Constituting a glutathione S-transferase-cocktail vaccine against tick infestation

Cocktail vaccines are proposed as an attractive way to increase protection efficacy against specific tick species. Furthermore, such vaccines made with different tick antigens have the potential of cross-protecting against a broad range of tick species. However, there are still limitations to the selection of immunogen candidates. Acknowledging that glutathione S-transferases (GSTs) have been exploited as vaccines against ticks and other parasites, this study aimed to analyze a GST-cocktail vaccine as a potential broad-spectrum tick vaccine. To constitute the GST-cocktail vaccine, five tick species of economic importance for livestock industry were studied (Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus microplus, Amblyomma variegatum, and Haemaphysalis longicornis). Tick GST ORF sequences were cloned, and the recombinant GSTs were produced in Escherichia coli. rGSTs were purified and inoculated into rabbits, and the immunological response was characterized. The humoral response against rGST-Rd and rGST-Av showed a stronger cross-reactivity against heterologous rGSTs compared to rGST-Hl, rGST-Ra, and rGST-Rm. Therefore, rGST-Rd and rGST-Av were selected for constituting an experimental rGST-cocktail vaccine. Vaccination experiment in rabbits showed that rGST-cocktail caused 35% reduction in female numbers in a Rhipicephalus sanguineus infestation. This study brings forward an approach to selecting immunogens for cocktail vaccines, and the results highlight rGST–Rd and rGST-Av as potentially useful tools for the development of a broad-spectrum tick vaccine.     

Novel tick glutathione transferase inhibitors as promising acaricidal compounds

Ticks are important ectoparasites with a worldwide distribution. The most commonly used method for tick control involves the use of acaricides. The main problem is that its indiscriminate use has led to the selection of resistant tick populations. Glutathione transferases (GSTs) are enzymes that play an important role in the detoxification of several types of compounds used in commercial tick control products. This work aims to find new bioactive molecules through in vitro assays with a panel of 160 molecules with putative inhibitory activity on the Rhipicephalus microplus GST enzyme (RmGST). Also, selected molecules were tested against GSTs from other tick species; Rhipicephalus decoloratus, Amblyomma variegatum, Rhipicephalus appendiculatus, and Haemaphysalis longicornis. The first screening on RmGST identified 30 compounds with the ability to modify the enzymatic activity of this enzyme. These compounds included different chemical families, like chalcones, diarylideneketones, flavone, thiazoles, thiourea, steroids, thiadiazines, indazoles, and hydrazine. The most potent compounds against RmGST belong to the diarylideneketones family with an inhibition concentration of 50% of activity (IC₅₀) between 7-50 μM. Interestingly, one of the most potent compounds was also an inhibitor of the GST from other tick species. Experiments with R. microplus adults and larvae showed toxicity at 150 μM, suggesting a potential acaricidal effect of these molecules.     

Mapping protective epitopes in the tick and mosquito subolesin ortholog proteins

The tick protective antigen, subolesin, is a structural and functional ortholog of insect akirins, an evolutionary conserved group of proteins that regulate gene expression thus affecting multiple cellular processes such as digestion, immune response, reproduction and development. In this study, we searched for common protective epitopes in tick and mosquito subolesin ortolog proteins. By combining the results of peptide and phage-display libraries scan analysis with sera from protected animals with computational modeling, three different epitope types (i) linear B-cell epitopes, (ii) conformational epitopes, and (iii) conformational discontinuous epitopes were identified. The determination of conserved protective epitopes in subolesin ortologs may lead to the development of a multi-target universal vaccine directed at the control of both arthropod infestations and reduction of vector capacity to transmit pathogens.     

Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus

Vaccines based on recombinant Bm86 gut antigen from Boophilus microplus are a useful component of integrated control strategies against B. microplus infestations of cattle. The capacity of such vaccines to control heterologous infestations by two African tick species was investigated. The mean weight of engorged female ticks and mean egg mass per tick were significantly reduced in B. decoloratus infestations, but there was no effect of the vaccine against adult Rhipicephalus appendiculatus. We cloned, sequenced and expressed two Bm86 homologues (Bd86) from B. decoloratus. Amino acid sequence identity between Bd86 homologues (Bd86-1 and Bd86-2) and Bm86 was 86% and 85%, respectively, compared to 93% identity between the variants. Native Bd86 protein in B. decoloratus tick mid-gut sections and recombinant Bd86-1 reacted strongly with sera from TickGARD vaccinated cattle. TickGARD can therefore protect against a heterologous tick species with multiple antigen sequences. Epitope mapping using sera from TickGARD-vaccinated cattle identified two linear peptides conserved between the Bd86 homologues and Bm86. These epitopes represent candidate synthetic peptide vaccines for control of Boophilus spp. and the pathogens transmitted by these tick vectors.     

