Immunized Groups Research Articles

Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1β, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

Investigating BB0405 as a novel Borrelia afzelii vaccination candidate in Lyme borreliosis

BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

Immunization with a novel recombinant protein (YidR) reduced the risk of clinical mastitis caused by Klebsiella spp. and decreased milk losses and culling risk after Escherichia coli infections

The primary objective of this study was to evaluate the protective efficacy of a novel recombinant subunit vaccine containing the protein YidR (rYidR) against clinical mastitis (CM) caused by Klebsiella spp. and Escherichia coli. Given that E. coli infection is known to cause metritis, we also evaluated the effect of rYidR vaccination on the incidence of metritis and conception at the first artificial insemination. Retained placenta and abortion incidence, milk production and composition, and serological responses to specific antigens were also evaluated. In total, 3,107 cows were blocked by parity and randomly allocated into 1 of 3 treatment groups: experimental recombinant subunit vaccine containing the YidR protein (rYidR); commercial vaccine composed of Klebsiella pneumoniae siderophore receptors and porin protein (Kleb-SRP; KlebVax, Epitopix, Willmar, MN); and sterile water adjuvanted with aluminum hydroxide (20%; placebo). Vaccinations were performed at the dry-off for cows, and at 223 ± 3 d of pregnancy for pre-fresh heifers. A second administration was given at 21 ± 3 d after the first injection. Vaccination with rYidR significantly reduced the incidence of CM caused by Klebsiella spp. (3.2%) when compared with the placebo (5.1%) group. No difference was observed on risk of Klebsiella CM between Kleb-SRP (5.9%) and placebo groups. Cows in the rYidR group that experienced E. coli CM had a lower risk of death or culling (12.5%) compared with the Kleb-SRP (27.6%) and placebo groups (27.8%). Furthermore, among cows that developed E. coli CM, rYidR-immunized cows produced more milk than did cows in the placebo and Kleb-SRP groups. Regardless of CM occurrence, rYidR-immunized cows tended to have higher milk production up to the eighth month of lactation than cows in the other groups. No significant effect of treatment was observed on the overall incidence of abortion and metritis; however, the risk of retained placenta tended to be lower for the rYidR group (4.7%) compared with the placebo group (6.7%). In addition, primiparous cows in the rYidR group had the highest conception risk at the first artificial insemination (48.3%) compared with the placebo (39.5%) group, and no significant difference was observed when the Kleb-SRP (40.1%) group was compared with the placebo group. Generally, higher antibody serum titers (IgM and IgG) were observed for the immunized groups compared with the placebo. In conclusion, the rYidR vaccine reduced the risk of CM caused by Klebsiella spp. and the mortality or culling of cows with E. coli infections. Other benefits of the novel vaccine include maintenance of milk production after CM caused by E. coli, and higher conception risk at the first service in primiparous cows compared with cows in the placebo and Kleb-SRP groups.

Elicitation of Highly Pathogenic Avian Influenza H5N1 M2e and HA2-Specific Humoral and Cell-Mediated Immune Response in Chicken Following Immunization With Recombinant M2e-HA2 Fusion Protein.

The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Sphingomyelin liposome bearing whole Leishmania lysate antigens induce strong Th2 immune response in BALB/c mice.

Objective(s):Whole Leishmania lysate antigens (WLL) has been shown to be effective to tackle leishmaniasis in murine models. Although liposomes can be considered as promising vaccines, the activity of phospholipase-A (PLA) in WLL, breeds difficulties to preparing stable liposomal WLL. One strategy to overcome this shortcoming is to use lipids such as sphingomyelin (SM) which is resistant against PLA. This study aim is formulating stable SM liposomes containing WLL and comparing their adjuvant effects with another first generation vaccine , i.e. solube Leishmania Antigen (SLA) liposomes in BALB/c mice. Materials and Methods:BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with Empty-liposome (E-lipo), Particulate WLL, Liposome-WLL, Liposome-SLA and control Buffer, three times every 2-week. Protection was assessed through measuring the swollen footpads and the load of parasites in the spleen. Other factors were used to assess the response of immune system by means of IgG subclasses, IL-4 and IFN-γ levels and intracellular cytokine assay in cultured splenocytes. Results:Although liposomal WLL were stable in terms of physicochemical properties, mice received Liposome-WLL did not reduce footpad swelling. The load of parasites in spleen and levels of IL-4- were also higher compared to other immunized groups. In terms of IgG isotypes, no considerable difference observed in mice received Liposome-WLL or other formulations. Conclusion:Liposome-WLL could be a suitable vaccine delivery system when a Th2 response is desired. Also, further studies are warranted to fully understand the role of sphingomyelin in inducing an immune response.

