Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5'-carboxyfluorosceinsuccidyl ester.

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. RESULTS: We observed that 20.3% (+/- 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. CONCLUSIONS: Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.

Oral antigen exposure in newborn piglets circumvents induction of oral tolerance in response to intraperitoneal vaccination in later life.

BACKGROUND: We previously determined that newborn piglets orally gavaged with Ovalbumin (OVA) responded to systemic OVA re-exposure with tolerance; if adjuvants were included in oral vaccine, piglets responded with antibody-mediated immunity (Vet Immunol Immunopathol 161(3-4):211-21, 2014). Here, we will investigate whether newborn piglets gavaged with a vaccine comprised of OVA plus unmethylated CpG oligodeoxynucleotides (CpG; soluble component; OVA/CpG) combined with OVA plus CpG encapsulated within polyphosphazene microparticles (MP; particulate component) responded with systemic and mucosal immunity. To monitor the response to systemic antigen re-exposure, piglets were i.p.-immunized with OVA plus Incomplete Freund's Adjuvant (IFA) one month later. RESULTS: Newborn piglets (n = 5/group) were gavaged with a combined soluble and particulate vaccine consisting of OVA (0.5-0.05 mg) plus 50 µg CpG and 0.5 mg OVA plus 50 µg CpG encapsulated within a polyphosphazene MP (0.5 mg) referred to as OVA/CpG + MP. Control piglets were gavaged with saline alone. Piglets were i.p. immunized with 10 mg OVA (or saline) in IFA at four weeks of age and then euthanized at eight weeks of age. We observed significantly higher titres of serum anti-OVA immunoglobulin (Ig) IgM, IgA, IgG, IgG1, IgG2 and IgG in piglets immunized with 0.05 mg OVA/CpG + MP relative to saline control animals. Thus, a single oral exposure at birth to a combined soluble and particulate OVA vaccine including adjuvants can circumvent induction of oral tolerance which impacts response to i.p. vaccination in later life. Further, piglets gavaged with 0.05 mg OVA/CpG + MP generated significant anti-OVA IgG and IgG1 titres in lung compared to saline control piglets but results were comparable to titres measured in parenteral control piglets. Peripheral blood mononuclear cells (PBMCs) ex vivo-stimulated with OVA showed markedly decreased production of IL-10 cytokine after 72 hours relative to animal-matched cells incubated with media alone. No production of IFN-γ was observed from any groups. CONCLUSION: Newborn piglets gavaged with low dose soluble and particulate OVA plus CpG ODN and polyphosphazene adjuvants produced antigen-specific antibodies in serum and lung after systemic re-exposure in later life. These data indicate circumvention of oral tolerance but not induction of oral immunity.

Vaccination with a Lawsonia intracellularis subunit water in oil emulsion vaccine mitigated some disease parameters but failed to affect shedding.

Lawsonia intracellularis is the causative agent of ileitis in swine that manifests as slower weight gain, mild or hemorrhagic diarrhea and/or death in severe cases. As an economically important swine pathogen, development of effective vaccines is important to the swine industry. In developing a subunit vaccine with three recombinant antigens - FliC, GroEL and YopN - we wanted to identify a formulation that would produce robust immune responses that reduce disease parameters associated with Lawsonia intracellularis infection. We formulated these three antigens with four adjuvants: Montanide ISA 660 VG, Montanide Gel 02 PR, Montanide IMS 1313 VG NST, and Montanide ISA 61 VG in an immunogenicity study. Groups vaccinated with formulations including Montanide ISA 660 VG or Montanide ISA 61 VG had significantly more robust immune responses than groups vaccinated with formulations including Montanide Gel 02 PR or Montanide IMS 1313 VG NST. In the challenge study, animals vaccinated with these antigens and Montanide ISA 61 VG had reduced lesion scores, reduced lesion lengths, and increased average daily gain, but no reduction in shedding relative to the control animals. This work shows that this vaccine formulation should be considered for future study in a field and performance trial.

A Single-Dose Intramuscular Nanoparticle Vaccine With or Without Prior Intrauterine Priming Triggers Specific Uterine and Colostral Mucosal Antibodies and Systemic Immunity in Gilts but Not Passive Protection for Suckling Piglets.