Immunization with phage virus-like particles displaying Zika virus potential B-cell epitopes neutralizes Zika virus infection of monkey kidney cells

Zika virus (ZIKV) is a mosquito-borne flavivirus that has re-emerged and is associated with many debilitating clinical manifestations. Research is currently being conducted to develop a prophylactic vaccine against the virus; however, there has not been any licensed ZIKV vaccine. Recent studies have identified potential B-cell epitopes (amino acids 241–259, 294–315, 317–327, 346–361, 377–388 and 421–437) on the envelope protein of ZIKV, which could be explored to develop peptide vaccines against ZIKV infection. Nevertheless, the immunogenicity of these epitopes has never been assessed. Here, we displayed these epitopes on highly immunogenic bacteriophage virus-like particles (VLPs; MS2, PP7 and Qβ) platforms and assessed their immunogenicity in mice. Mice immunized with a mixture of VLPs displaying ZIKV envelope B-cell epitopes elicited anti-ZIKV antibodies. Although, immunized mice were not protected against a high challenge dose of ZIKV, sera – albeit at low titers – from immunized mice neutralized (in vitro) a low dose of ZIKV. Taken together, these results show that these epitopes are B-cell epitopes and they are immunogenic when displayed on a Qβ VLP platform. Furthermore, the results also show that immunization with VLPs displaying a single B-cell epitope minimally reduces ZIKV infection whereas immunization with a mixture of VLPs displaying a combination of the B-cell epitopes neutralizes ZIKV infection. Thus, immunization with a mixture of VLPs displaying multiple ZIKV B-cell epitopes is a good strategy to enhance ZIKV neutralization.     

Immunodominant T cell peptides from four candidate malarial antigens as biomarkers of protective immunity against malaria

A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.     

The generation of a simian adenoviral vectored HCV vaccine encoding genetically conserved gene segments to target multiple HCV genotypes

BackgroundHepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity.MethodsWe developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000–1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1–6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-ϒ ELISpot and intracellular cytokine assays.ResultsChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/10⁶ splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/10⁶ against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans.ConclusionsSimian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.     

Identification and functional analysis of ferritin 2 from the Taiga tick Ixodes persulcatus Schulze

Ferritin 2 (FER2) is an iron storage protein, which has been shown to be critical for iron homeostasis during blood feeding and reproduction in ticks and is therefore suitable as a component for anti-tick vaccines. In this study, we identified the FER2 of Ixodes persulcatus, a major vector for zoonotic diseases such as Lyme borreliosis and tick-borne relapsing fever in Japan, and investigated its functions. Ixodes persulcatus-derived ferritin 2 (Ip-FER2) showed concentration-dependent iron-binding ability and high amino acid conservation, consistent with FER2s of other tick species. Vaccines containing the recombinant Ip-FER2 elicited a significant reduction of the engorgement weight of adult I. persulcatus. Interestingly, the reduction of engorgement weight was also observed in Ixodes ovatus, a sympatric species of I. persulcatus. In silico analyses of FER2 sequences of I. persulcatus and other ticks showed a greater similarity with I. scapularis and I. ricinus and lesser similarity with Hyalomma anatolicum, Haemaphysalis longicornis, Rhipicephalus microplus, and R. appendiculatus. Moreover, it was observed that the tick FER2 sequences possess conserved regions within the primary structures, and in silico epitope mapping analysis revealed that antigenic regions were also conserved, particularly among Ixodes spp ticks. In conclusion, the data support further protective tick vaccination applications using the Ip-FER2 antigens identified herein.     

