge and reactivation compared with those with P2-gD/gC/gB plus BoIL-6 or P2-gD/gC/gB-RaIL-6. P2-gD/gC/gB-BoIL-6 provided protective effects against viral shedding and tissue pathogenesis similar to those of the inactivated vaccine. The data confirmed the safety and immunogenicity of multiple-epitope recombinant protein and a potential vaccine candidate to control the disease, especially for pregnant cattle.

Shingrix for Herpes Zoster: A Review

Herpes zoster (HZ), also known as shingles, results from reactivation of the latent varicella-zoster virus (VZV), which commonly causes chickenpox in childhood. Greater than 90% of adults are infected with this virus, putting them at risk for reactivation. HZ presents as a painful, vesicular rash distributed in a unilateral and dermatomal pattern along dorsal root or cranial nerve ganglia. The rash often presents with prodromal symptoms and progresses to include clear vesicular clusters, evolving through stages of pustulation, ulceration, and crusting. HZ therapy currently involves the use of antiviral agents and pain management; however, HZ prophylaxis has been strongly recommended in older adults through vaccination with a live attenuated vaccine, Zostavax®. A new recombinant subunit vaccine, HZ/su (Shingrix®), is the subject of this review. In clinical trials, HZ/su demonstrated an overall vaccine efficacy of 97.2% among participants 50 years of age or older, indicating a significantly reduced risk of HZ in these individuals. Shingrix® was approved by the US FDA in October 2017 as HZ prophylaxis.

Vaccination of sows with a dendritic cell-targeted porcine epidemic diarrhea virus S1 protein-based candidate vaccine reduced viral shedding but exacerbated gross pathological lesions in suckling neonatal piglets.

Porcine epidemic diarrhea virus (PEDV) poses a serious threat to swine worldwide as evidenced by its recent introduction into the USA and the devastating economic impact it caused to the USA swine industry. Commercial vaccines against PEDV are available but their efficacies are inadequate. Therefore, vaccines with improved efficacy are needed to effectively control PEDV infections. We previously determined the immunogenicity of a novel dendritic cell (DC)-targeted PEDV S1 protein-based subunit vaccine in weaned piglets in which the PEDV antigen was targeted to DCs through a porcine Langerin-specific antibody. In this study, we evaluated the protective efficacy of this DC-targeting vaccine by immunizing sows at 5 and 2 weeks prior to farrowing and by challenging the 5-day-old piglets with PEDV. The results showed that immunization of sow with DC-targeted PEDV vaccine did not eliminate faecal virus shedding in piglets but significantly reduced faecal viral RNA levels in the early days after virus challenge. The vaccine also reduced the amount of PEDV antigen in intestinal tissues presented with intestinal villi regrowth. However, the DC-targeted vaccine neither mitigated PEDV clinical signs nor affected viral RNA loads in intestinal tissues of piglets. In the vaccinated sow, DC-targeted PEDV vaccine enhanced T helper 1-like cluster of differentiation (CD)4 T cell responses and induced IgG but not IgA-specific immune responses. The suckling piglets in the DC-targeted vaccine group showed increased gross pathological lesions in the small intestine. Results in this study provide insights into the effects of sow cellular immune responses to PEDV infection in suckling piglets.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC.

OBJECTIVE: In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. METHODS: A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. RESULTS: Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. CONCLUSION: An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

Safety and immunogenicity of an AS01-adjuvanted varicella-zoster virus subunit candidate vaccine against herpes zoster in adults >=50 years of age.

BACKGROUND: An adjuvanted varicella-zoster virus glycoprotein E (gE) subunit vaccine candidate for herpes zoster is in development. In this trial we compared the safety, reactogenicity, and immunogenicity of the vaccine antigen combined with different adjuvant doses. METHODS: This was a phase II, observer-blind, randomized, multinational study. Adults ≥50 years old were randomized 4:4:2:1 to be vaccinated at months 0 and 2 with gE combined with a higher (AS01B) or lower (AS01E) dose adjuvant, unadjuvanted gE, or saline. Following each dose, solicited events were recorded for 7 days and unsolicited adverse events for 30 days. Serious adverse events were collected for 1 year. Cell-mediated and humoral immune responses were assessed at baseline and following each dose. RESULTS: No vaccine-related severe adverse events were reported. Solicited adverse events were generally mild to moderate and transient. For all gE-based vaccines, pain was the most common local symptom and fatigue the most common general symptom. Immune responses were significantly enhanced by AS01B and AS01E compared to unadjuvanted gE and were significantly stronger for gE/AS01B than for gE/AS01E. CONCLUSIONS: AS01 improved the immunogenicity of gE while retaining acceptable safety and reactogenicity profiles. The enhancement of gE-specific cellular and humoral responses was adjuvant dose dependent. CLINICAL TRIALS REGISTRATION: NCT00802464.

