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.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E. coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L. rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A. hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGF-ß), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A. hydrophila.

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.

Oral vaccination of Macrobrachium rosenbergii with baculovirus-expressed M. rosenbergii nodavirus (MrNV) capsid protein induces protective immunity against MrNV challenge.

White Tail Disease (WTD) is one of the important viral diseases of fresh water giant prawn Macrobrachium rosenbergii, which is caused by Macrobrachium rosenbergii nodavirus (MrNV). In the present study, the capsid protein gene of MrNV containing a His-tag was cloned into a baculovirus vector pVL1393 and expressed the recombinant MrNV protein in insect cells, using a baculovirus expression system. A band corresponding to the MrNV protein of 43 kDa was characterized after fractionating the proteins of baculovirus-infected cell lysates by SDS-polyacrylamide gel, and immunostaining with His-tag monoclonal antibody. Furthermore, purified MrNV capsid protein assembled into virus-like particles (VLPs) of ∼30 nm in diameter, when examined by transmission electron microscopy (TEM). To vaccinate the larvae by oral route, the recombinant MrNV (r-MrNV) protein was coated with artificial prawn feed and fed to M. rosenbergii larvae (90 ±â€¯10 mg) for 60 days. After 30 and 60 days of vaccine treatment, group of prawns were challenged with virulent MrNV orally. Samples were collected at different time intervals to evaluate the survival of larvae and to analyze the presence of MrNV by double-step PCR and expression of immune/ toll-like receptor (TLR) genes. Non-vaccinated group of M. rosenbergii larvae succumbed to death and had 90% mortality, whereas the r-MrNV protein treated groups exhibited 65 and 80% survival (P  ≤  0.001) for 30 and 60 days post-vaccination (dpv), respectively. Double-step PCR diagnosis revealed that there was 100% positive signals observed in non-vaccinated prawn group, whereas the infection was reduced significantly (P < 0.001) to 32 and 17% respectively in 30 and 60 dpv. Among the four different immune/ TLR genes such as antimicrobial peptide (Mramp), lysozyme (MrLY), proPhenol Oxidase (MrPPO) and Toll-Like Receptor (MrToll) expression screening, Mramp was successfully expressed in the MrNV subunit protein vaccinated prawns, whereas the non-vaccinated prawn had no immune/TLR gene expression. Taken together, our results demonstrate that oral vaccination of M. rosenbergii larvae with baculovirus-expressed MrNV capsid protein confer up to 78% protection against MrNV infection.

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.

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.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Process development and preclinical evaluation of a major Plasmodium falciparum blood stage vaccine candidate, Cysteine-Rich Protective Antigen (CyRPA).

Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) is an essential, highly conserved merozoite antigen that forms an important multi-protein complex (RH5/Ripr/CyRPA) necessary for erythrocyte invasion. CyRPA is a promising blood-stage vaccine target that has been shown to elicit potent strain-transcending parasite neutralizing antibodies. Recently, we demonstrated that naturally acquired immune anti-CyRPA antibodies are invasion-inhibitory and therefore a correlate of protection against malaria. Here, we describe a process for the large-scale production of tag-free CyRPA vaccine in E. coli and demonstrate its parasite neutralizing efficacy with commonly used adjuvants. CyRPA was purified from inclusion bodies using a one-step purification method with high purity (>90%). Biochemical and biophysical characterization showed that the purified tag-free CyRPA interacted with RH5, readily detected by a conformation-specific CyRPA monoclonal antibody and recognized by sera from malaria infected individuals thus indicating that the recombinant antigen was correctly folded and retained its native conformation. Tag-free CyRPA formulated with Freund's adjuvant elicited highly potent parasite neutralizing antibodies achieving inhibition of >90% across diverse parasite strains. Importantly, we identified tag-free CyRPA/Alhydrogel formulation as most effective in inducing a highly immunogenic antibody response that exhibited efficacious, cross-strain in vitro parasite neutralization achieving ~80% at 10 mg/ml. Further, CyRPA/Alhydrogel vaccine induced anti-parasite cytokine response in mice. In summary, our study provides a simple, scalable, cost-effective process for the production of tag-free CyRPA that in combination with human-compatible adjuvant induces efficacious humoral and cell-mediated immune response.

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.

Lyophilization Process Engineering and Thermostability of ID93 + GLA-SE, a Single-Vial Adjuvanted Subunit Tuberculosis Vaccine Candidate for Use in Clinical Studies.

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.

CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization.

The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralizing antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.