An effective single-dose vaccine that protects the dam and her suckling offspring against infectious disease would be widely beneficial to livestock animals. We assessed whether a single-dose intramuscular (i.m.) porcine epidemic diarrhea virus (PEDV) vaccine administered to the gilt 30 days post-breeding could generate mucosal and systemic immunity and sufficient colostral and mature milk antibodies to protect suckling piglets against infectious challenge. The vaccine was comprised of polymeric poly-(lactide-co-glycolide) (PGLA)-nanoparticle (NP) encapsulating recombinant PEDV spike protein 1 (PEDVS1) associated with ARC4 and ARC7 adjuvants, a muramyl dipeptide analog and a monophosphoryl lipid A (MPLA) analog, respectively (NP-PEDVS1). To establish whether prior mucosal exposure could augment the i.m. immune response and/or contribute to mucosal tolerance, gilts were immunized with the NP-PEDVS1 vaccine via the intrauterine route at breeding, followed by the i.m. vaccine 30 days later. Archived colostrum from gilts that were challenged with low-dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. On day 100 of gestation (70 days post i.m. immunization), both vaccinated groups showed significant PEDVS1-specific IgG and IgA in the serum, as well as in uterine tissue collected on the day of euthanasia. Anti-PEDVS1 colostral IgG antibody titers collected at farrowing were significantly higher relative to the negative control gilts indicating that the NP vaccine was effective in contributing to the colostral antibodies. The PEDVS1-specific colostral IgA and anti-PEDVS1 IgG and IgA antibodies in the mature milk collected 6 days after farrowing were low for both vaccinated groups. No statistical differences between the vaccinated groups were observed, suggesting that the i.u. priming vaccine did not induce mucosal tolerance. Piglets born to either group of vaccinated gilts did not receive sufficient neutralizing antibodies to protect them against infectious PEDV at 3 days of age. In summary, a single i.m. NP vaccine administered 30 days after breeding and a joint i.u./i.m. vaccine administered at breeding and 30 days post-breeding induced significant anti-PEDVS1 immunity in systemic and mucosal sites but did not provide passive protection in suckling offspring.

Evaluation of immunogenicity and protection mediated by Lawsonia intracellularis subunit vaccines.

Lawsonia intracellularis is an economically important bacterium that causes ileitis in pigs. Current vaccines for L. intracellularis do not allow for differentiation between infected and vaccinated animals (DIVA), which is beneficial for disease tracking and surveillance. Previously, we identified five putative surface L. intracellularis proteins that were targeted by antibodies from pigs infected with L. intracellularis which could serve as antigens in a subunit vaccine. We conducted two trials to determine whether these antigens were immunogenic and provided protection against infectious challenge and whether truncated glycoprotein D could be used as a DIVA antigen. For Trial 1, 5 week-old piglets were administered intramuscular monovalent vaccines comprised of a recombinant (r) flagella subunit protein (rFliC,) and DIVA antigen (truncated glycoprotein D (TgD), a herpes virus antigen) both formulated with a combination adjuvant consisting of polyinosinic:polycytidylic acid(poly I:C), host defense peptide 1002 and polyphosphazene, referred to as Triple Adjuvant (TriAdj). Relative to control animals, animals vaccinated with rFliC and rTgD had significantly elevated antigen-specific humoral immunity in sera suggesting that rFliC and TgD are immunogenic. Control animals had negligible anti-TgD titres suggesting that TgD may be a suitable DIVA antigen for pigs. For Trial 2, piglets were immunized with a trivalent vaccine (FOG vaccine consisting of rFLiC, rOppA protein (a ABC Type dipeptide transport system) and rGroEL (a stress response protein)) and a divalent vaccine (CM vaccine consisting of rClpP (an ATP-dependent Clp protease proteolytic subunit) and rMetK (a S-adenosyl methionine synthase)) formulated with Emulsigen®. Relative to the control pigs, pigs immunized with the FOG vaccine produced robust and significantly higher serum IgG antibodies against rFliC and rGroEL, and significantly higher anti-FliC and anti-GroEL IgA antibodies in jejunal (GroEL only) and ileal intestinal mucosa. Pigs immunized with CM vaccine produced significantly higher serum antibodies against rClpP and rMetK and significantly higher anti-rClpP IgA antibodies in the ileum relative to the control pigs. Quantitative polymerase chain reaction (qPCR) analysis showed that 18 days after challenge with infectious L. intracellularis, challenged/control pigs and pigs that received the CM vaccine, but not the pigs vaccinated with the FOG vaccine, shed significantly more bacteria in feces than the unchallenged controls pigs. These data suggest that the FOG vaccinated pigs showed limited protection. While promising, more work is needed to enhance the efficiency of the intramuscular vaccine to show significant disease protection.

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice.

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/- ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/- ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species.

Assessment of Immunological Response and Impacts on Fertility Following Intrauterine Vaccination Delivered to Swine in an Artificial Insemination Dose.

To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo. Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response.

Use of flow cytometry and PCR analysis to detect 5-carboxyfluoroscein-stained obligate intracellular bacteria Lawsonia intracellularis invasion of McCoy cells.

