IFITM3-specific antibody reveals IFN preferences and slow IFN induction of the antiviral factor IFITM3 in humans.

Using a specific antibody, we found that expression of the viral restriction factor IFITM3 differs across cell types within the immune compartment with higher expression in myeloid rather than lymphoid cells. IFITM3 expression was increased following IFN stimulation, mostly type I, in immune cells, with the exception of T cells.

Identification of genetic loci associated with higher resistance to pancreas disease (PD) in Atlantic salmon (Salmo salar L.).

BACKGROUND: Pancreas disease (PD) is a contagious disease caused by salmonid alphavirus (SAV) with significant economic and welfare impacts on salmon farming. Previous work has shown that higher resistance against PD has underlying additive genetic components and can potentially be improved through selective breeding. To better understand the genetic basis of PD resistance in Atlantic salmon, we challenged 4506 smolts from 296 families of the SalmoBreed strain. Fish were challenged through intraperitoneal injection with the most virulent form of the virus found in Norway (i.e., SAV3). Mortalities were recorded, and more than 900 fish were further genotyped on a 55 K SNP array. RESULTS: The estimated heritability for PD resistance was 0.41 ± 0.017. The genetic markers on two chromosomes, ssa03 and ssa07, showed significant associations with higher disease resistance. Collectively, markers on these two QTL regions explained about 60% of the additive genetic variance. We also sequenced and compared the cardiac transcriptomics of moribund fish and animals that survived the challenge with a focus on candidate genes within the chromosomal segments harbouring QTL. Approximately 200 genes, within the QTL regions, were found to be differentially expressed. Of particular interest, we identified various components of immunoglobulin-heavy-chain locus B (IGH-B) on ssa03 and immunoglobulin-light-chain on ssa07 with markedly higher levels of transcription in the resistant animals. These genes are closely linked to the most strongly QTL associated SNPs, making them likely candidates for further investigation. CONCLUSIONS: The findings presented here provide supporting evidence that breeding is an efficient tool for increasing PD resistance in Atlantic salmon populations. The estimated heritability is one of the largest reported for any disease resistance in this species, where the majority of the genetic variation is explained by two major QTL. The transcriptomic analysis has revealed the activation of essential components of the innate and the adaptive immune responses following infection with SAV3. Furthermore, the complementation of the genomic with the transcriptomic data has highlighted the possible critical role of the immunoglobulin loci in combating PD virus.

Lack of Truncated IFITM3 Transcripts in Cells Homozygous for the rs12252-C Variant That is Associated With Severe Influenza Infection.

Interferon-induced transmembrane 3 (IFITM3) is known to restrict the entry of a range of enveloped viruses. The single nucleotide polymorphism rs12252-C within IFITM3 has been shown to be associated with severe influenza A virus infection. It has been suggested that rs12252-C results in expression of a truncated IFITM3 protein lacking the first 21 amino acids. By performing high-throughput RNA sequencing on primary dendritic cells and peripheral blood mononuclear cells isolated from pandemic H1N1 influenza and human immunodeficiency virus-1 (HIV-1) infected patients we show that full-length IFITM3 mRNA is dominantly expressed (>99%) across all rs12252 genotypes. Full-length IFITM3 protein can be detected in all genotypes.

Gene expression profile of HCT-8 cells following single or co-infections with Cryptosporidium parvum and bovine coronavirus.

Among the causative agents of neonatal diarrhoea in calves, two of the most prevalent are bovine coronavirus (BCoV) and the intracellular parasite Cryptosporidium parvum. Although several studies indicate that co-infections are associated with greater symptom severity, the host-pathogen interplay remains unresolved. Here, our main objective was to investigate the modulation of the transcriptome of HCT-8 cells during single and co-infections with BCoV and C. parvum. For this, HCT-8 cells were inoculated with (1) BCoV alone, (2) C. parvum alone, (3) BCoV and C. parvum simultaneously. After 24 and 72 h, cells were harvested and analyzed using high-throughput RNA sequencing. Following differential expression analysis, over 6000 differentially expressed genes (DEGs) were identified in virus-infected and co-exposed cells at 72 hpi, whereas only 52 DEGs were found in C. parvum-infected cells at the same time point. Pathway (KEGG) and gene ontology (GO) analysis showed that DEGs in the virus-infected and co-exposed cells were mostly associated with immune pathways (such as NF-κB, TNF-α or, IL-17), apoptosis and regulation of transcription, with a more limited effect exerted by C. parvum. Although the modulation observed in the co-infection was apparently dominated by the virus, over 800 DEGs were uniquely expressed in co-exposed cells at 72 hpi. Our findings provide insights on possible biomarkers associated with co-infection, which could be further explored using in vivo models.

