Specific IgM Levels Research Articles

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

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

Adjuvant Effects of a CC Chemokine for Enhancing the Efficacy of an Inactivated Streptococcus agalactiae Vaccine in Nile Tilapia (Oreochromis niloticus).

In this study, the ability of a CC chemokine (On-CC1) adjuvant to enhance the efficacy of a formalin-killed Streptococcus agalactiae vaccine (WC) in inducing immune responses against S. agalactiae in Nile tilapia was investigated through immune-related gene expression analysis, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing, and challenge tests. Significantly higher S. agalactiae-specific IgM levels were detected in fish in the WC+CC group than in the WC alone or control groups at 8 days postvaccination (dpv). The WC vaccine group exhibited increased specific IgM levels at 15 dpv, comparable to those of the WC+CC group, with sustained higher levels observed in the latter group at 29 dpv and after challenge with S. agalactiae for 14 days. Immune-related gene expression analysis revealed upregulation of all target genes in the control group compared to those in the vaccinated groups, with notable differences between the WC and WC+CC groups at various time intervals. Additionally, transcriptome analysis revealed differential gene expression profiles between the vaccinated (24 and 96 hpv) and control groups, with notable upregulation of immune-related genes in the vaccinated fish. Differential gene expression (DGE) analysis revealed significant upregulation of immunoglobulin and other immune-related genes in the control group compared to those in the vaccinated groups (24 and 96 hpv), with distinct patterns observed between the WC and WC+CC vaccine groups. Finally, challenge with a virulent strain of S. agalactiae resulted in significantly higher survival rates for fish in the WC and WC+CC groups compared to fish in the control group, with a notable increase in survival observed in fish in the WC+CC group.

Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus (SGIV) infection in grouper.

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp

Aeromonas veronii is an important aquatic zoonotic, which elicits a range of diseases, such as haemorrhagic septicemia. To develop an effective oral vaccine against Aeromonas veronii infection in carp, the Aeromonas veronii adhesion (Aha1) gene was used as a target molecule to attach to intestinal epithelial cells. Two anchored recombinant. Lactic acid bacteria strains (LC-pPG-Aha1 1038 bp and LC-pPG-Aha1-LTB 1383 bp) were constructed by fusing them with the E. coli intolerant enterotoxin B subunit (LTB) gene and using Lactobacillus casei as antigen delivery vector to evaluate immune effects of these in carp. Western blotting and immunofluorescence were used to confirm that protein expression was successful. Additionally, levels of specific IgM in serum and the activities of ACP, AKP, SOD, LYS, C3, C4, and lectin enzymes-were assessed. Cytokines IL-10, IL-1β, TNF-α, IgZ1, and IgZ2 were measured in the liver, spleen, kidney, intestines, and gills tissue by qRT-PCR, which showed an increasing trend compared with the control group (P < 0.05). A colonization assay showed that the two L. casei recombinants colonized the middle and hind intestines of immunized fish. When immunized carp were experimentally challenged with Aeromonas veronii the relative percentage protection of LC-pPG-Aha1 was 53.57%, and LC-pPG-Aha1-LTB was 60.71%. In conclusion, these results demonstrate that Aha1 is a promising candidate antigen when it is displayed on lactic acid bacteria (Lc-pPG-Aha1 and Lc-pPG-Aha1-LTB) seems promising for a mucosal therapeutic approach. We plan to investigate the molecular mechanism of the L. casei recombinant in regulating the intestinal tissue of carp in future studies.

Assessment of antibody responses against SARS-CoV-2 in unvaccinated individuals and vaccinees from Omicron-BA.2 infection in Zhaoqing, Guangdong Province, China

Currently, the majority of the global population has been vaccinated with the COVID-19 vaccine, and characterization studies of antibodies in vivo from Omicron breakthrough infection and naive infection populations are urgently needed to provide pivotal clues about accurate diagnosis, treatment, and next-generation vaccine design against SARS-CoV-2 infection. We showed that after infection with Omicron-BA.2, the antibody levels of specific IgM against the Wuhan strain and specific IgG against Omicron were not significantly elevated within 27 days of onset. Interestingly, in this study, the levels of humoral immunity against Omicron-specific IgM were significantly increased after breakthrough infection, suggesting that the detection of Omicron-specific IgM antibodies can be used as a test criterion of Omicron breakthrough infection. In addition, we observed that serums from unvaccinated individuals and the majority of vaccinated infections possessed only low or no neutralizing activity against Omicron at the onset of Omicron breakthrough infections, and at the later stage of Omicron-BA.2 breakthrough infection, levels of neutralization antibody against the Wuhan and Omicron strains were elevated in infected individuals. The findings of this study provide important clues for the diagnosis of Omicron breakthrough infections, antibody characterization studies and vaccine design against COVID-19.