In this study, we describe a method to quantify invasion of obligate intracellular bacteria, Lawsonia intracellularis, inside McCoy cells. In immunological research, the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE) is commonly used to quantify eukaryotic cellular proliferation. Instead of using it in this traditional way, we stained L. intracellularis with CFSE dye prior to infection of McCoy cells. Flow cytometry was performed to quantify the percentage of eukaryotic cells which had taken up or were associated with fluorescent bacteria. As obligate intracellular bacteria, they cannot replicate outside of eukaryotic cells and thus qPCR analysis was used to quantify bacterial growth. Indirectly, PCR analysis confirmed invasion rather than adherence to the McCoy cell surface. Fluorescent activated cell sorting (FACS) was used to sort the CFSE(+) (i.e. infected) McCoy cells from the CFSE(-) (i.e. non-infected) McCoy cells and confocal microscopy was used to confirm bacterial invasion and cytosolic localization of CFSE-L. intracellularis. To show that this approach could be used in conjunction with functional assays, we investigated the effect that serum antibodies had on CFSE-bacterial invasion and growth. Instead of blocking invasion, rabbit hyperimmune serum augmented invasion of the bacteria inside McCoy cells and qPCR analysis confirmed bacterial growth over the course of 5days. We conclude that CFSE-labeling of bacteria and qPCR can be used to track and quantify bacterial invasion and may be a valuable tool for studying the invasive properties of bacteria, especially if commercial antibodies are not available. This approach may be adapted for use in other obligate intracellular bacteria and intracellular pathogens.

Rapamune does not attenuate high cholesterol-induced atherosclerosis in rabbits.

Solid-organ transplant recipients are prone to develop atherosclerosis. The objectives of this study were to investigate the effects of Rapamune (Wyeth Canada, Saint-Larent, QC, Canada) on the rabbit model of atherosclerosis. The rabbits were assigned to four groups: group I, regular diet (control); group II, 1% cholesterol diet; group III, control with Rapamune (1 mg/kg/d orally); and group IV, high cholesterol diet with Rapamune. Blood samples for serum lipids (triglycerides [TG], total cholesterol [TC], low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C]), as well as malondialdehyde, and protein carbonyls, the indices of oxidative stress were collected at the end of 2 months on the respective diet regimen. Aortic tissue for atherosclerotic changes were also collected for oxidative stress indices were also collected. Rapamune reduced serum levels of TG, TC, LDL-C, and HDL-C. Rapamune elevated the oxidative stress in rabbits on high cholesterol diet. Rapamune did not attenuate extent of atherosclerosis (group II vs. group IV, 45.00 ± 12.00 vs. 57.28 ± 2.99%); intimal thickness (group II vs. group IV, 32.38 ± 7.14 × 10(3) vs. 21.90 ± 11.98 × 10(3) µm(2)); intimal/medial ratio (group II vs. group IV, 0.50 ± 0.06 vs. 0.35 ± 0.06); and macrophage accumulation (group II vs. group IV, 69.72 ± 5.02 vs. 61.52 ± 8.94%) in the intima of rabbits on high cholesterol diet. The data suggest that (1) Rapamune increased the oxidative stress in rabbits on high cholesterol diet and (2) Rapamune did not attenuate the hypercholesterolemic atherosclerosis in the rabbit model.

A single, low dose oral antigen exposure in newborn piglets primes mucosal immunity if administered with CpG oligodeoxynucleotides and polyphosphazene adjuvants.

By definition, soluble antigens ingested orally trigger mucosal tolerance such that any subsequent re-exposure by a systemic route results in suppression of immunity. We propose that antigens introduced in extreme early life can readily traverse the gut wall and therefore circumvent induction of mucosal tolerance and instead induce immunity. Piglets were drenched with low-doses of ovalbumin (OVA; 5mg or 0.05 mg) alone, OVA plus adjuvants (CpG oligodeoxynucleotides and PCEP polyphosphazene) or saline within 6h of birth. At 28 days of age, they were administered 10mg OVA plus 1:1 Montanide adjuvant (or saline) via the intraperitoneal (i.p.) route or via the oral route. Serum was obtained on day 28 and day 49 to measure OVA-specific antibodies titres. All piglets boosted orally with OVA plus Montanide, regardless of prior OVA exposure, failed to induce immunity. As expected, piglets drenched with saline but boosted via the i.p. route with OVA plus Montanide showed significant induction of anti-OVA IgA, IgG, IgG1 and IgG2 relative to saline control piglets. Newborn animals drenched with 5mg or 0.05 mg OVA failed to induce oral immunity. A second intramuscular injection in adulthood triggered immunity in the piglets that were drenched with 0.05 mg OVA and boosted initially by the i.p. route suggesting that some systemic lymphocytes were primed despite initial lack of induction of humoral immunity. In contrast, piglets orally immunized with 5mg or 0.05 mg OVA plus adjuvants resulted in significant induction of anti-OVA IgA (5mg only), IgM, IgG, IgG1 and IgG2 in serum relative to saline control piglets as well as significant induction of anti-OVA IgA, IgM (5mg only) IgG, IgG1 (5mg only) or IgG2 relative to piglets drenched with OVA alone. These data clearly show that the response was sensitive to the oral vaccine components and was not simply a response to the i.p. immunization at day 28. This work demonstrates that newborn piglets respond to oral antigens with immunity if re-exposure to the antigen occurs via a systemic route and if adjuvants are included with the oral vaccine administered at birth. These results should be further explored to establish whether early life oral vaccination can be exploited to protect this susceptible population against infectious diseases.