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep.

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host's gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3-8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8-14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Abrupt rise in atmospheric CO2 overestimates community response in a model plant-soil system.

Attempts to understand the ecological effect of increasing atmospheric CO2 concentration, [CO2], usually involve exposing today's ecosystems to expected future [CO2] levels. However, a major assumption of these approaches has not been tested--that exposing ecosystems to a single-step increase in [CO2] will yield similar responses to those of a gradual increase over several decades. We tested this assumption on a mycorrhizal fungal community over a period of six years. [CO2] was either increased abruptly, as is typical of most [CO2] experiments, or more gradually over 21 generations. The two approaches resulted in different structural and functional community responses to increased [CO2]. Some fungi were sensitive to the carbon pulse of the abrupt [CO2] treatment. This resulted in an immediate decline in fungal species richness and a significant change in mycorrhizal functioning. The magnitude of changes in fungal diversity and functioning in response to gradually increasing [CO2] was smaller, and not significantly different to those with ambient [CO2]. Our results suggest that studies may overestimate some community responses to increasing [CO2] because biota may be sensitive to ecosystem changes that occur as a result of abrupt increases.

Clinical and immunological responses in sheep after inoculation with Himar1-transformed Anaplasma phagocytophilum and subsequent challenge with a virulent strain of the bacterium.

In Norway, the tick-transmitted bacterium Anaplasma phagocytophilum is estimated to cause tick-borne fever (TBF) in 300 000 lambs on pastures each year, resulting in economic and animal welfare consequences. Today, prophylactic measures mainly involve the use of acaricides, but a vaccine has been requested by farmers and veterinarians for decades. Several attempts have been made to produce a vaccine against A. phagocytophilum including antigenic surface proteins, inactivated whole cell vaccines and challenge followed by treatment. In the current study, a virulent wild type strain of A. phagocytophilum named Ap.Norvar1 (16S rRNA sequence partial identical to sequence in GenBank acc.no M73220) was subject to genetic transformation with a Himar1-transposon, which resulted in three bacterial mutants, capable of propagation in a tick cell line (ISE6). In order to test the immunogenicity and pathogenicity of the live, mutated bacteria, these were clinically tested in an inoculation- and challenge study in sheep. One group was inoculated with the Ap.Norvar1 as an infection control. After inoculation, the sheep inoculated with mutated bacteria and the Ap.Norvar1 developed typical clinical signs of infection and humoral immune response. After challenge with Ap.Norvar1, 28 days later all groups inoculated with mutated bacteria showed clinical signs of tick-borne fever and bacteremia while the group initially inoculated with the Ap.Norvar1, showed protection against clinical disease. The current study shows a weak, but partial protection against infection in animals inoculated with mutated bacteria, while animals that received Ap.Norvar1 both for inoculation and challenge, responded with homologues protection.

Specific human cytomegalovirus signature detected in NK cell metabolic changes post vaccination.

Effective vaccines for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) remain a significant challenge for these infectious diseases. Given that the innate immune response is key to controlling the scale and nature of developing adaptive immune responses, targeting natural killer (NK) cells that can promote a T-helper type 1 (Th1)-type immune response through the production of interferon-γ (IFNγ) remains an untapped strategic target for improved vaccination approaches. Here, we investigate metabolic and functional responses of NK cells to simian adenovirus prime and MVA boost vaccination in a cohort of healthy volunteers receiving a dual HCV-HIV-1 vaccine. Early and late timepoints demonstrated metabolic changes that contributed to the sustained proliferation of all NK cells. However, a strong impact of human cytomegalovirus (HCMV) on some metabolic and functional responses in NK cells was observed in HCMV seropositive participants. These changes were not restricted to molecularly defined adaptive NK cells; indeed, canonical NK cells that produced most IFNγ in response to vaccination were equally impacted in individuals with latent HCMV. In summary, NK cells undergo metabolic changes in response to vaccination, and understanding these in the context of HCMV is an important step towards rational vaccine design against a range of human viral pathogens.

Genome-Wide Association Study Confirms Previous Findings of Major Loci Affecting Resistance to Piscine myocarditis virus in Atlantic Salmon (Salmo salar L.).