Enhanced epitope immunoreactivity of the dominant epitope of Toxoplasma gondii fused at the "N terminus" of HPV16L1

For improving epitope immunogenicity and achieving the co-immunization, late protein 1 (L1) of HPV type 16 (HPV16L1) was selected as the vector to carry the dominant epitope of Toxoplasma gondii because of the shared common population between Toxoplasma gondii and human papillomavirus (HPV). RSepitope-HPV16L1 (RSepitope fused at the "N-terminus" of HPV16L1) and HPV16L1-RSepitope (RSepitope fused at the "C-terminus" of HPV16L1) chimeras were constructed. After transfection of COS-7 cells with the recombinants, Western blot, RT-PCR, and immunofluorescence experiments confirmed that RSepitope-HPV16L1 could successfully express the corresponding mRNA and protein of RSepitope and HPV16L1, but the HPV16L1-RSepitope construct could not. A "prime-boost" immunization program was applied in mice to further evaluate the immune response elicited by the constructs, and the RSepitope-HPV16L1 immunization group produced the most significantly increased humoral and cellular immune responses (the highest RSepitope-specific IgG antibody level and the highest IFN-γ production, respectively), in which both elevated Th1 and Th2 immune responses were obtained. Moreover, the advantage of HPV16L1 as an epitope carrier was remarkable for RSepitope-HPV16L1, which induced a more prominent immunological response than RSepitope alone (without fusion with HPV16L1). Our research indicated that the N-terminus of HPV16L1 could be a better insertion site for enhancing target epitope immunogenicity, and our study offers a design for epitope vaccine of reasonable combination.

Immunogenicity of in-silico designed multi-epitope DNA vaccine encoding SAG1, SAG3 and SAG5 of Toxoplasma gondii adjuvanted with CpG-ODN against acute toxoplasmosis in BALB/c mice

The causative agent of toxoplasmosis, Toxoplasma gondii (T. gondii), is able to influence the health of humans and other vertebrates. Toxoplasma may cause severe illness in the fetus and immunocompromised individuals. The high incidence and intense damages of Toxoplasma infection clearly shows the need to achieve the safe and suitable vaccine. In this study, an immunoinformatics approach was employed to design a multi-epitope DNA vaccine encoding the T. gondii SAG1, SAG3 and SAG5. The bioinformatic outputs supported the immunogenic and non-allergic natures of multi-epitope vaccine. Thereafter, the protective efficacy of the vaccine was evaluated with/without CpG-ODN adjuvant in a laboratory animal model. BALB/c mice were immunized subcutaneously with multi-epitope DNA vaccine. The in vivo findings indicated that the multi-epitope DNA vaccine elicited significant production of IgG antibodies (472.90 ± 2.74 ng/ml) as well as IFN-γ (173.71 ± 26.39 pg/ml) (p < 0.001). Moreover, a significant reduced parasite-burden (17,470 per mg of spleen) and prolonged survival time (9 days) were observed in the immunized groups compared to the controls (p < 0.05). The low values of IL-4 (22.5 ± 0.16 pg/ml) were detected in vaccinated mice compared to the control (PBS) (p > 0.05). In addition, CpG-ODN as an adjuvant increased the immune efficacy of the multi-epitope DNA vaccine. In multi-epitope vaccine+CpG-ODN group, the values of IgG antibodies (535.90 ±7.29 ng/ml) and IFN-γ (358.21 ± 32.70 pg/ml) were significanly higher than the multi-epitope vaccine group. Meanwhile, an increased survival time (10 days) and fewer parasite load (15,485 per mg of spleen) were observed in multi-epitope vaccine+CpG-ODN group. The results revealed that the DNA vaccine containing epitopes of SAG1, SAG3 and SAG5 adjuvanted with CpG-ODN might be a new model for further investigations against acute T. gondii infection.