The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats

Summary Background Glutathione S-transferases (GSTs)* are an important class of phase II, predominantly detoxifying, enzymes. The supergene family is composed of several isoenzymes, hetero- and homodimers, with tissue specific distribution and levels of expression. The hypothesis is that a higher expression of individual proteins within a specific tissue may be associated with a decreased burden of exposure to reactive carcinogens and ultimately with a decreased cancer risk in this tissue. Aims of the study Since nutrition is expected to contribute to the gene expression, it was the aim of this study to investigate the impact of dietary factors, especially resistant starch, and of the gut microflora, which may be influenced by diet, on the GSTs in colon cells of rats. Methods For this, a technique using high pressure liquid chromatography was established with which for the first time GST isoenzymes were analysed in colon cells and compared to the levels of the corresponding proteins in the liver of the same rat. Results It was found that colon cells contain mainly GST π and low amounts of μ but not GST α. In contrast, the predominant form of GSTs in the liver was α, then μ and hardly any π. Altogether, liver cells had approximately tenfold more total GSTs than colon cells. The feeding of “Crystalean”, a retrograded, high amylose starch which alters the fermentation profile and the composition of the microflora, led to higher levels of GST π in the colon. Furthermore, the comparison of GSTs in colon cells of germ-free rats revealed they were much lower than those observed in rats with conventional microflora. Conclusions These findings clearly demonstrate that the gut bacteria, or their metabolic products, enhance GST expression. The studies support the hypothesis that nutrition – by affecting the gut flora – may induce this potentially protective and important class of phase II enzymes in important tumor target cells.     

Identification of linear B epitopes of pertactin of Bordetella pertussis induced by immunization with whole and acellular vaccine

Pertussis is a serious infectious disease of the respiratory tract caused by the gram-negative bacteria Bordetella pertussis. There has been a reemergence of this disease within the population of several countries that have well established vaccination programs. Analyzes of clinical isolates suggest an antigenic divergence between the vaccine-based strains to the circulating strains. Although antibodies against P.69 are involved in the observed protective immunity, the sequences recognized as antigenic determinants in P.133, the precursor for P.69, P.3.4 and P.30, have not be determined. Here, the precise mapping of linear B-cell epitopes within the predicted P.133 pertactin sequences was accomplished using the SPOT-synthesis of peptide arrays onto cellulose membranes and screening with murine sera generated by vaccination with either the Pertussis cellular (miPc) or Pertussis acellular (miPa) vaccine. A total of 23 major epitopes were identified by sera from miPc vaccinated mice, while thirteen were identified by sera from miPa vaccinated mice. Of these epitopes, 12 epitopes were specifically identified by antibodies produced in response to the miPc vaccine and two were specific to the miPa vaccine. These epitopes were distributed throughout the pertactin sequence but a significant number were concentrated to the P.30 Prn segment. An analysis of the epitope correlation homologies indicated that the variations from the observed mutations in pertactin would not constitute a problem using these vaccines. In addition, the mapping of epitopes demonstrated a higher number of linear B-cell epitopes immunized with the Pc vaccine than the Pa vaccine.     

EB病毒LMP-1、BALF4及BARF1片段的B细胞表位预测

目的:预测EB病毒LMP-1、BALF4及BARF1片段的B细胞表位。方法:采用Protean软件分析EBV的LMP1、BALF4及BARF1三个氨基酸片段的亲水性、表面可能性、抗原指数及其二级结构中的柔性区域,并结合吴玉章的抗原指数预测法预测其B细胞表位。结果:B细胞表位可能位于LMP-1 N端第356-358,2-19,249-314区段,BARF1N端第8-11,18-25,41-49,203-211区段,BALF4N端第385-387,833-845,398-415,427-435,453-458,23-32,466-473,234-250,642-652区段;另外LMP-1第185-223、BARF1第265-272,132-135以及BALF4第827-832区段内或附近也可能存在B细胞表位。结论:用多参数同时预测LMP-1、BALF4及BARF1的B细胞表位,为制备高效的鼻咽癌血清学诊断试剂和表位疫苗奠定了理论基础。     

Designing the epitope flanking regions for optimal generation of CTL epitopes

The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.     

Overlapping CD8 + and CD4 + T-cell epitopes identification for the progression of epitope-based peptide vaccine from nucleocapsid and glycoprotein of emerging Rift Valley fever virus using immunoinformatics approach