Cardiomyopathy syndrome is a viral disease of Atlantic salmon, mostly affecting fish during the late stages of production, resulting in significant losses to the industry. It has been shown that resistance to this disease has a strong genetic component, with quantitative trait loci (QTL) on chromosomes 27 (Ssa27) and Ssa12 to explain most of the additive genetic variance. Here, by analysing animals from a different year-class and a different population, we further aimed to confirm and narrow down the locations of these QTL. The data support the existence of the two QTL and suggest that the causative mutation on Ssa27 is most likely within the 10-10.5 Mbp segment of this chromosome. This region contains a cluster of major histocompatibility complex class I (MHC I) genes with the most strongly associated marker mapped to one of these loci. On Ssa12, the data confirmed the previous finding that the location of the causative mutation is within the 61.3 to 61.7 Mbp region. This segment contains several immune-related genes, but of particular interest are genes related to MHC II. Together, these findings highlight the likely key role of MHC genes in Atlantic salmon following infection with Piscine myocarditis virus (PMCV) and their potential impact on influencing the trajectory of this disease.

Genomic and Transcriptomic Analysis of Amoebic Gill Disease Resistance in Atlantic Salmon (Salmo salar L.).

Amoebic gill disease (AGD) is one of the most important parasitic diseases of farmed Atlantic salmon. It is a source of major economic loss to the industry and poses significant threats to animal welfare. Previous studies have shown that resistance against this disease has a moderate, heritable genetic component, although the genes and the genetic pathways that contribute to this process have yet to be elucidated. In this study, to identify the genetic mechanisms of AGD resistance, we first investigated the molecular signatures of AGD infection in Atlantic salmon through a challenge model, where we compared the transcriptome profiles of the naïve and infected animals. We then conducted a genome-wide association analysis with 1,333 challenged tested fish to map the AGD resistance genomic regions, supported by the results from the transcriptomic data. Further, we investigated the potential of incorporating gene expression analysis results in genomic prediction to improve prediction accuracy. Our data suggest thousands of genes have modified their expression following infection, with a significant increase in the transcription of genes with functional properties in cell adhesion and a sharp decline in the abundance of various components of the immune system genes. From the genome-wide association analysis, QTL regions on chromosomes ssa04, ssa09, and ssa13 were detected to be linked with AGD resistance. In particular, we found that QTL region on ssa04 harbors members of the cadherin gene family. These genes play a critical role in target recognition and cell adhesion. The QTL region on ssa09 also is associated with another member of the cadherin gene family, protocadherin Fat 4. The associated genetic markers on ssa13 span a large genomic region that includes interleukin-18-binding protein, a gene with function essential in inhibiting the proinflammatory effect of cytokine IL18. Incorporating gene expression information through a weighted genomic relationship matrix approach decreased genomic prediction accuracy and increased bias of prediction. Together, these findings help to improve our breeding programs and animal welfare against AGD and advance our knowledge of the genetic basis of host-pathogen interactions.

Assessing the clinical and bacteriological outcomes of vaccination with recombinant Asp14 and OmpA against A. phagocytophilum in sheep.

Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4+, CD8+, γδ TcR+, λ-Light chain+, CD11b+, CD14+ and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.

A Novel Vaccine Strategy Employing Serologically Different Chimpanzee Adenoviral Vectors for the Prevention of HIV-1 and HCV Coinfection.

Background: Nearly 3 million people worldwide are coinfected with HIV and HCV. Affordable strategies for prevention are needed. We developed a novel vaccination regimen involving replication-defective and serologically distinct chimpanzee adenovirus (ChAd3, ChAd63) vector priming followed by modified vaccinia Ankara (MVA) boosts, for simultaneous delivery of HCV non-structural (NSmut) and HIV-1 conserved (HIVconsv) region immunogens. Methods: We conducted a phase I trial in which 33 healthy volunteers were sequentially enrolled and vaccinated via the intramuscular route as follows: 9 received ChAd3-NSmut [2.5 × 1010 vp] and MVA-NSmut [2 × 108 pfu] at weeks 0 and 8, respectively; 8 received ChAdV63.HIVconsv [5 × 1010 vp] and MVA.HIVconsv [2 × 108 pfu] at the same interval; 16 were co-primed with ChAd3-NSmut [2.5 × 1010 vp] and ChAdV63.HIVconsv [5 × 1010 vp] followed at week 8 by MVA-NSmut and MVA.HIVconsv [both 1 × 108 pfu]. Immunogenicity was assessed using peptide pools in ex vivo ELISpot and intracellular cytokine assays. Vaccine-induced whole blood transcriptome changes were assessed by microarray analysis. Results: All vaccines were well tolerated and no vaccine-related serious adverse events occurred. Co-administration of the prime-boost vaccine regimens induced high magnitude and broad T cell responses that were similar to those observed following immunization with either regimen alone. Median (interquartile range, IQR) peak responses to NSmut were 3,480 (2,728-4,464) and 3,405 (2,307-7,804) spot-forming cells (SFC)/106 PBMC for single and combined HCV vaccinations, respectively (p = 0.8). Median (IQR) peak responses to HIVconsv were 1,305 (1,095-4,967) and 1,005 (169-2,482) SFC/106 PBMC for single and combined HIV-1 vaccinations, respectively (p = 0.5). Responses were maintained above baseline to 34 weeks post-vaccination. Intracellular cytokine analysis indicated that the responding populations comprised polyfunctional CD4+ and CD8+ T cells. Canonical pathway analysis showed that in the single and combined vaccination groups, pathways associated with antiviral and innate immune responses were enriched for upregulated interferon-stimulated genes 24 h after priming and boosting vaccinations. Conclusions: Serologically distinct adenoviral vectors encoding HCV and HIV-1 immunogens can be safely co-administered without reducing the immunogenicity of either vaccine. This provides a novel strategy for targeting these viruses simultaneously and for other pathogens that affect the same populations. Clinical trial registration: https://clinicaltrials.gov, identifier: NCT02362217.