Immunogenicity of Newcastle disease virus strain ZG1999HDS applied oculonasally or by means of nebulization to day-old chicks

Newcastle disease (ND) is one of the classic viral infections of poultry which resists all the efforts of eradication. Newcastle disease virus (NDV) strain ZG1999HDS was isolated during the outbreak in 1,999 at a broiler farm in Croatia. Previous trials in chickens confirmed it to be a lentogenic pathotype and immunogenic by stimulating humoral and cell mediated immunity. Further characterization by deduced amino acid sequence at the cleavage site of fusion protein confirmed its lentogenic nature, and in vitro tests its oncolytic capacity. Owing to its immunogenicity, strain ZG1999HDS is considered for vaccine development. In this study, 1-day-old chicks were vaccinated using strain ZG1999HDS oculonasally or by nebulization. Strain ZG1999HDS induced humoral immune response in both immunized groups The cell-mediated immune response occurred earlier in the group immunized by nebulization, as shown by a higher frequency rate of T and B lymphocytes, and significantly higher expression of IFN-α in respiratory organs and IFN-γ expression in the spleen. Viral genomic RNA was not detected in investigated organs. Thus, NDV strain ZG1999HDS is immunogenic when administered by means of nebulization or oculonasally without any adverse effects and is therefore suitable for further research and vaccine development. Further research is needed regarding its tropism.

Oral Vaccination of Grass Carp (Ctenopharyngodon idella) with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody interference in field farm applications

BackgroundClassical swine fever (CSF) is one of the most devastating pig diseases that affect the swine industry worldwide. Besides stamping out policy for eradication, immunization with vaccines of live attenuated CSF or the CSF-E2 subunit is an efficacious measure of disease control. However, after decades of efforts, it is still hard to eliminate CSF from endemically affected regions and reemerging areas. Most of previous studies demonstrated the efficacy of different CSF vaccines in laboratories under high containment conditions, which may not represent the practical performance in field farms. The inadequate vaccine efficacy induced by unrestrained factors may lead to chronic or persistent CSF infection in animals that develop a major source for virus shedding among pig populations. In this study, a vaccination-challenge-cohabitation trial on specific-pathogen-free (SPF) pigs and long-term monitoring of conventional sows and their offspring were used to evaluate the efficacy and the impact of maternally derived antibody (MDA) interference on CSF vaccines in farm applications.ResultsThe trials demonstrated higher neutralizing antibody (NA) titers with no clinical symptoms and significant pathological changes in the CSF-E2 subunit vaccine immunized group after CSFV challenge. Additionally, none of the sentinel pigs were infected during cohabitation indicating that the CSF-E2 subunit vaccine could provoke adequately acquired immunity to prevent horizontal transmission. In field farm applications, sows immunized with CSF-E2 subunit vaccine revealed an average of higher and consistent antibody level with significant reduction of CSF viral RNA detection via saliva monitoring in contrast to those of live attenuated CSF vaccine immunized sows possessing diverse antibody titer distributions and higher viral loads. Furthermore, early application of the CSF-E2 subunit vaccine in 3-week-old piglets illustrated no MDA interference on primary immunization and could elicit consistent and long-lasting adequate antibody response suggesting the flexibility of CSF-E2 subunit vaccine on vaccination program determination.ConclusionsThe CSF-E2 subunit vaccine demonstrated significant efficacy and no MDA interference for immunization in both pregnant sows and piglets. These advantages provide a novel approach to avoid possible virus shedding in sow population and MDA interference in piglets for control of CSF in field farm applications.

The dry powder formulation of mixed cross-linked dextran microspheres and tetanus toxoid-loaded trimethyl chitosan nanospheres as a potent adjuvant for nasal delivery system.

Objective(s):The present study aimed to determine the immunoadjuvant efficacy of mixed cross-linked dextran microspheres (CDM) and tetanus toxoid (TT)-loaded trimethyl chitosan (TMC) nanospheres in dry powder form. Materials and Methods:The TMC nanoparticles (NPs) containing TT were produced using the ionic gelation method. Co-administration of TT-loaded TMC NPs and CDM as an absorption enhancer was performed to improve immunity against the antigen. Dry powder formulations were delivered via the nasal route in a rabbit model. Results:Among immunization groups, mixing of CDM with TT encapsulated in TMC NPs could elicit the highest titer of systemic IgG antibody. Furthermore, the addition of CDM to TT-loaded TMC enhanced the sIgA response relative to the TT solution. Conclusion:The TMC NPs had a considerable effect on mucosal and systemic immunity against the TT antigen. Therefore, the CDM excipient can be utilized for nasal immunization to elevate systemic and mucosal responses.

A practical method for evaluating the in vivo efficacy of EVA-71 vaccine using a hSCARB2 knock-in mouse model

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5-6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titer of antibodies was measured after one week. Mice at four weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17 and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model

ABSTRACT Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

The combined vaccination protocol of DNA/MVA expressing Zika virus structural proteins as efficient inducer of T and B cell immune responses

ABSTRACT Zika virus (ZIKV) is a mosquito-borne pathogen with public health importance due to the high risk of its mosquito vector dissemination and the severe neurological and teratogenic sequelae associated with infection. Vaccines with broad immune specificity and control against this re-emerging virus are needed. Here, we described that mice immunized with a priming dose of a DNA plasmid mammalian expression vector encoding ZIKV prM-E antigens (DNA-ZIKV) followed by a booster dose of a modified vaccinia virus Ankara (MVA) vector expressing the same prM-E ZIKV antigens (MVA-ZIKV) induced broad, polyfunctional and long-lasting ZIKV-specific CD4+ and CD8+ T-cell immune responses, with high levels of CD4+ T follicular helper cells, together with the induction of neutralizing antibodies. All those immune parameters were significantly stronger in the heterologous DNA-ZIKV/MVA-ZIKV immunization group compared to the homologous prime/boost immunizations regimens. Collectively, these results provided an optimized immunization protocol able to induce high levels of ZIKV-specific T-cell responses, as well as neutralizing antibodies and reinforce the combined use of DNA-based vectors and MVA-ZIKV as promising prophylactic vaccination schedule against ZIKV.

Immunogenicity and protection efficacy of a Salmonella enterica serovar Typhimurium fnr, arcA and fliC mutant

Salmonella enterica serovar Typhimurium is a major food-borne pathogen that can cause self-limited gastroenteritis or life-threatening invasive diseases in humans. There is no licensed S. Typhimurium vaccine for humans to date. In this study, we attempted to construct a live attenuated vaccine strain of S. Typhimurium based on three genes, namely, the two global regulator genes fnr and arcA and the flagellin subunit gene fliC. The S. Typhimurium three-gene mutant, named SLT39 (ΔfnrΔarcAΔfliC), exhibited a high level of attenuation with a colonization defect in mouse tissues and approximately 104-fold decreased virulence compared with that of the wild-type strain. To evaluate the immunogenicity and protection efficacy of STL39, mice were inoculated twice with a dose of 107 CFU or 108 CFU at a 28-day interval, and the immunized mice were challenged with a lethal dose of the wild-type S. Typhimurium strain one month post second immunization. Compared with mock immunization, SLT39 immunization with either dose elicited significant serum total IgG, IgG1 and IgG2a and faecal IgA responses against inactivated S. Typhimurium antigens at a comparable level post second immunization, whereas the 108 CFU group induced higher levels of duodenal and caecal IgA than the 107 CFU group. Furthermore, the bacterial loads in mouse tissues, including Peyer’s patches, spleen and liver, significantly decreased in the two SLT39 immunization groups compared to those in the control group post challenge. Additionally, all mice in the SLT39 (108 CFU) group and 80% of the mice in the SLT39 (107 CFU) group survived the lethal challenge, suggesting full protection and 80% protection efficacy, respectively. Thus, the S. Typhimurium fnr, arcA and fliC mutant proved to be a potential attenuated live vaccine candidate for prevention of homologous infection.

Oral immunization with soybean storage protein containing amyloid β4‐10 prevents spatial learning decline

AbstractBackgroundAmyloid‐β protein (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Because AD pathologies begin two decades before the onset of dementia, prevention of Aβ amyloidosis has been proposed as a mean to block the pathological cascade. We generated a transgenic plant‐based vaccine, a soybean storage protein containing Aβ4‐10, named Aβ+, for oral Aβ immunization.MethodOne mg of Aβ+ or control protein (Aβ‐) with 10 mg cholera toxin subunit B was orally administered via a catheter to TgCRND8 mice once a week from 9 weeks. Extended Morris water maze tests for 10 consecutive days were examined every 4 weeks. Mice were analyzed at 23 weeks (Aβ+ n = 9, Aβ– n = 11), 43 weeks (Aβ+ n = 7, Aβ– n = 6), and 59 weeks (Aβ+ n = 6, Aβ– n = 7).ResultAβ+ immunization raised both anti‐Aβ antibodies and cellular immune responses. Plasma from Aβ+ treated mice showed staining of Aβ oligomers. Spatial learning decline was prevented in the Aβ+ immunized group from 21 weeks. In Tris‐buffered saline (TBS), sodium dodecyl sulfate (SDS), and formic acid (FA) serial extractions, all sets of Aβ species from Aβ monomer, low to high molecular weight Aβ oligomers, and Aβ smears had different solubility in TgCRND8 brains. Aβ oligomers decreased in TBS fractions, corresponding to an increase in high molecular weight Aβ oligomers in SDS extracts and Aβ smears in FA fraction of the Aβ+ treated group. There was significant inhibition of histological Aβ burden, especially in diffuse plaques, and suppression of microglial inflammation. Processing of amyloid‐β protein precursor was not different between Aβ+ and Aβ‐ groups. No evidence of amyloid‐related inflammatory angiopathy was observed.ConclusionAβ+ oral immunization could be a promising, cheap, and long‐term safe disease‐modifying therapy to prevent the pathological process in AD.

The novel pathogenic Citrobacter freundii (CFC202) isolated from diseased crucian carp (Carassius auratus) and its ghost vaccine as a new prophylactic strategy against infection

Citrobacter freundii, a common pathogen of freshwater fish, severely restricts the sustainable development of the aquaculture industry. In this study, the novel C. freundii strain CFC202 was isolated from the intestines of crucian carp, which was annotated with three virulence factors related to promoting bacterial colonization and causing anemia by the whole genome analysis. We found that the strain adhered to the surface of hepatic L8824 cells, and actin aggregation occurred at the adhesion site of the strain. Then, C. freundii was fluorescently labelled and imaged the distribution of crucian carp under an In Vivo Imaging System (IVIS) during natural infection. The results showed that this strain mainly gathered in the intestines of crucian carp, and the intestinal villi were severely damaged by TEM. From the results of histopathological examination, the intestinal mucosa was obviously shed and various organs were damaged. Hence, we speculated that C. freundii could cause attaching and effacing (A/E) lesions to fish cells, which in turn caused pathological changes to various organs. Moreover, we constructed C. freundii ghost (CFG) and evaluated their immune effects by oral and injection immunization in crucian carp. Compared with the non-immunized group, non-specific immune indicators (ACP, AKP, SOD, and LZM), agglutination antibody titres of the serum, and the expression of immune-related genes (TNF-α and IL-6) of the head kidney and spleen were significantly increased in the immunized group (P < 0.01), indicating that innate and specific immune responses were induced by the CFG. In addition, the immunized fish showed higher protection than the non-immunized fish after challenge: oral control (20.00% survival); oral test (80.21% survival); injected control (6.67% survival); injected test (87.50% survival). Taken together, our research was the first to demonstrate that the constructed CFG might be developed as either oral or injected vaccines against C. freundii in aquaculture.

Self-Assembled Multi-Epitope Peptide Amphiphiles Enhance the Immune Response against Enterovirus 71.