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.

Evaluation of Protective Efficacy of Recombinant Toxoplasma gondii DDX39 Protein Vaccine against Acute and Chronic T. gondii Infection in Mice.

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+ T cells rising by 21.9% and CD8+ T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response. Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15-20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

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.

Development and characterization of HCMV recombinant subunit vaccines based on T-cell epitopes.

Human cytomegalovirus (HCMV), a ubiquitous ß-herpes virus, mostly causes asymptomatic infections in adults with healthy immune systems. Due to immunosuppressive therapy, solid organ transplantation (SOT) recipients are at increased risk of HCMV infection. In recent years, the interdisciplinary, filed of immunoinformatics, based on computer science, and modern immunology, has emerged. In this study, we designed three types of recombinant subunit vaccines, which are expressed by the E. coli BL21 strain according to immunoinformatics prediction. Subsequently, we evaluated the innate and cellular immune responses of recombinant subunit vaccines in vivo and/or in vitro. Flow cytometry analysis, revealed that recombinant subunit vaccines enhanced both innate and cellular immune responses in vivo and/or in vitro. We also found that the novel herb adjuvant hesperetin (HES) increased memory T cell inflation. Overall, we developed three types of recombinant subunit vaccines based on HCMV antigen fragments containing multiple T-cell epitopes and assessed the innate and cellular immune responses in vivo and/or in vitro.

Recombinant Toxoplasma gondii Calreticulin protein provides partial protection against acute and chronic toxoplasmosis.

Toxoplasma gondii, a highly prevalent apicomplexan pathogen, can cause serious or even fatal toxoplasmosis in both animals and humans. Immunoprophylaxis is considered a promising strategy for controlling this disease. Calreticulin (CRT) is known as a pleiotropic protein, which is critical for calcium storage and phagocytosis of apoptotic cells. Our study examined the protective effects of recombinant T. gondii Calreticulin (rTgCRT) as a recombinant subunit vaccine against the T. gondii challenge in mice. Here, rTgCRT was successfully expressed in vitro using prokaryptic expression system. Polyclonal antibody (pAb) has been prepared by immunizing Sprague Dawley rats with rTgCRT. Western blotting showed that rTgCRT and natural TgCRT protein were recognized by serum of T. gondii infected mice and rTgCRT pAb, respectively. T lymphocyte subsets and antibody response were monitored using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The results showed that ISA 201 rTgCRT could stimulate lymphocyte proliferation and induce high levels of total and subclasses of IgG. After the RH strain challenge, a longer survival period was given by the ISA 201 rTgCRT vaccine compared to the control groups; after infection with the PRU strain, we observed a 100% survival rate and a significant reduction in cysts load and size. In the neutralization test, high concentrations of rat-rTgCRT pAb provided 100% protection, while in the passive immunization trial, only weak protection was observed after RH challenge, indicating that rTgCRT pAb needs further modification to improve its activity in vivo. Taken together, these data confirmed that rTgCRT can trigger strong cellular and humoral immune responses against acute and chronic toxoplasmosis.

Extracellular polysaccharide of Lactobacillus plantarum enhance immune efficacy of oprH gene recombinant subunit vaccine from Pseudomonas aeruginosa.

To evaluate the immune enhancement effect of the extracellular polysaccharide of Lactobacillus plantarum on oprH recombinant subunit vaccine from Pseudomonas aeruginosa, a recombinant subunit vaccine of oprH (rOprH vaccine) was developed. The EP-rOprH vaccine was prepared with the extracellular polysaccharide of L. plantarum as an adjuvant. Mice were vaccinated with the rOprH and EP-rOprH vaccines, and the outer membrane protein (OMP) and inactivated vaccines were used as controls. The levels of serum antibody, interferon-γ (IFN-γ), interleukin (IL-2), and IL-4 were determined after vaccination. Finally, the protective efficacy of the vaccine was evaluated after challenge with virulent P. aeruginosa. Following vaccination, the serum antibody levels were significantly higher in mice vaccinated with the EP-rOprH vaccine than in those vaccinated with the rOprH vaccine (P<0.05). Moreover, the serum antibody levels detected in the EP-rOprH vaccine group were similar to those detected in the OMP vaccine group when P. aeruginosa suspension was used as the coating antigen. However, the levels in the EP-rOprH vaccine group were higher than those in the OMP vaccine and inactivated vaccine groups when the purified rOprH protein was used as the coating antigen (P<0.05). The level of IFN-γ, IL-2, and IL-4 in mice vaccinated with the EP-rOprH vaccine was significantly higher than that in mice vaccinated with the rOprH vaccine (P<0.05) and comparable to that in mice vaccinated with the OMP vaccine. The protective rates were 65%, 80%, 80%, and 95% with the rOprH, EP-rOprH, OMP, and inactivated vaccines, respectively. Thus, the extracellular polysaccharide of L. plantarum significantly enhanced the immune response and protection provided by the recombinant subunit vaccine of oprH.