The Relation between Immunological Features and the Positive SARS-CoV-2 Nucleic Acid in Patients with Nonsevere COVID⁃19.

Objective The novel coronavirus nucleic acid results are the core indicators of illness monitoring. This study aimed to evaluate the relationship between immunological features and positive SARS-CoV-2 nucleic acid by analyzing the clinical and immunological features in nonsevere COVID-19 cases. Methods Data from nonsevere COVID-19 patients admitted to Haihe Hospital from May 2020 to June 2021 were retrospectively reviewed and analyzed. Results (1) A total of 122 cases were reviewed in the present study, including 38 mild and 84 moderate cases. The average age of mild cases was significantly different from moderate cases (P < 0.001). Eight patients complained of hyposmia and it was more frequent in mild cases (P < 0.001). The nucleic acid positive duration (NPD) of nonsevere novel coronavirus was 20.49 (confidence interval (CI) 17.50–3.49) days. (2) The levels of specific IgM and IgG for COVID-19 were higher in mild cases than in moderate cases (P=0.023 and P=0.047, respectively). (3) The correlation analysis with antibodies and T-cell subtypes showed that the lymphocyte (LYM) count, T cells, CD4+T cells, and CD8+T cells had a linear correlation with NPD. (4) Among the 93 patients monitored, 62 COVID-19 cases presented a progressive rise of specific IgM and IgG. The daily increase rates of IgM and IgG were 38.42% (CI 28.22–48.61%) and 24.90% (CI 0.23–29.58%), respectively. Conclusion The levels and daily increase rates of specific IgM and IgG against the virus can vary between cases. The NPD presented a linear correlation with the LYM, T cells, CD4+T cells, and CD8+T cells. Hence, more attention should be paid to these indicators in clinical practice.

Immunogenicity of the drug "Live intranasal vaccine for the prevention of pertussis" (GamLPV) with a single use in healthy volunteers

Introduction. A significant increase in the incidence of pertussis in the world, including among adolescents and adults, the prevalence of mild forms of the disease and asymptomatic carrier of bacteria B. pertussis, and the resulting need for mass revaccination of different age groups determine the demand for new vaccines against B. pertussis. In N.F. Gamaleya Federal Research Center for Epidemiology and Microbiology, a live intranasal pertussis vaccine for the prevention of pertussis (GamLPV) has been developed. The GamLPV vaccine underwent preclinical studies that proved its safety and effectiveness in experiments on small laboratory animals and nonhuman monkeys. Safety of vaccine is shown in clinical studies on healthy volunteers.The aim of the study is to assess the immunogenicity of different doses of the drug GamLPV when first used in healthy volunteers.Materials and methods. The study was conducted as randomized placebo-controlled, blind trial with consistent volunteer inclusion and dose escalation. Study ID in clinicaltrials.gov database: NCT03137927 (A Phase I Clinical Study of a GamLPV, a Live Intranasal Bordetella Pertussis Vaccine). The following parameters of humoral and cellular immune responses were assessed in dynamics: levels of specific IgM, IgG and IgA antibodies in blood serum of volunteers and the number of cytokines interleukin-17, tumor necrosis factor-α, interferon-γ produced after specific induction in vitro of blood mononuclears of vaccinated volunteers. Dynamics of attenuated bacteria persistence in nasopharynx of vaccinated volunteers was evaluated.Results. Intranasal vaccination of volunteers with the drug Gam LPV resulted in the formation of a specific humoral (IgG and IgA) and cellular immune response. The dose-dependent nature of immunoglobulin and cytokine production was shown. Attenuated bacteria persisted for a long time in the nose/oropharynx of vaccinated volunteers.Discussion. Good tolerability of all tested doses of the drug justifies the choice for further investigation of a vaccine dose equal to 4 × 109 CFU. At the next stage, the safety and immunogenicity of two-time vaccination of volunteers will be studied.