Subunit vaccines consist of non-genetic material, such as peptides or proteins. They are considered safe because they have fewer side effects; however, they have low immunogenicity when used alone. We aimed to enhance the immune response of peptide-based vaccines by using self-assembled multimeric peptide amphiphiles (PAs). We designed two epitope PAs by conjugating epitope peptides from Enterovirus 71 (EV71) virus particle (VP) 1 and VP3 capsid proteins with different fatty acid chain lengths (VP1PA and VP3PA). These PAs self-assembled into supramolecular structures at a physiological pH, and the resulting structures were characterized using atomic force microscopy. Multi-epitope PAs (m-PAs) consisted of a 1:1 mixture of VP1PA and VP3PA solutions. To evaluate immunogenicity, m-PA constructs were injected with adjuvant subcutaneously into female Balb/c mice. Levels of antigen-specific immunoglobulin G (IgG) and IgG1 in m-PA-injected mice serum samples were analyzed using ELISA and Western blotting. Additionally, cytokine production stimulated by each antigen was measured in splenocytes cultured from immunized mice groups. We found that m-PA showed improved humoral and cellular immune responses compared to the control and peptide groups. The sera from m-PA immunized mice group could neutralize EV71 infection and protect host cells. Thus, self-assembled m-PAs can promote a protective immune response and can be developed as a potential platform technology to produce peptide vaccines against infectious viral diseases.

Eimeria tenella: IMP1 protein delivered by Lactococcus lactis induces immune responses against homologous challenge in chickens

Avian coccidiosis leads to severe economic losses on the global poultry industry. Immune mapped protein-1 (IMP1) is a novel membrane protein, and was reported to be a candidate protective antigen. However, production and utilization modes of IMP1 using Lactococcus lactis as delivery vector were not reported untill now. In the present study, Eimeria tenella IMP1 (EtIMP1) protein was expressed in L. lactis under the nisin-inducible promoter, and EtIMP1 protein was produced in cytoplasmic, cell wall-anchored and secreted forms. Each chicken was orally immunized with one of the three live EtIMP1-expressing lactococci three times at 2 weeks intervals (immunized group), or with live bacteria harboring empty vector (immunized control group). Chickens in immunized and immunized control group were challenged with E. tenella sporulated oocysts to assess the immune responses. The results showed that proliferative responses of peripheral blood T lymphocytes, mRNA expression levels of IL-2, IL-4, IL-10 and IFN-γ in spleen tissues, levels of serum IgG and secretory IgA (sIgA) in cecal lavage fluids from chickens in immunized group were all significantly elevated compared to that in immunized control group. All three the live EtIMP1-expressing lactococci significantly decreased oocyst shedding, alleviated pathological damage in cecum and improved weight gain compared with bacteria harboring empty vector. These results suggested EtIMP1 protein delivered by L. lactis might be a promising candidate in developing novel vaccines against Eimeria infection.

Immunogenicity and duration of immunity of the polyvalent vaccine against chicken salmonellosis

Poultry salmonellosis causes serious economic damage and must be prevented by disinfection, zoohygienic measures or by vaccination. To develop a new polyvalent vaccine against poultry salmonellosis, we used bacterial strains of Salmonella enteritidis, S. typhimurium and S. gallinarum. Antigenic and immunogenic efficacy of the vaccine was tested on specific-pathogen free chickens, which were divided into five groups of 10 birds in each group and were vaccinated intramuscularly at 8 and 12 weeks: group A (non-immunized control), group B (S. enteritidis mono-vaccine), group C (S. typhimurium mono-vaccine), group D (S. gallinarum mono-vaccine) and group E (trivalent vaccine Polimun Salmo). None of the immunized birds showed such adverse reactions as abnormal behaviour, mortality or signs of anorexia, depression or diarrhea. Two weeks after the revaccination, 5 birds in each group were challenged by watering 3 cm3 of working suspensions of S. gallinarum, S. typhimurium and S. enteritidis control strains at a concentration of 1 × 109 CFU. 72 h after the challenge, faeces were collected from all chickens in each group to identify Salmonella excretion with faeces, and the chickens were euthanized. Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared with the non-immunized control group. Blood samples were selected weekly from 5 chickens of each group for 184 days. The antigenic efficacy of the vaccines was studied by reaction of haemagglutination in the obtained serum. The potent antigen-specific response to lymphocyte activation found in all immunized groups indicated the induction of immune responses. Overall, the results showed that persistent immunity is formed in 4 weeks after the revaccination and lasts for a productive period. Immune response of chickens on day 184 after vaccination with Polimun Salmo was 1: 647, indicating that the developed polyvalent vaccine against common serovars of S. enterica in poultry is effective and immunogenic and can be further used in field studies.