How does the humoral response to HIV-2 infection differ from HIV-1 and can this explain the distinct natural history of infection with these two human retroviruses?

A substantial proportion of people infected with HIV-2, the second causative agent of acquired immune deficiency syndrome (AIDS), behave as long-term non-progressors (LTNP) and are able to control the infection more effectively than most HIV-1-infected patients. A better understanding of the differences in the natural history of HIV-1 and HIV-2 infection, and how these relate to the relative immunogenicity and evolution of the two virus strains, could provide important insights into the mechanisms of protective immunity in HIV infection. One of the most striking differences is that most people infected with HIV-2 generate high titers of broadly neutralizing antibodies, whereas this is relatively uncommon in HIV-1 infection. In this review we compare the underlying structural differences of the envelope (Env) between HIV-1 and HIV-2, and examine how these might affect the antibody responses as well as their impact on Env evolution and control of viral replication.

Interferon-induced transmembrane protein-3 rs12252-C is associated with rapid progression of acute HIV-1 infection in Chinese MSM cohort.

BACKGROUND: The interferon-inducible transmembrane protein-3 (IFITM3) is a protein that restricts multiple pathogenic viruses such as influenza virus. The single-nucleotide polymorphism rs12252-C, which is rare in Caucasian populations, but much more common in the Han Chinese population, has been found in much higher homozygous frequency in patients with severe acute influenza. Until now, there has been no study on the effect of this genetic variant on the clinical control of other viral infections. OBJECTIVES: To investigate the impact of IFITM3-rs12252 genotypes on primary HIV-1 infection progression in an acute HIV-1-infected cohort in Beijing (PRIMO), China. DESIGN AND METHODS: We identified IFITM3-rs12252 genotypes of 178 acute HIV-1-infected patients and 196 HIV-negative candidates from the PRIMO cohort. HIV-1 viral load and CD4(+) T-cell counts were monitored at multiple time points during the first year of infection, and the association between IFITM3-rs12252 genotype and disease progression was evaluated. RESULTS: The current study shows that the IFITM3-rs12252 genetic variant affects the progression of HIV-1 infection, but not the acquisition. A significantly higher frequency of the CC/CT genotypes was found in rapid progressors compared to nonprogressors. Patients with CC/CT genotypes showed an elevated peak viremia level and significantly lower CD4(+) T-cell count at multiple time points during the first year of primary infection, and a significantly higher risk of rapid decline of the CD4(+) T-cell count to below 350  cells/µl. CONCLUSION: A novel association between IFITM3 gene polymorphism and rapid disease progression is reported in an acute HIV-1-infected MSM cohort in China.

From the laboratory to clinical trials and back again: lessons learned from HIV prevention trials.

PROBLEM: Inadequate, irrelevant, or inappropriate timing of biological specimen collection during clinical trials is a cause for delay in understanding and explaining correlates of protection and/or effectiveness, particularly at the portal of entry in the context of sexual HIV transmission and its prevention. METHODS: We present examples of HIV prevention trials to illustrate the impact of preplanned versus unplanned laboratory science program on the interpretation of trial results and advancement of the field. RESULTS: Of the five completed pre-exposure prophylaxis trials, only two announced main outcome results simultaneously with data on correlates of drug-related effectiveness. In four of the vaccine trials completed, the only one that showed a protective effect presented data on protection correlates significantly later. CONCLUSION: Clinical trials must preplan collaborative immunophysiological research and prioritize biological specimen collection and storage for enhancement of research on correlates of protection. Similarly appropriate specimens should be prioritized for pathogenesis research.

Four loci explain 83% of size variation in the horse.

Horse body size varies greatly due to intense selection within each breed. American Miniatures are less than one meter tall at the withers while Shires and Percherons can exceed two meters. The genetic basis for this variation is not known. We hypothesize that the breed population structure of the horse should simplify efforts to identify genes controlling size. In support of this, here we show with genome-wide association scans (GWAS) that genetic variation at just four loci can explain the great majority of horse size variation. Unlike humans, which are naturally reproducing and possess many genetic variants with weak effects on size, we show that horses, like other domestic mammals, carry just a small number of size loci with alleles of large effect. Furthermore, three of our horse size loci contain the LCORL, HMGA2 and ZFAT genes that have previously been found to control human height. The LCORL/NCAPG locus is also implicated in cattle growth and HMGA2 is associated with dog size. Extreme size diversification is a hallmark of domestication. Our results in the horse, complemented by the prior work in cattle and dog, serve to pinpoint those very few genes that have played major roles in the rapid evolution of size during domestication.

Selection and expression of recombinant single domain antibodies from a hyper-immunized library against the hapten azoxystrobin.

Three V(H)Hs against the model hapten, azoxystrobin (MW 403), were isolated from a hyper-immunized phage-displayed V(H)H library. This library was constructed by isolating the V(H)H-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing V(H)Hs specific to the hapten. After screening 95 clones, three V(H)Hs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized metal affinity chromatography. Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) showed that A27 and A85 were specific to azoxystrobin while A72 was not. The IC(50) values of A27 and A85 V(H)Hs were 7.2 and 2.0µM, respectively. To our knowledge A85 is one of the highest affinity V(H)Hs that has yet been isolated against a hydrophobic hapten such as azoxystrobin.

Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin.

Concerns over the occurrence of the veterinary antibiotic monensin (MW 671Da) in animal food products and water have given rise to the need for a sensitive and rapid detection method. In this study, four monensin-specific single chain variable fragments (scFvs) were isolated from a hyperimmunized phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to bovine serum albumin (BSA). The coding sequences of the scFvs were engineered in the order 5'-V(L)-linker-V(H)-3', where the linker encodes for Gly(10)Ser(7)Arg. Three rounds of selection were performed against monensin conjugated to chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH), alternately. In the third round of selection, two different strategies, which differed in the number of washes and the concentration of the coating conjugates, were used to select for specific binders to monensin. A total of 376 clones from round two and three were screened for their specific binding to monensin conjugates and positive clones were sequenced. It was found that 80% of clones from round three contained a stop codon. After removing the stop codon by site-directed mutagenesis, ten binders with different amino acid sequences were subcloned into the vector pMED2 for soluble expression in Escherichia coli HB2151. Four of these scFvs bound to free monensin as determined using competitive fluorescence polarization assays (C-FPs). IC(50) values ranged from 0.031 and 231 microM. A cross-reactivity assay against salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best binders were highly specific to monensin.

Pentabody-mediated antigen delivery induces antigen-specific mucosal immune response.

An efficient immunization system is essential for the development of mucosal vaccine. Cholera toxin (CT) and Escherichia coli heat labile toxin (LT) are among the strongest adjuvants tested in experimental animals but their use in humans has been hindered by their toxicity. On the other hand, the role of their non-toxic B-subunits, CTB or LTB, in enhancing mucosal immune response is not clear. We propose here a novel strategy for the induction of mucosal immune responses. Single domain antibodies (sdAbs) against a model antigen bovine serum albumin (BSA) were raised from the antibody repertoire of a llama immunized with BSA, pentamerized by fusing the sdAbs to CTB, generating the so-called pentabodies. These pentabodies were used to deliver the antigen by mixing the two components and administering the mixture to mice intranasally. One construct was equivalent to CT in helping induce mucosal immune response. It was also found that this ability was probably due to its high affinity to BSA, providing some insight into the controversial role of CTB in mucosal immunization: at least for BSA, the model antigen BSA employed in this study, CTB has to be tightly linked to the antigen to have adjuvant/immune-enhancing effect.

Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B.

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T(0)) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 mug of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T(1) plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T(0) plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T(1) generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.