Bacillus subtilis spores as an adjuvant to enhance the protection efficacy of the SVCV subunit vaccine (SVCV‐M protein) in German mirror carp ( Cyprirnus Carpio Songpa Linnaeus Mirror )

The present study evaluated the adjuvant effects of Bacillus subtilis (B. subtilis) spores on the immune response induced by an SVCV-M protein, which was equivalent to a subunit vaccine in German mirror carp (Cyprirnus Carpio Songpa Linnaeus Mirror). The following results were observed: in vitro, there were no toxic effects of B. subtilis spores and SVCV-M protein on the viability of epithelioma papulosum cyprini (EPC) cells. In addition, B. subtilis spores can significantly reduce the oxidative damage of the spring viraemia of carp virus (SVCV) on EPC cells, but protein-M had no significant effects on the oxidative damage of EPC cells. In vivo, higher levels of specific IgM could be detected in the protein-M+B. subtilis spores group, compared with that in PBS, B. subtilis spores and Protein-M groups. Immune-related indexes including the activities of LZM and AKP and the content of C3 and C4 in the protein-M+B. subtilis spores group were strongly upregulated. Immune-related genes had the same trend as the immune-related indexes. Furthermore, the antioxidant capacity of the protein-M+B. subtilis spores group was also better than that of PBS, B. subtilis spores and Protein-M groups. Finally, the highest survival rate (59.69%) was observed in the protein-M+B. subtilis spores group, while the survival rate was 8.89% in protein-M group, and 0% in the PBS and B. subtilis spores groups. This study suggests B. subtilis spores as an adjuvant that can enhance the protection efficacy of the SVCV subunit vaccine (SVCV-M protein) in common carp.

Detectable antibodies against SARS-CoV-2 in newborns from mothers infected with COVID-19 at different gestational ages

Detectable antibodies against SARS-CoV-2 in newborns from mothers infected with COVID-19 at different gestational ages

Passive Immunization Delays Disease Outcome in Gilthead Sea Bream Infected With Enteromyxum leei (Myxozoa), Despite the Moderate Changes in IgM and IgT Repertoire.

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.

Surface display of spring viremia of carp virus glycoprotein on Lactococcus lactis and its protection efficacy in common carp (Cyprinus carpio L.)

Spring viremia of carp virus (SVCV) causes devastating disease in aquaculture, resulting in significant economic impact. To develop an effective means against SVCV infection, a Lactococcus lactis (L.lactis) based subunit vaccine (pNZ-UGA) was developed based on surface displaying of SVCV glycoprotein using anchoring motif of the cA (C terminus of the peptidoglyvsn-binding) domains of AcmA, a major autolysin from L.lactis. The surface expression of SVCV glycoprotein was verified by indirect immunofluorescence assay. The efficacy of the constructed vaccine was further evaluated in common carp. The results showed that the higher levels of specific IgM could be detected in fish vaccinated with pNZ-UGA, compared with that in PBS and L.lactis groups. Immune-related genes including TNF-α, IL-6b, IL-1β, Cxcr 1, Cxca, IFNg2b, I-IFN, and IgM expression in pNZ-UGA group were strongly up-regulated, revealing that robust innate immune response was induced. Notably, the lowest cumulative mortality (13.46%) was observed in fish vaccinated with pNZ-UGA vaccine after SVCV challenge, whereas the cumulative mortality were 100.00% and 92.31% in PBS and L.lactis groups, respectively. This study suggests the potential use of the recombinant L.lactis with surface displaying antigen proteins as effective vaccines against SVCV and other fish virus infection.