Two formulations of coronavirus disease-19 recombinant subunit vaccine candidate made up of S1 fragment protein P1, S2 fragment protein P2, and nucleocapsid protein elicit strong immunogenicity in mice.

INTRODUCTION: Coronavirus disease (COVID-19) is ongoing as a global epidemic and there is still a need to develop much safer and more effective new vaccines that can also be easily adapted to important variants of the pathogen. In the present study in this direction, we developed a new COVID-19 vaccine, composed of two critical antigenic fragments of the S1 and S2 region of severe acute respiratory syndrome coronavirus 2 as well as the whole nucleocapsid protein (N), which was formulated with either alum or alum plus monophosphoryl lipid A (MPLA) adjuvant combinations. METHODS: From within the spike protein S1 region, a fragmented protein P1 (MW:33 kDa) which includes the receptor-binding domain (RBD), another fragment protein P2 (MW:17.6) which contains important antigenic epitopes within the spike protein S2 region, and N protein (MW:46 kDa) were obtained after recombinant expression of the corresponding gene regions in Escherichia coli BL21. For use in immunization studies, three proteins were adsorbed with aluminum hydroxide gel and with the combination of aluminum hydroxide gel plus MPLA. RESULTS: Each of the three protein antigens produced strong reactions in enzyme-linked immunosorbent assays and Western blot analysis studies performed with convalescent COVID-19 patient sera. In mice, these combined protein vaccine candidates elicited high titer anti-P1, anti-P2, and anti-N IgG and IgG2a responses. These also induced highly neutralizing antibodies and elicited significant cell-mediated immunity as demonstrated by enhanced antigen-specific levels of interferon-γ (INF-γ) in the splenocytes of immunized mice. CONCLUSION: The results of this study showed that formulations of the three proteins with Alum or Alum + MPLA are effective in terms of humoral and cellular responses. However, since the Alum + MPLA formulation appears to be superior in Th1 response, this vaccine candidate may be recommended mainly for the elderly and immunocompromised individuals. We also believe that the alum-only formulation will provide great benefits for adults, young adolescents, and children.

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine / 中华微生物学和免疫学杂志

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

A randomized lot-to-lot immunogenicity consistency study of the candidate zoster vaccine HZ/su.

BACKGROUND: The risk of developing herpes zoster (HZ) increases with age and is thought to be associated with a decrease in cell-mediated immunity in older adults. The adjuvanted varicella-zoster virus (VZV) glycoprotein E (gE) recombinant subunit vaccine (HZ/su) showed >90% efficacy in the prevention of HZ when administered in adults ≥50 years of age. Here we aim to evaluate immunogenicity consistency of 3 different HZ/su vaccine lots and to assess safety of these lots. METHODS: This multicenter, phase III, double-blind, randomized study (NCT02075515), assessed lot-to-lot consistency in terms of immunogenicity of HZ/su and also assessed safety of these lots. Participants aged 50 years or older were randomized (1:1:1) to receive 2 doses of HZ/su, 2 months apart, from 1 out of 3 randomized HZ/su lots (Lots A, B and C). Humoral immunogenicity was assessed pre-vaccination and 1 month post-second vaccination by anti-gE antibody enzyme-linked immunosorbent assay. Lot-to-lot consistency was demonstrated if the 2-sided 95% confidence intervals of the anti-gE geometric mean concentration ratio between all lot pairs were within 0.67 and 1.5. Solicited symptoms were recorded within 7 days and unsolicited adverse events (AEs) within 30 days after each vaccination. Serious AEs (SAEs) and potential immune-mediated diseases (pIMDs) were reported until study end (12 months post-second vaccination). RESULTS: Of 651 participants enrolled in the study, 638 received both doses of the HZ/su vaccine and 634 completed the study. Humoral immune responses were robust and consistency between 3 manufacturing lots was demonstrated. The incidence of solicited symptoms, unsolicited AEs and SAEs was comparable between all lots. Three fatal SAEs, 1 in each lot, were reported, none of which were considered vaccine-related by investigator assessment. Two out of the 8 reported pIMDs were considered vaccine-related by the investigator. CONCLUSION: The three HZ/su manufacturing lots demonstrated consistent immunogenicity. No safety concerns were identified. Clinical trial registry number: NCT02075515 (ClinicalTrials.gov).