Rift Valley fever virus (RVFV) is an emergent arthropod-borne zoonotic infectious viral pathogen which causes fatal diseases in the humans and ruminants. Currently, no effective and licensed vaccine is available for the prevention of RVFV infection in endemic as well as in non-endemic regions. So, an immunoinformatics-driven genome-wide screening approach was performed for the identification of overlapping CD8 + and CD4 + T-cell epitopes and also linear B-cell epitopes from the conserved sequences of the nucleocapsid (N) and glycoprotein (G) of RVFV. We identified overlapping 99.39% conserved 1 CD8 + T-cell epitope (MMHPSFAGM) from N protein and 100% conserved 7 epitopes (AVFALAPVV, LAVFALAPV, FALAPVVFA, VFALAPVVF, IAMTVLPAL, FFDWFSGLM, and FLLIYLGRT) from G protein and also identified IL-4 and IFN-γ induced (99.39% conserved) 1 N protein CD4 + T-cell epitope (HMMHPSFAGMVDPSL) and 100% conserved 5 G protein CD4 + T-cell epitopes (LPALAVFALAPVVFA, PALAVFALAPVVFAE, GIAMTVLPALAVFAL, GSWNFFDWFSGLMSW, and FFLLIYLGRTGLSKM). The overlapping CD8 + and CD4 + T-cell epitopes were bound with most conserved HLA-C*12:03 and HLA-DRB1*01:01, respectively with the high binding affinity (kcal/mol). The combined population coverage analysis revealed that the allele frequencies of these epitopes are high in endemic and non-endemic regions. Besides, we found 100% conserved and non-allergenic 2 decamer B-cell epitopes, GVCEVGVQAL and RVFNCIDWVH of G protein had the sequence similarity with the nonamer CD8 + T-cell epitopes, VCEVGVQAL and RVFNCIDWV, respectively. Consequently, these epitopes may be used for the development of epitope-based peptide vaccine against emerging RVFV. However, in vivo and in vitro experiments are required for their efficient use as a vaccine.     

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus microplus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host–parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZαA vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l^?1. qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host–parasite interaction to overcome the immune responses in resistant cattle.     

Control of tick infestations in wild roe deer (Capreolus capreolus) vaccinated with the Q38 Subolesin/Akirin chimera

Ticks (Acari: Ixodidae) are considered to be the most important vectors of disease-causing pathogens in domestic and wild animals, and emerging and re-emerging tick-borne diseases (TBD) exert an enormous impact on them. Wild ungulates are hosts for a wide variety of tick species and tick-borne pathogens that affect human and animal health. Consequently, the control of tick infestations and tick-borne pathogen prevalence is essential in some regions. Acaricides and animal management or culling have been used for the control of tick infestations and TBD, but tick vaccines constitute the best alternative to reduce the impact of acaricides on tick resistance and the environment. Previous results of controlled vaccination trials have shown that the Q38 Subolesin/Akirin chimera containing conserved protective epitopes could be a candidate universal antigen to control multiple tick species infestations. Thus, vaccination trials are necessary to validate these results under field conditions. In this study, we characterized the effect of Q38 vaccine on a wild population of European roe deer (Capreolus capreolus) in the Andalusian roe deer Reference Station (Junta de Andalucía, Cádiz, Spain). In this location, roe deer suffer especially severe parasitic conditions in some periods and commercial pesticides and ixodicides that are authorized to control ticks without specificity are frequently applied in the field, posing a threat to the environment. Animals vaccinated over a three-year period showed an antibody response to the vaccine antigen and a reduction in tick infestations by multiple species including Hyalomma marginatum, H. lusitanicum, Rhipicephalus bursa and Ixodes ricinus previously identified in roe deer, when compared to untreated controls. These results suggest the efficacy of Q38 for the control of tick infestations in wildlife.     

Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control

Rhipicephalus microplusis the most important ectoparasite of livestock in tropical and subtropical areas around the world. Research focused on developing an efficient vaccine for cattle tick control is a high priority. The aim of this study was to evaluate the rSBm7462® peptide (Bm86-B and T cell epitopes) regarding its properties of immunogenicity, protective effect in cattle and efficacy against R. microplus. This peptide was produced by a fermentative process in the yeast culture system of Komagataella (Pichia) pastoris strain Km 71. The vaccination assay was conducted in a tick-free area using non-splenectomised Holstein Friesian calves, separated into immunisation and control groups. These animals individually received the recombinant peptide and the inoculum without peptide using saponin as an adjuvant at three time points. The calves were challenged 21 days after the last immunisation using 4500 larvae per animal. An indirect ELISA was used to identify the IgG kinetics of serum samples from the calves studied. The qPCR was performed to determine the cytokine gene expression from the total RNA of the cultured PBMCs. Histomorphometry of the germinal centres (GCs) was performed measuring slides with haematoxylin-eosin staining of surgically removed lymph nodes from immunised calves. The antibody response showed a significant induction of high-affinity IgGs in calves immunised with the recombinant peptide in comparison to the controls. The kinetics of antibodies in immunised calves showed a significant increase during the experiment. This increase in high-affinity IgGs correlated with a gradual increase of the GC diameter following each peptide vaccination. Cytokine expression profiles demonstrating an adaptive immune response in calves immunised with rSBm7462® confirmed the T-dependent response. Vaccine efficacy was calculated at 72.4 % following the analysis and fecundity of collected adult female ticks, compared between control and vaccinated groups. These findings demonstrate that this new recombinant peptide is an option for control of R. microplus infestations.     