Многофакторный анализ клинико-лабораторных показателей, уровней IL-17a, IL-23, IL-33, IL-35 и cпецифических антител в сыворотке крови у больных безэритемной формой иксодового клещевого боррелиоза

Aim: to determine the association between clinical laboratory parameters, the production of cytokines (IL-17A, -23, -33, -35), and specific IgM and IgG in the serum of patients with Lyme borreliosis without erythema migrans. Patients and Methods: complete blood count, the concentrations of IL-17A, -23, -33, -35, and the levels of specific IgM and IgG were measured during acute infection and convalescence (n=30). The control group included age- and sex-matched healthy individuals (n=30). Statistical analysis was performed using the StatSoft Statistica v 10.0 software (parametric and non-parametric methods and multifactorial analysis, i.e., principal component analysis). Results: most (80%) patients with Lyme borreliosis without erythema migrans are the people of working age. In most patients, the combination of the specific antibodies against Borrelia afzelii and Borrelia garinii (76.7%) and severe intoxication and inflammatory process (100%) were detected. Moderate and severe disease associated with meningism was diagnosed in 90% and 10%, respectively. The mean duration of hectic period was 8.3±1.27 days. Abnormal ECG was reported in 40% of patients, i.e., conduction abnormalities in 20%, sinus bradycardia in 16.7%,and sinus tachycardia in 3.3%. The clinical laboratory signs of hepatitis without jaundice were identified in 26.7%. During treatment, the significant reduction in band and segmented neutrophil counts as well as the significant increase in platelet count were revealed compared to these parameters at admission. Abnormal cytokine levels (i.e., the increase in IL-17A, -23, -33 and the deficiency of IL-35) were detected. Conclusions: multifactorial analysis has demonstrated that the severity of immunological abnormalities in patients with Lyme borreliosis without erythema migrans is associated with fever, cardiac and liver disorders, the high levels of IL-23 and IL-33, and the lack of IL-35 and specific IgM and IgG. KEYWORDS: tick-borne borreliosis, Lyme disease without erythema migrans, clinical laboratory signs, cytokines, specific antibodies, multifactorial analysis, principal component analysis. FOR CITATION: Sapozhnikova V.V., Bondarenko A.L. Multifactorial analysis of clinical laboratory signs, the levels of IL-17A, IL-23, IL-33, IL-35, and specific antibodies in the serum of patients with Lyme borreliosis without erythema migrans. Russian Medical Inquiry. 2020;4(11):676–681. DOI: 10.32364/2587-6821-2020-4-11-676-681.

Added value of IgM detection and low avidity index as markers of acute bovine besnoitiosis

Early in vivo diagnosis of bovine besnoitiosis is crucial for the success of control programmes. However, diagnosis in acutely infected animals is hindered by the low sensitivity of the available serological tools.In this study, a novel ELISA to detect specific anti-Besnoitia besnoiti IgM antibodies was developed. The usefulness of this tool together with an avidity ELISA were studied with a well-coded sera panel from experimentally and naturally infected cattle. First, the kinetics of specific IgM levels were determined in experimentally infected calves during the acute and chronic infection. Next, IgM levels were determined in naturally infected cattle with either acute or chronic infection. Finally, the IgG avidity index was monitored in both experimentally and naturally infected cattle.Specific IgM antibodies were detected prior to specific IgG antibodies (7-19 days vs. 17–26 days post-infection). A prompt IgM response was associated with the end of the febrile stage in experimentally infected calves. Naturally and experimentally infected animals with acute clinical signs tested IgM-positive but IgG-negative, followed by IgG seroconversion 2–3 weeks later. Chronically infected cattle developed both IgM and IgG specific antibodies. Moreover, a progressive increase in the avidity index (AI) was observed in all experimentally infected calves during the course of the experimental trials. However, a low AI coincided with visible tissue cysts. Low avidity values were also detected when naturally infected cattle with acute clinical signs seroconverted, in contrast to a high AI detected in chronically infected cattle.In summary, IgM and avidity ELISAs improved the early in vivo diagnosis of bovine besnoitiosis. IgM-positive but IgG-negative results were indicative of an acute infection, whereas IgG positive results accompanied by low avidity values confirmed a recent infection.

