Immunodominant Protein Research Articles

Identification and Characterization of Immunodominant Proteins from Tick Tissue Extracts Inducing a Protective Immune Response against Ixodes ricinus in Cattle.

Ixodes ricinus is the main vector of tick-borne diseases in Europe. An immunization trial of calves with soluble extracts of I. ricinus salivary glands (SGE) or midgut (ME) previously showed a strong response against subsequent tick challenge, resulting in diminished tick feeding success. Immune sera from these trials were used for the co-immunoprecipitation of tick tissue extracts, followed by LC-MS/MS analyses. This resulted in the identification of 46 immunodominant proteins that were differentially recognized by the serum of immunized calves. Some of these proteins had previously also drawn attention as potential anti-tick vaccine candidates using other approaches. Selected proteins were studied in more detail by measuring their relative expression in tick tissues and RNA interference (RNAi) studies. The strongest RNAi phenotypes were observed for MG6 (A0A147BXB7), a protein containing eight fibronectin type III domains predominantly expressed in tick midgut and ovaries of feeding females, and SG2 (A0A0K8RKT7), a glutathione-S-transferase that was found to be upregulated in all investigated tissues upon feeding. The results demonstrated that co-immunoprecipitation of tick proteins with host immune sera followed by protein identification using LC-MS/MS is a valid approach to identify antigen–antibody interactions, and could be integrated into anti-tick vaccine discovery pipelines.

Lack of detail in population-level data impedes analysis of SARS-CoV-2 variants of concern and clinical outcomes

The SARS-CoV-2 lineage B.1.1.7 is characterised by a suite of defining mutations in the immunodominant spike protein, including a signature Asp501Tyr substitution in the receptor-binding domain.1 First reported in December 2020, in the UK, the variant's discovery coincided with a substantial surge in case numbers and fatalities in the UK, raising concerns that this variant was both more infectious and virulent than previous variants. Epidemiological and modelling studies have yielded good evidence that B.1.1.7 is more transmissible than other variants.

Levels of anti-B13 antibodies over time in a cohort of chronic infected by Trypanosoma cruzi. Its relationship with specific treatment and clinical status

The immunodominant B13 protein of Trypanosoma cruzi is found on the surface of trypomastigotes and exhibits cross-reactivity with the human cardiac myosin heavy chain; for which antibodies against this parasitic antigen may be involved in the development of disease pathology. In a cohort of chronically T. cruzi-infected adults, undergoing trypanocidal treatment, or not, we, therefore, decided to evaluate the levels of anti-B13 antibodies (ELISA-B13) and its eventual relationship with heart complaints. Two hundred twenty-eight serum samples from 76 chronically infected adults with an average follow-up of 24 years were analyzed. Thirty of them had received trypanocidal treatment. Among treated patients, anti-B13 Ab levels in successive samples showed a significant decrease in reactivity as the years after treatment increased (ANOVA test, p=0.0049). At the end of the follow-up, 36.7% became non-reactive for ELISA B13. Untreated patients did not have significant variations in the level of anti-B13 antibodies during follow-up. None of the treated patients had electrocardiographic changes compatible with chronic chagasic cardiomyopathy, whereas 21.7% of those undergoing no treatment did show such kind of pathological electrocardiogram tracings. ELISA-B13 was reactive in all cases with heart involvement. Among untreated patients, there were no significant differences in anti-B13 antibodies when comparing individuals without proven pathology with those with chronic chagasic cardiomyopathy. Although treatment with trypanocidal drugs was followed by decreased anti-B13 antibody levels, such assessment was unhelpful in differentiating the evolution of chronic chagasic heart disease.

Immunoreactive Proteins of Dormant Mycobacterium tuberculosis Cells

The protein profile of dormant Mtb obtained after the gradual acidification of Mtb culture was studied to find antigenic proteins for humans that are expressed by M. tuberculosis (Mtb) cells in vitro under conditions close to the situation of persistence in vivo. According to 2D electrophoresis, a significant diversity of proteins in dormant cells was found. However, the representation of individual proteins in dormant versus active cells differed substantially. Immunoblotting in different protein fractions of dormant cells revealed ten proteins that are able to bind antibodies in pooled sera of TB patients. Two proteins (Rv2018 and Rv0341) are new immunogenics that were not previously found in other studies. Four proteins (Rv0341, Rv2018, Rv1509, Rv2986) with the maximal structural specificity for Mtb due to their unique extended domains were selected for further analysis. These proteins were expressed in E. coli cells and studied via enzyme-linked immunosorbent assay (ELISA) for the immunogenicity of individual sera of TB patients and healthy donors. All proteins were found to have the ability to react with individual sera of TB patients. In TB patients, 5–45% (depending on the particulate protein) have a titer that is higher than the average titers of healthy donors +SD; the most immunogenic was protein Rv2986. Thus, the application of phenotypically changed (dormant) Mtb cells makes it possible to identify a specific repertoire of immunodominant proteins that could be used in the construction of polypeptides that are useful for the serodiagnosis of active/latent TB.

Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis.

Background and Aim:The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis.Materials and Methods:Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done.Results:All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden’s index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85).Conclusion:BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

Identification of Immunodominant Outer Membrane Proteins of Fusobacterium necrophorum from Severe Ovine Footrot By MALDI-TOF Mass Spectrometry

The aim of this study was to identify the immunodominant outer membrane proteins (OMPs) of Fusobacterium necrophorum from sheep affected with severe foot-rot. The OMP profile of ovine strains of F. necrophorum has not been well studied. We analyzed the OMP profile of the most frequent lktA variant JKS-F3 of F. necrophorum associated with severe ovine foot-rot with lesion score 4 in order to identify its major immunodominant OMPs. Electrophoretic separations of extracted OMPs showed a number of spots in two-dimensional electrophoretic gels. Two immunoreactive proteins of size around 43 kDa were identified through western blotting using hyperimmune sera raised in rabbits. These two immunogenic OMPs were analyzed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) which revealed that these two OMPs of lktA variant JKS-F3 of F. necrophorum showed 46 and 42 percent protein sequence coverage and scores of 125 and 114, respectively, with the reported 43 kDa outer membrane protein of F. necrophorum strain H05, a putative porin having properties similar to pore-forming proteins of anaerobic Gram-negative bacteria. These identified immunogenic OMPs will contribute to our understanding of the pathogenic role played by this organism in ovine foot-rot and could be exploited to devise an effective control strategy through development of an OMP-based recombinant vaccine to mitigate foot-rot in sheep and goats.

Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism.

ObjectivesTo identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis.ResultsIn the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome.ConclusionsIdentified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10529-021-03091-4.

Multilocus sequence typing and phylogenetic analysis revealed two distinct almond witches'‐broom phytoplasma subpopulations in Iran

AbstractDuring a field survey conducted in different geographical regions of Iran, phytoplasma‐like symptoms were observed in peach, almond, wild almond and GF‐677 rootstock. Based on 16S rDNA amplification, followed by nucleotide sequence and phylogenetic analyses, almond witches'‐broom (AlmWB) phytoplasma strains were found in association with symptomatic plants. Based on the single‐nucleotide polymorphisms within 16S rDNA sequence, most of the Iranian AlmWB phytoplasma strains were distinguished from the Lebanese strains. Further molecular typing, based on sequence and phylogenetic analyses of rplV‐rpsC (ribosomal proteins) and imp (immunodominant membrane protein) genes, indicated that AlmWB phytoplasma strains can be differentiated in two subpopulations: Ir, including exclusively Iranian strains; Lb, comprising strains from Lebanon and Iran. Moreover, the selection pressure analysis of imp gene sequences suggested that these two AlmWB phytoplasma subpopulations had some distinct biological or ecological properties. Finally, results of the sequence and phylogenetic analyses performed on rplV‐rpsC and imp genes showed a considerably high genetic distance between AlmWB phytoplasma strains (16SrIX‐B and variants) and phytoplasmas belonging to other 16SrIX subgroups. The significance of genetic variation in phytoplasmas of 16SrIX group is discussed in relation to their biological characteristics and geographical distinction.

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

SummaryThe race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.