Immunogenicity and protective efficacy of a multi-epitope recombinant toxin antigen of Pasteurella multocida against virulent challenge in mice

Pasteurella multocida (P. multocida) infection frequently results in porcine atrophic rhinitis and swine plague, leading to large economic losses for the swine industry worldwide. P. multocida toxin (PMT, 146 kDa) is a highly virulent key virulence factor that plays a vital role in causing lung and turbinate lesions. This study developed a multi-epitope recombinant antigen of PMT (rPMT) that showed excellent immunogenicity and protection in a mouse model. Using bioinformatics to analyse the dominant epitopes of PMT, we constructed and synthesized rPMT containing 10 B-cell epitopes, 8 peptides with multiple B-cell epitopes and 13 T-cell epitopes of PMT and a rpmt gene (1,974 bp) with multiple epitopes. The rPMT protein (97 kDa) was soluble and contained a GST tag protein. Immunization of mice with rPMT stimulated significantly elevated serum IgG titres and splenocyte proliferation, and serum IFN-γ and IL-12 were upregulated by 5-fold and 1.6-fold, respectively, but IL-4 was not. Furthermore, the rPMT immunization group exhibited alleviated lung tissue lesions and a significantly decreased degree of neutrophil infiltration compared with the control groups post-challenge. In the rPMT vaccination group, 57.1% (8/14) of the mice survived the challenge, similar to the bacterin HN06 group, while all the mice in the control groups succumbed to the challenge. Thus, rPMT could be a suitable candidate antigen for developing a subunit vaccine against toxigenic P. multocida infection.     

An ingenious design for peptide vaccines

For humoral immunization, it may be possible to make effective and safe peptide vaccines for various diseases by selection of proper B-cell epitopes. However, a lack of T-cell epitopes on short peptides, such as those associated with major histocompatibility complex (MHC)-restriction, is a major problem for peptide vaccine development. We propose a solution for the design of peptide vaccines that involves induction of broadly reactive T-cell epitopes via agretopes. The strategy involves positioning multi-agretope type peptides on the N-terminal side of a di-lysine linker and B-cell epitopes on the C-terminal side. The addition of the arginine-glysine-aspartate (RGD)-motif to the N terminus of the peptide enhances its immunogenicity, and enables nasal immunization without adjuvants.     

Insights into the biochemical features and immunogenic epitopes of common bradyzoite markers of the ubiquitous Toxoplasma gondii

The widespread distribution of Toxoplasma gondii (T. gondii) infection and its harsh outcomes in pregnant women and immunocompromised patients lead researchers towards vaccination strategies. The present in silico investigation was done to reveal biophysical properties and immunogenic epitopes of six bradyzoite markers for rational vaccine design in future. For this purpose, different web servers were used to predict antigenicity, allergenicity, solubility, physicochemical properties, post-translational modification sites (PTMs), the presence of signal peptide and transmembrane domains. Moreover, the secondary and tertiary structures of the proteins were revealed followed by refinement and validation. Finally, NetCTL server was used to predict cytotoxic T-lymphocyte (CTL) epitopes, with subsequent immunogenicity analysis. Also, IEDB server was utilized to predict helper T-lymphocyte (HTL) epitopes, followed by IFN-γ and IL-4 induction, antigenicity and population coverage analysis. As well, several linear antigenic B-cell epitopes were found, with good water solubility and without allergenicity. Totally, these proteins showed appropriate antigenicity, abundant PTMs as well as many CTL, HTL and B-cell epitopes, which could be directed for future vaccination studies in the context of multi-epitope vaccine design.     

A V3 loop haptenic peptide sequence, when tandemly repeated, enhances immunogenicity by facilitating helper T-cell responses to a covalently linked carrier protein.

Subunit immunogens containing tandemly repeated copies of T- and B-cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. It has been unclear, however, whether the increased immunogenicity of a tandemly repeated B-cell epitope necessarily results from increased helper T-cell responses to intrinsic T-cell epitopes in the tandemly repeated sequences, or to neodeterminant T-cell epitopes created at the junction of adjacent repeat sequences. We examined this question by comparing the immunogenicity in mice of two immunogens containing one or eight tandemly repeated copies of an HIV-1 V3 loop haptenic sequence. Our results show that the tandemly repeated haptenic sequence potentiates the immunogenicity of the protein construct, likely through the facilitation of enhanced B-cell interaction with the tandem repeat construct and the consequent elicitation of increased carrier protein-specific helper T-cell responses.