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)

BackgroundHirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.ResultsGlycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 109 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 104–105 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 102–103L. lactis cells could still be recovered.ConclusionsHIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

Oral vaccination of tilapia against Streptococcus agalactiae using Bacillus subtilis spores expressing Sip

Streptococcus agalactiae infections are becoming an increasing problem in aquaculture because of significant morbidity and mortality, which restricts the healthy development of tilapia aquaculture. To seek safe and effective prevention measures, a Bacillus subtilis GC5 surface displayed vaccine was prepared and applied orally in tilapia. The study first showed that recombinant spores can engraft in the tilapia intestine. Then, the effect of protection and the immune responses were evaluated. The results of ELISA showed that Sip-specific antibody in the sera of GC5-Sip-immunized fish can be detected after the first oral administration when compared to the phosphate buffer saline (PBS) control group, and the levels of specific IgM gradually strengthened with boosting, so does the specific antibody against bacteria, proving that humoral immunity was induced. Quantitative real-time PCR (qRT-PCR) results showed that the immune-related gene expression of the gut and spleen exhibited a different rising trend in the GC5-Sip group, revealing that innate immune response and local as well as systemic cellular immunity were induced. The outcome of fish immunized with GC5-Sip spores provided a relative percent survival (RPS) of 41.7% against S. agalactiae and GC5 group had an RPS of 24.2%, indicating that GC5-Sip was safe and effective in protecting tilapia against bacterial infection. Our study demonstrated that the oral administration of B. subtilis spores expressing Sip could cause an effective immune response and offer good resistance to bacterial infection. Our work may lead to the development of new ideas for immunoprophylaxis against S. agalactiae infection.

Oral delivery of Bacillus subtilis spores expressing grass carp reovirus VP4 protein produces protection against grass carp reovirus infection

Grass carp (Ctenopharyngodon idellus) hemorrhagic disease (GCHD), caused by grass carp reovirus (GCRV), has given rise to an enormous loss in grass carp industry during the past years. Up to date, vaccination remained to be the most effective way to protect grass carp from GCHD. Oral vaccination is of major interest due to its advantages of noninvasive, time-saving, and easily-operated. The introduction of oral vaccination has profound impact on aquaculture industry because of its feasibility of extensive application for fish in various size and age. However, the main challenge in developing oral vaccine is that antigens are easily degraded and are easy to induce tolerance. Bacillus subtilis (B. subtilis) spores would be an ideal oral vaccine delivery system for their robust specialty, gene operability, safety and adjuvant property. VP4 protein is the major outer capsid protein encoded by GCRV segment 6 (S6), which plays an important role in viral invasion and replication. In this study, we used B. subtilis spores as the oral delivery system and successfully constructed the B. subtilis CotC-VP4 recombinant spores (CotC-VP4 spores) to evaluate its protective efficacy in grass carp. Grass carp orally immunized with CotC-VP4 spores showed a survival rate of 57% and the relative percent survival (RPS) of 47% after the viral challenge. Further, the specific IgM levels in serum and the specific IgZ levels in intestinal mucus were significantly higher in the CotC-VP4 group than those in the Naive group. The immune-related genes including three innate immune-related genes (IL-4/13A, IL-4/13B, CSF1R), four adaptive immune-related genes (BAFF, CD4L, MHC-II, CD8), three inflammation-related genes (IL-1β, TNF-α, TGF-β) and interferon type I (IFN-I) related signaling pathway genes were significantly up-regulated in the CotC-VP4 group. The study demonstrated that the CotC-VP4 spores produced protection in grass carp against GCRV infection, and triggered both innate and adaptive immunity post oral immunization. This work highlighted that Bacillus subtilis spores were powerful platforms for oral vaccine delivery, and the combination of Bacillus subtilis spores with GCRV VP4 protein was a promising oral vaccine.

Identity and validity of conserved B cell epitopes of filovirus glycoprotein: towards rapid diagnostic testing for Ebola and possibly Marburg virus disease