Obtaining Recombinant Antigens for the Development of Serological Diagnosis of Marburg Fever

Aim. Production of recombinant viral antigens of the main immunodominant proteins: glycoprotein (GPΔMLD), nucleoprotein (NP) and matrix protein (VP40) of the Marburg virus, as well as the study of their antigenic and immunogenic properties.Materials and methods. To create recombinant proteins GPΔMLD, NP and VP40 of the Marburg virus, synthesized nucleotide sequences encoding these proteins cloned into the pET21a expression vector were used. The immunogenic and antigenic properties of the obtained recombinant proteins were tested using a number of biomodels (mice, chickens, and guinea pigs).Results and discussion. Recombinant plasmids containing genes encoding proteins GPΔMLD, NP, VP40 of the Marburg virus, as well as Escherichia coli producing strains, with the yield of purified preparations of recombinant proteins GPΔMLD, NP, VP40 from one liter of culture fluid – 5, 10, and 10 μg were obtained, respectively. When mice are immunized, recombinant proteins GP, NP, and VP40 MARV induce the synthesis of high titer antibodies (recombinant proteins NP and VP40 – more than 409600, and recombinant protein GPΔMLD – 12800). Mouse antibodies specific to recombinant proteins interact in an enzyme-linked immunosorbent assay (ELISA) with the antigen of inactivated MARV. Antibodies of chickens immunized with virus-like particles containing the surface glycoprotein of the Marburg virus and antibodies of guinea pigs immunized with an experimental DNA vaccine containing the GPΔMLD MARV gene recognize the recombinant GPΔMLD protein and the viral protein in the inactivated MARV. The resulting recombinant proteins are immunogenic/antigenic and can be used for the development of enzymelinked immunosorbent assay systems.

Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA)

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Immunological Characterization of Proteins Expressed by Genes Located in Mycobacterium tuberculosis-Specific Genomic Regions Encoding the ESAT6-like Proteins.

The 6 kDa early secreted antigen target (ESAT6) is a low molecular weight and highly immunogenic protein of Mycobacterium tuberculosis with relevance in the diagnosis of tuberculosis and subunit vaccine development. The gene encoding the ESAT6 protein is located in the M. tuberculosis-specific genomic region known as the region of difference (RD)1. There are 11 M. tuberculosis-specific RDs absent in all of the vaccine strains of BCG, and three of them (RD1, RD7, and RD9) encode immunodominant proteins. Each of these RDs has genes for a pair of ESAT6-like proteins. The immunological characterizations of all the possible proteins encoded by genes in RD1, RD7 and RD9 have shown that, besides ESAT-6 like proteins, several other proteins are major antigens useful for the development of subunit vaccines to substitute or supplement BCG. Furthermore, some of these proteins may replace the purified protein derivative of M. tuberculosis in the specific diagnosis of tuberculosis by using interferon-gamma release assays and/or tuberculin-type skin tests. At least three subunit vaccine candidates containing ESAT6-like proteins as antigen components of multimeric proteins have shown efficacy in phase 1 and phase II clinical trials in humans.

Prime-boost with Chikungunya virus E2 envelope protein combined with Poly (I:C) induces specific humoral and cellular immune responses

Chikungunya virus (CHIKV) is an arbovirus transmitted to humans mainly by the bite of infected Aedes aegypti and Aedes albopictus mosquitoes. CHIKV illness is characterized by fever and long-lasting arthritic symptoms, and in some cases it is a deadly disease. The CHIKV envelope E2 (E2CHIKV) glycoprotein is crucial for virus attachment to the cell. Furthermore, E2CHIKV is the immunodominant protein and the main target of neutralizing antibodies. To date, there is no available prophylactic vaccine or specific treatment against CHIKV infection. Here, we designed and produced a DNA vaccine and a recombinant protein containing a consensus sequence of E2CHIKV. C57BL/6 mice immunized twice with the E2CHIKV recombinant protein in the presence of the adjuvant Poly (I:C) induced the highest E2CHIKV-specific humoral and cellular immune responses, while the immunization with the homologous DNA vaccine pVAX-E2CHIKV was able to induce specific IFN-γ producing cells. The heterologous prime-boost strategy was also able to induce specific cellular and humoral immune responses that were, in general, lower than the responses induced by the homologous E2CHIKV recombinant protein immunization. Furthermore, recombinant E2CHIKV induced the highest titers of neutralizing antibodies. Collectively, we believe this is the first report to analyze E2CHIKV-specific humoral and cellular immune responses after immunization with E2CHIKV recombinant protein and DNA pVAX-E2CHIKV vaccine platforms.

Surface Layer Protein A Expressed in Clostridioides difficile DJNS06-36 Possesses an Encephalitogenic Mimotope of Myelin Basic Protein.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Recent studies suggest that migration of Th1 and Th17 cells specific for enteric bacteria from the gut to the CNS may lead to the initiation and/or exacerbation of autoimmune diseases including MS. Human leukocyte antigen (HLA)-DR15 is an MHC class II (MHCII) haplotype highly associated with the development of MS that contains the two HLA-DRB* genes, DRB1*1501 (DR2b) and DRB5*0101 (DR2a). To identify enteric bacteria which harbor antigenic epitopes that activate myelin-specific T cells and drive CNS inflammation, we screened for enteric bacteria which express cross-reactive epitopes (‘mimotopes’) of an immunodominant myelin basic protein 89–98 (MBP89-98) epitope. Based on known MHCII HLA-DR2a amino acid binding motifs and cultivation with splenic T cells isolated from MBP-T cell receptor (TCR)/DR2a transgenic (Tg) mice, we discovered that a certain variant of surface layer protein A (SLPA), which is expressed by a subtype of Clostridioides difficile, contains an amino acid sequence that activates MBP89-98-reactive T cells. Furthermore, activation of MBP-specific T cells by SLPA upon active immunization induced experimental autoimmune encephalomyelitis (EAE) in MBP-TCR/DR2a Tg mice. This study suggests that a unique strain of C. difficile possesses an encephalitogenic mimotope of MBP that activates autoreactive, myelin-specific T cells.

HCMV infection induces AD pathology in astrocytes in vitro

AbstractBackgroundInfectious agents have long been suspected to be risk factors for Alzheimer’s disease (AD). Some studies have identified a link between neurotropic viruses and AD. While these studies demonstrated an association between AD and neuroviruses, most do not establish a causal link. However, many AD pathological hallmarks, including beta amyloid plaques (Aβ), astrogliosis, neuroinflammation, and neuronal damage, overlap with pathologies caused by neuroviruses. Approximately 80% of adults are seropositive for human cytomegalovirus (HCMV), a virus that is neuroinvasive, productively infects brain cells, and is immune‐dominant in aging populations. Using post‐mortem tissues from the Rush Alzheimer’s Disease Center we previously reported that HCMV infection is associated with AD clinical markers. In the present study, we assessed the ability of HCMV infection to induce two AD pathological hallmarks in astrocytes: Aβ production and astrocyte reactivity.MethodTo determine the impact of HCMV infection on astrocytes, we generated low‐passage clinical HCMV isolates for infection of human fetal astrocytes (HFA) and astroglioma (U251) cells. To assess the ability of HCMV to produce Aβ, we infected HFAs and U251s with HCMV for 3‐9 days then immunostained for Aβ and HCMV proteins. To test whether HCMV infection results in astrocyte reactivity, we infected HFAs with HCMV for 5 days then quantified levels of reactivity‐associated transcripts via quantitative PCR.ResultHFA and U251 infected cells showed HCMV cytopathic effects characteristic of permissive cells, including enlargement and plaque formation. HCMV infection of HFA and U251 cells induced the expression of Aβ as observed by co‐staining of Aβ and HCMV proteins. Aβ production was observed within 3 days and persisted until at least 9 days post‐infection. To assess the amyloidogenicity of HCMV, we employed the Waltz algorithm, which predicted two amyloidogenic sequences within immunodominant HCMV proteins pp65 and IE1 (average score: 98.3). Further, HCMV infection of HFAs mediated a 2‐ to 4‐ fold induction in CXCL10, CD44, and IRGM, reactivity‐associated genes also involved in immune and inflammatory pathways.ConclusionOur data demonstrate that HCMV infection can contribute to AD pathology in cultured astrocytes. These findings could ultimately inform the use of antiviral therapy to prevent and/or reduce AD severity.

Immune Response to Tick-Borne Hemoparasites: Host Adaptive Immune Response Mechanisms as Potential Targets for Therapies and Vaccines.

Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T- and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens.

Development of Human Vectored Brucellosis Vaccine Formulation: Assessment of Safety and Protectiveness of Influenza Viral Vectors Expressing Brucella Immunodominant Proteins in Mice and Guinea Pigs.

In this paper, we first used recombinant influenza viral vector (rIVV) subtype H5N1 expressing from the open reading frame of NS1 80 and NS1 124 amino acids of Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7/L12, and Cu-Zn superoxide dismutase (SOD) proteins to develop a human brucellosis vaccine. We made 18 combinations of IVVs in mono-, bi-, and tetravalent vaccine formulations and tested them on mice to select the safest and most effective vaccine samples. Then, the most effective vaccine candidates were further tested on guinea pigs. Safety of the rIVV-based vaccine candidate was evaluated by a mouse weight-gain test. Mice and guinea pigs were challenged with the virulent strain B. melitensis 16M. The protective effect of the rIVV-based vaccine candidate was assessed by quantitation of Brucella colonization in tissues and organs of challenged animals. All vaccine formulations were safe in mice. Tested vaccine formulations, as well as the commercial B. melitensis Rev.1 vaccine, have been found to protect mice from B. melitensis 16M infection within the range of 1.6 to 2.97 log10 units (P < 0.05). Tetravalent vaccine formulations from the position of NS1 80 amino acids (0.2 ± 0.4), as well as the commercial B. melitensis Rev.1 vaccine (1.2 ± 2.6), have been found to protect guinea pigs from B. melitensis 16M infection at a significant level (P < 0.05). Thus, tetravalent vaccine formulation Flu-NS1-80-Omp16+Flu-NS1-80-L7/L12+Flu-NS1-80-Omp19+Flu-NS1-80-SOD was chosen as a potential vaccine candidate for further development of an effective human vaccine against brucellosis. These results show a promising future for the development of a safe human vaccine against brucellosis based on rIVVs.

Multigene characterization of a ‘Candidatus Phytoplasma australasia’ strain associated with Roystonea regia in Oman

Florida or Cuban royal palm (Roystonea regia (Kunth) O.F. Cook) is an important ornamental plant with many uses for humans. Royal palm trees grown in Muscat, Oman, were found exhibiting leaf yellowing, leaf decay starting on the mature leaves, and leaf stunting. Trunk tissue samples were collected and analysed with nested and semi-nested PCR assays for the 16S rRNA, translation elongation factor Tu (tuf), and immunodominant membrane protein (imp) genes, in order to characterize the phytoplasma associated with the affected trees. Sequence and phylogenetic analysis of the 16S rRNA, tuf, and imp genes indicated that the phytoplasma strains detected in the trees were identical and belonged to the 16SrII-D subgroup. This is the first report of an association between ‘Candidatus Phytoplasma asutralasia’-related strain and R. regia.

Exoantigens of Paracoccidioides spp. Promote Proliferation and Modulation of Human and Mouse Pulmonary Fibroblasts.

Paracoccidioidomycosis (PCM) is a systemic granulomatous fungal infection caused by thermally dimorphic fungi of the genus Paracoccidioides. Endemic in Latin America, PCM presents with high incidence in Brazil, Colombia, and Venezuela, especially among rural workers. The main clinical types are acute/subacute (AF) form and chronic form (CF). Even after effective antifungal treatment, patients with CF usually present sequelae, such as pulmonary fibrosis. In general, pulmonary fibrosis is associated with dysregulation wound healing and abnormal fibroblast activation. Although fibrogenesis is recognized as an early process in PCM, its mechanisms remain unknown. In the current study, we addressed the role of Paracoccidioides spp. exoantigens in pulmonary fibroblast proliferation and responsiveness. Human pulmonary fibroblasts (MRC-5) and pulmonary fibroblasts isolated from BALB/c mice were cultivated with 2.5, 5, 10, 100, and 250 µg/ml of exoantigens produced from P. brasiliensis (Pb18 and Pb326) and P. lutzii (Pb01, Pb8334, and Pb66) isolates. Purified gp43, the immunodominant protein of P. brasiliensis exoantigens, was also evaluated at concentrations of 5 and 10 µg/ml. After 24 h, proliferation and production of cytokines and growth factors by pulmonary fibroblasts were evaluated. Each exoantigen concentration promoted a different level of interference of the pulmonary fibroblasts. In general, exoantigens induced significant proliferation of both murine and human pulmonary fibroblasts (p < 0.05). All concentrations of exoantigens promoted decreased levels of IL-6 (p < 0.05) and VEGF (p < 0.05) in murine fibroblasts. Interestingly, decreased levels of bFGF (p < 0.05) and increased levels of TGF-β1 (p < 0.05) and pro-collagen I (p < 0.05) were observed in human fibroblasts. The gp43 protein induced increased TGF-β1 production by human cells (p = 0.02). In conclusion, our findings showed for the first time that components of P. brasiliensis and P. lutzii interfered in fibrogenesis by directly acting on the biology of pulmonary fibroblasts.