BackgroundEbolavirus and Marburgvirus are genera of the virus family Filoviridae. Filoviruses cause rare but fatal viral hemorrhagic fevers (VHFs) in remote villages of equatorial Africa with potential for regional and international spread. Point-of-care (POC) rapid diagnostic tests (RDTs) are critical for early epidemic detection, reponse and control. There are 2 RDTs for Zaire ebolavirus (EBOV), but not other Ebolavirus spp. or Marburg marburgvirus (MARV). We validate 3 conserved B cell epitopes of filovirus glycoprotein (GP) using ebola virus diseases (EVD) survivor samples, towards devising pan-filovirus RDTs.MethodsIn-silico Immuno-informatics:- (a) multiple and basic local alignments of amino-acid sequences of filovirus (4 Ebolavirus spp. & MARV) Gp1, 2 and epitope prediction and conservation analyses within context of ClusterW, BLAST-P and the immune epitope database analysis resource (IEDB-AR); alongside (b) in-vitro enzyme immuno-assays (EIAs) for SUDV Gp1, 2 antigen and host-specific antibodies (IgM and IgG) among 94 gamma irradiated EVD survivor serum and 9 negative controls.ResultsLinear B cell epitopes were present across the entire length of all Gp1, 2, most lying in the region between amino acids positioned 350 and 500. Three seperate epitopes 97/80_GAFFLYDRLAST, 39_YEAGEWAENCY and 500_CGLRQLANETTQALQLFLRATTELR (designated UG-Filo-Peptide− 1, 2 and 3 respectively) were conserved within all studied filovirus species Gp1, 2. Gp1, 2 host specific IgM levels were comparably low (av. ODs < 0.04 [95% CI: 0.02837 to 0.04033]) among the 9 negative controls and 57 survivor samples analyzed. Host specific IgG levels, on the other hand, were elevated (av. ODs > 1.7525 [95% CI: 0.3010 to 3.1352]) among the 92 survivor samples relative to the 9 negative controls (av. ODs < 0.2.321 [95% CI: -0.7596 to 0.5372]). Filovirus Gp1, 2 antigen was not detected [av. ODs < 0.20] within EVD survivor serum relative to recombinant protein positive controls [av. ODs = 0.50].ConclusionsThese conserved B cell epitopes of filovirus Gp1, 2 and their derivative antibodies are promising for research and development of RDTs for EVD, with potential for extension to detect MVD.

Leptospiroza w Polsce w latach 2014-2017 – charakterystyka zakażeń i dane z nadzoru

The aim of the study was the analysis of current Leptospira spp. infections in Poland on the basis of blood serum samples tests results and clinical data collected from clinicians in the Laboratory NIPH-NIH. Clinical materials from 48 patients with clinical symptoms suggesting Leptospira spp suspected of leptospirosis from the years 2014-2017 were included to the study. Blood serum samples collected from patients were tested in Laboratory of Rickettsiae, Chlamydiae and Spirochaetes (currently Laboratory of Vector-borne Diseases) of NIPH-NIH. Levels of specific IgM and IgG antibodies to Leptospira spp. antigens were detected with the enzyme-linked immunosorbent assay (ELISA). Specific antibodies to Leptospira spp. were detected in 18 patients (37.5%). IgM antibodies were found in 6 patients (12.5%) and IgG antibodies were identified in 7 patients (14.6%). Both classes of antibodies of were detected in 5 patients (10.4%). The most samples for study were sent to laboratory from Masovian (13 samples) and Kuyavian-Pomeranian (11 samples) Voivodeships. Not any samples from the Lower Silesia, Lublin, Łódź, Podlaskie and Warmian-Masurian Voivodeships were received. In these patients the most common symptoms of disease were: fever, hepatitis with jaundice and renal failure. The number of diagnosed human leptospirosis in Poland is low in comparison to the number of cases in other countries, although the Leptospira spp. spirochetes occur in animals in the environment.

HORIZONTAL TRANSMISSION OF PAROTITIS VACCINE STRAIN FROM VACCINATED TO CLOSE CONTACTS

Aim. A controlled study was carried out regarding horizontal transmission of the vaccine strain of parotitic virus (PV). Materials and methods. 20 couples took part in the study. Monitoring of both spouses was carried out for 42 days after a single vaccination of one of them, levels of specific IgM and IgG in sera and virus-neutralizing IgG in sera and saliva were studied in dynamics, PV RNA was also determined in sera and saliva samples. Amplified fragments of F, SH, NP and HN genes of PV were sequenced. Results. Transfer of PV vaccine strain was registered in all the couples except for one. Except a single contact spouse, all the rest demonstrated formation of a specific immune response. At month 4 of the observation, group of vaccinated spouses differed significantly from a group of contact spouses only by total content of specific IgG and their avidity. Conclusion. Horizontal transmission of PV strain was demonstrated in couples after vaccination of one of the spouses and resulted in formation of specific immunity in 95% of contact individuals.

Immune response induced by oral delivery of Bacillus subtilis spores expressing enolase of Clonorchis sinensis in grass carps (Ctenopharyngodon idellus)

Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection.