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Immunomodulatory Activity ofMoringa oleiferain Albino Rats

The study was conducted to evaluate immunomodulatory property of hot aqueous and ethanolic extracts of Moringa oleifera in albino rats. This study comprised of six groups containing six rats in each group. Group I served as control, received standard feed and water. Group II and IV received hot aqueous extract of Moringa oleifera @ 400 mg/kg b.wt, orally for 45 days. Group III and V received ethanolic extract of Moringa oleifera @ 400 mg/kg b.wt, orally for 45 days. Group VI received standard immunomodulatory drug Levamisole @ 50 mg/kg b.wt, orally for 45 days. A significant increase (P< 0.05) in HA titre was observed in levamisole treated group VI, ethanolic extract treated group V and hot aqueous extract treated group IV of Moringa oleifera as compared to group I. In delayed hypersensitivity test a significant increase (P< 0.05) in paw volume was observed in response to Sheep Red Blood Cells (SRBC's) in group II (hot aqueous extract), group III (ethanolic extract) and group VI (levamisole). Phagocytic index was also found to be non-significantly increased in levamisole treated group VI, ethanolic extract treated group III and hot aqueous extract treated group II as compared to group I. Thus the results showed that Moringa oleifera possess immunomodulatory property.

Examination of serological memory in rabbits injected with Bacillus anthracis protective antigen adsorbed to Alhydrogel

Serological memory after inoculation of protective antigen (PA) combined with Alhydrogel adjuvant (PA/Alhydrogel) was examined in New Zealand white rabbits, an animal model for anthrax. A threshold dose of 0.1μg of PA/Alhydrogel was identified which resulted in an ELISA titer 2weeks after a primary immunization of only 0.168μg anti-PA IgG per ml and a toxin-neutralizing antibody titer (TNA ED50) of 1.8 (n=40). A significant increase in anti-PA IgG and TNA ED50 titers were measured (p<0.0001) 2weeks after a booster immunization with 0.1μg of PA/Alhydrogel at 14days (n=10; 40.9μg anti-PA IgG per ml; 522 TNA ED50) and 28days (n=10; 63.8μg anti-PA IgG per ml; 501 TNA ED50). At this threshold dose of PA/Alhydrogel, protection against an aerosol exposure to Bacillus anthracis Ames spores improved as the booster immunization was administered from 4days (40% survival), to 8days (50% survival), and to 12days (80% survival) before challenge. The partial protection of rabbits, even in the absence of protective antibody titers (0.9μg anti-PA IgG per ml and 26 TNA ED50) when the booster immunization was administered 4days before challenge, suggested a protective potential for serologic memory.

Quantitative and Qualitative Antibody Responses to Immunization With the Pneumococcal Polysaccharide Vaccine in HIV-Infected Patients After Initiation of Antiretroviral Treatment: Results From a Randomized Clinical Trial.

Pneumococcal vaccination is recommended for human immunodeficiency virus-infected (HIV+) persons; the best timing for immunization with respect to initiation of antiretroviral therapy (ART) is unknown. Double-blind, placebo-controlled trial in HIV+ with CD4(+) T cells/µL (CD4) ≥ 200 randomized to receive the 23-valent pneumococcal polysaccharide vaccine (PPV23) or placebo at enrollment, followed by placebo or PPV23, respectively, 9-12 months later (after ≥6 months of ART). Capsular polysaccharide-specific immunoglobin (Ig) G and IgM levels to serotypes 1, 3, 4, 6B, and 23F, and opsonophagocytic killing activity (OPA) to serotypes 6B and 23F were evaluated 1 month postvaccination. One hundred seven subjects were enrolled, 72 (67.3%) were evaluable (36/group). Both groups had significant increases in pre- to 1-month postvaccination IgG levels, but negligible to IgM, and significant increases in OPA titers to serotype 6B but not to 23F. There were no significant differences between groups in serotype-specific IgM or IgG levels or OPA titers. For the combined groups, there was a significant correlation between serotype-specific IgG and OPA titers to 23F but not to 6B. There was no correlation between CD4, viral load and IgG responses. In HIV+ with CD4 ≥ 200, delaying PPV23 until ≥6 months of ART does not improve responses and may lead to missed opportunities for immunization.

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax

BackgroundAnthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2–2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans. MethodsTo optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants. ResultsImmunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits. ConclusionsAn LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax.

Toxoplasma Ig M positive in pregnancy: what does it mean from the perspective of the gynecologists?

Toxoplasma gondii infection in pregnancy occurs with maternal ingestion of cysts in undercooked meat or with maternal ingestion of oocysts found in water, food and soil (1). Contaminated under cleaned green salads (like garden rocket, parsley) and water may be important sources for women in Turkey. Although routine screening for toxoplasmosis in pregnancy is not recommended, this is not the case in daily practice and due to unnecessary screening and confounding test results, gynecologists should be also familiar with patients having the results of Toxoplasma IgM(+). One key-point for evaluation the toxoplasmosis-suspected pregnant woman is to know that congenital infection risk for the fetus is higher in third trimester; however, the risk of the injury for the fetus is greater in the first trimester (2). Pregnant women with IgM(+)/IgG(-) should be screened in 1 to 3 weeks again. If seroconversion in Ig G occurs, then infection is probably acquired during pregnancy and there is risk for congenital infection (3). Treatment is to be initiated with Spiramycin. Amniocentesis for amniotic fluid PCR should be performed after 16 weeks. A detailed fetal ultrasonography should be also performed. In case of IgM(+)/IgG(+), the results should be confirmed. Toxoplasma Gondii avidity testing is reasonable at this stage (4). High-avidity IgG antibodies are detected at least 12-16 weeks after infection, so high-avidity IgG results show that the infection was started 16 weeks before. Low-avidity results may persist for a long period (even more than one year after acute infection), so only avidity tests should not be used (1, 5). In our daily practice, we account the gestational week, the Ultrasonograhic findings with maternal serum results into account. In case of IgM(+)/IgG(+); we also perform IgG avidity test. We initiate medical treatment and repeat the tests in 3 weeks. A significant increase in IgG titers (3-4 times) together with low-avidity IgG is suggestive for an acute infection. Fetal infection should be checked with amniocentesis

On the connection between autoimmunity, tic disorders and obsessive-compulsive disorders: a meta-analysis on anti-streptolysin O titres.

Anti-streptolysin O (ASO) titration is useful in the context of autoimmune pathologies, including specific cases of tic and obsessive-compulsive disorders occurring after streptococcal infections. There is currently a lack of consensus on the use of ASO titres; therefore we performed a meta-analysis to systematise available data and clarify the role of ASO titres in the context of neuropsychiatric disorders. A meta-analysis was performed on ASO titration in neuropsychiatric patients, including tic disorders and obsessive-compulsive disorders. Included studies reported numbers of positive subjects, depending on a chosen threshold, or detailed ASO titrations. Three hundred and twenty nine studies were identified, of which 13 were eligible for meta-analysis. Due to limited available data, only tic disorders were evaluated. The odds ratio of finding an abnormal ASO titre in patients was 3.22 (95% C.I. 1.51-6.88) as compared to healthy controls and 16.14 (95% C.I. 8.11-32.11) as compared to non-psychiatric patients. Studies using different thresholds were generally concordant. ASO titres were also compared quantitatively, finding an overall difference of the means of 70.50 U/ml (95% C.I. 25.21-115.80) in favour of patients with tic disorders. Based on current evidence, tic disorders are associated with a significant increase in ASO titres, evident both in a threshold-level perspective and on a quantitative level. These results encourage the systematisation of ASO titration in the context of tic disorders.

Safety and immunogenicity in man of a cell culture derived trivalent live attenuated seasonal influenza vaccine: A Phase I dose escalating study in healthy volunteers

Live attenuated influenza vaccine (LAIV) offers the promise of inducing a variety of immune responses thereby conferring protection to circulating field strains. LAIVs are based on cold adapted and temperature sensitive phenotypes of master donor viruses (MDVs) containing the surface glycoprotein genes of seasonal influenza strains. Two types of MDV lineages have been described, the Ann Arbor lineages and the A/Leningrad/17 and B/USSR/60 lineages. Here the safety and immunogenicity of a Madin Darby Canine Kidney – cell culture based, intranasal LAIV derived from A/Leningrad/17 and B/USSR, was evaluated in healthy influenza non-naive volunteers 18–50 years of age. In a double-blind, randomized, placebo-controlled design, single escalating doses of 1×105, 1×106, or 1×107 tissue culture infectious dose 50% (TCID50) of vaccine containing each of the three influenza virus re-assortants recommended by the World Health Organization for the 2008–2009 season were administered intranasally. A statistically significant geometric mean increase in hemagglutination inhibition titer was reached for influenza strain A/H3N2 after immunization with all doses of LAIV. For the A/H1N1 and B strains, the GMI in HI titer did not increase for any of the doses. Virus neutralization antibody titers showed a similar response pattern. A dose–response effect could not be demonstrated for any of the strains, neither for the HI antibody nor for the VN antibody responses. No influenza like symptoms, no nasal congestions, no rhinorrhea, or other influenza related upper respiratory tract symptoms were observed. In addition, no difference in the incidence or nature of adverse events was found between vaccine and placebo treated subjects. Overall, the results indicated that the LAIV for nasal administration is immunogenic (i.e. able to provoke an immune response) and safe both from the perspective of the attenuated virus and the MDCK cell line from which it was derived, and it warrants further development.

Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Adenovirus Type 4 and 7 Vaccination or Adenovirus Type 4 Respiratory Infection Elicits Minimal Cross-Reactive Antibody Responses to Nonhuman Adenovirus Vaccine Vectors

Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd). Small statistically significant increases in titers to ChAd63, ChAd3, human Ad35, and human Ad5 were observed. Neutralizing antibodies elicited by Ad4 infection or immunization results in a small amount of adenovirus cross-reactivity.

Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase.

Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.

Augmented primary humoral immune response and decreased cell-mediated immunity by Murraya koenigii in rats

Murraya koenigii (Rutaceae) (curry patta: Hindi) of the family Rutaceae is used in the traditional Indian system of medicine for its immunomodulatory properties. The essential oil of the leaves of M. koenigii possesses antimicrobial, antifungal, and pesticidal activities and is used for the treatment of amebiasis, diabetes, and hepatitis. The present study was performed to evaluate the effect of M. koenigii on humoral and cell-mediated immune responses in rats. Aqueous extract of M. koenigii leaves was administered orally in a dose of 350 mg/kg. Cell-mediated immunity was assessed by measuring foot pad thickness following sensitization by injection of keyhole limpet hemocyanin and subsequent challenge by the same. Humoral immunity was assessed by measurement of hemagglutination titer to sheep red blood cells (SRBCs). In the humoral immune response, the administration of M. koenigii [350 mg/kg per os (p.o.)] from day 1 to day 7 after sensitization with SRBC on day 0 caused a significant increase in the primary anti-SRBC titer. However, the secondary immune response was decreased significantly (p<0.05) as shown by a decrease in secondary anti-SRBC titer measured on day 11 following a booster dose of antigen on day 8. In the delayed-type hypersensitivity test, M. koenigii (350 mg/kg, p.o.), when administered for 14 days, produced a significant (p<0.05) decrease in foot pad thickness when compared with the control group. Thus, these results suggest that oral administration of M. koenigii augments primary humoral immune response and decreases cell-mediated immunity.

Protective Humoral Immunity Induced by Lipopolysaccharide Incorporated Liposome in Mice Against Shigella flexneri Infection

This study was conducted to determine the role of lipopolysaccahride-liposome conjugate (LPS-LIP) as a potential vaccine against shigellosis in mice by determining the IgG titer. One hundred stool samples were collected from patients with diarrhea from different hospitals in Baghdad city during the period December 2011-May 2012. Thirty isolates were suspected to be Shigella. Four isolates of Shigella flexneri were obtained after performing some biochemical tests and Api System. Antibiotic sensitivity test was carried out and results revealed that all isolates were resist to some antibiotics used in this study. One isolate was selected for LPS extraction by phenol hot water extraction method. Chemical characterization of the extracted LPS revealed that the carbohydrate content was 2.34 mg/ml, while the protein concentration was 0.52µg/ml. Partial purification of the extracted LPS was carried out by using gel- filtration chromatography on Sephacryl S-200 and results showed that three peaks were obtained and protein, carbohydrate concentration were estimated for each peak. The second peak observed to have the highest carbohydrate content was (25%). and the lowest contaminated protein was (0.001%). The partial purified LPS was analyzed by Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE) and results reveled that two bands with molecular weights 100 and 150 KDa were present. Band with MW 100 kDa represented LPS. IgG titer was estimated by Elisa technique. A significant increase in IgG titer was recorded in mice treated with conjugate after infection with S. flexneri.

CHARACTERIZATION OF SERUM IMMUNOGLOBULIN IN CHANNA STRIATA (BLOCH) AND KINETICS OF ITS RESPONSE TO AEROMONAS HYDROPHILA ANTIGEN

The immunoglobulin (Ig) from the serum of Channa striata was isolated by gel electroelution and characterized further to understand its nature and subsequent applications in studying the immune response. The purity of the sample was confirmed with the presence of a single band on native gradient PAGE and the molecular weight of ∼897 kDa was determined from the gel. In SDS-PAGE, C. striata Ig was reduced to produce two bands corresponding to H (heavy) (∼72 kDa) and L (light) (∼27 kDa) chain subunits. Polyclonal antiserum against the purified Ig was raised in a rabbit and adsorbed with 10% liver tissue homogenate of C. striata to enhance its specificity. By an indirect ELISA standardized using the adsorbed rabbit antiserum, the normal serum Ig concentration in C. striata was estimated to be 3.48 mg/mL. Further, a kinetic study of specific immunoglobulin response to formalin-killed Aeromonas hydrophila antigen was undertaken using another indirect ELISA, which showed a significant increase in serum immunoglobulin titer from day 2 onwards and reached its peak at day 14. Subsequently, the Ig titer was dropped from day 21 onwards till the completion of the experiment at day 42, although it was at a significantly higher level than the control.

Hibiscus tiliaceus: A possible immunomodulatory agent

ObjectiveThe objective of the study is to evaluate the immunomodulatory effect of methanolic leaf extract of Hibiscus tiliaceus (MLHT) in pyrogallol induced immunosuppressed Wistar rats. MethodsThe methanolic extract of leaves of H. tiliaceus was administered orally at the dosage levels of 250 mg/kg/day and 500 mg/kg/day b.w in Wistar rats for 28 days. The assessment of immunomodulatory activity, humoral and cellular immunity was studied by hemagglutination antibody (HA) titer, delayed type hypersensitivity (DTH), Neutrophil adhesion test and carbon clearance test. In order to induce immunosuppression in rats pyrogallol (100 mg/kg/day, p.o.) is used and septilin syrup (1ml/100 g/day, p.o.) used as standard as it is immunostimulating agent. Hematological and biochemical were estimated by standard methods. ResultsOral administration of MLHT showed a significant increase in the production of circulating antibody titer in response to sheep red blood cells (SRBCs). A significant (P < 0.001) increase in both primary and secondary HA titer was observed when compared to control group, whereas in H. tiliaceus showed significant (P < 0.001) increase in HA titer. MLHT significantly (P < 0.001) potentiated the DTH reaction by facilitating the footpad thickness response to SRBCs in sensitized rats. Also MLHT evoked a significant (P < 0.001) increase in percentage neutrophil adhesion to nylon fibers and phagocytic activity. It also enhanced the production of RBC, WBC and hemoglobin. It does not much affect the biochemical parameters. ConclusionAn oral administration of the MLHT showed immunomodulatory effect in Wistar rats in a dose dependent manner. From the results obtained and reported phytochemical studies H. tiliaceus has a significant effect on both humoral and cellular immunity in experimental animals, this may be attributed to the polyphenols and flavonoid content of the plant extract.

Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia

BackgroundWe addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs).ResultsWe examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after – but not before – any SHIV challenge. With this “subtractive biopanning” approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus.ConclusionsAnti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of “vaccine only” responses in clinical vaccine trials.

Chlamydia Pneumoniae Infection-Associated Erythema Multiforme

There is a well-known correlation between Herpes simplex (HSV) infection and erythema multiforme (EM). More recently, in Japan, it was found that Chlamydia pneumoniae (Cp) may promote the development of EM. All cases of Cp infection-associated EM that had been diagnosed in our clinic over the past two years (from 2011 to 2012) were analyzed. Cp infection was diagnosed on the basis of a significant increase (>2.00) in anti-Cp IgM titers, as measured by the HITAZYME-ELISA test. There were 7 cases of Cp-EM, one male and 6 females. Median age was 13 years (range 3-29 years). It is recommended that the possible involvement of Cp infection, besides HSV or Mycoplasma pneumoniae infections, should be considered in all cases of EM.

Challenges of Culturing Human Norovirus in Three-Dimensional Organoid Intestinal Cell Culture Models

Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

PLGA nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs

Porcine reproductive and respiratory syndrome (PRRS) is a chronic viral disease of pigs, has been posing a huge economic concern to pig industry worldwide. In this study, we developed biodegradable PLGA [poly(d,l-lactide-co-glycolide)] nanoparticle-entrapped killed PRRSV vaccine (Nano-KAg), and administered intranasally to pigs once and evaluated the immune correlates. In Nano-KAg vaccinated homologous virus challenged pigs, complete clearance of viremia was observed in 2 weeks, associated with a significant increase in virus neutralizing titers only in the lungs, compared to both unvaccinated and killed vaccine vaccinated pigs. The lung homogenate and sera of Nano-KAg vaccinated pigs had higher levels of IFN-γ and lower levels of TGF-β than control groups. Restimulation of mononuclear cells isolated from the lungs, blood, BAL, and TBLN of Nano-KAg vaccinated pigs’ secreted significantly increased levels of Th1 cytokines, IFN-γ and IL-12. In addition, higher frequencies of CD3+CD8+, CD4+CD8+, and γδ T cells, and reduced frequency of Foxp3+ T-regulatory cells were observed in Nano-KAg vaccinated pigs. Thus, intranasal delivery of Nano-KAg vaccine may be a suitable strategy to elicit anti-PRRSV immune response required to better clear viremia in pigs.

Altered Response to A(H1N1)pnd09 Vaccination in Pregnant Women: A Single Blinded Randomized Controlled Trial

BackgroundPregnant women were suspected to be at particular risk when H1N1pnd09 influenza became pandemic in 2009. Our primary objective was to compare the immune responses conferred by MF59®-adjuvanted vaccine (Focetria®) in H1N1pnd09-naïve pregnant and non-pregnant women. The secondary aims were to compare influences of dose and adjuvant on the immune response.MethodsThe study was nested in the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2010) pregnancy cohort in 2009-2010 and conducted as a single-blinded block-randomised [1∶1∶1] controlled clinical trial in pregnant women after gestational week 20: (1) 7.5 µg H1N1pnd09 antigen with MF59-adjuvant (Pa7.5 µg); (2) 3.75 µg antigen half MF59-adjuvanted (Pa3.75 µg); (3) 15 µg antigen unadjuvanted (P15 µg); and in non-pregnant women receiving (4) 7.5 µg antigen full adjuvanted (NPa7.5 µg). Blood samples were collected at baseline, 3 weeks, 3 and 10 months after vaccination, adverse events were recorded prospectively.Results58 pregnant women were allocated to Pa7.5 µg and 149 non-pregnant women were recruited to NPa7.5 µg. The sero-conversion rate was significantly increased in non-pregnant (NPa7.5 µg) compared with pregnant (Pa7.5 µg) women (OR = 2.48 [1.03–5.95], p = 0.04) and geometric mean titers trended towards being higher, but this difference was not statistically significant (ratio 1.27 [0.85–1.93], p = 0.23). The significant titer increase rate showed no difference between pregnant (Pa7.5 µg) and non-pregnant (NPa7.5 µg) groups (OR = 0.49 [0.13–1.85], p = 0.29).ConclusionOur study suggests the immune response to the 7.5 µg MF59-adjuvanted Focetria® H1N1pnd09 vaccine in pregnant women may be diminished compared with non-pregnant women.Trial RegistrationClinicalTrials.gov NCT01012557.

Comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus

In order to better understand how immunization against porcine reproductive and respiratory syndrome virus (PRRSV) can be improved using commercial vaccines, different strategies of immunization were applied in the field using an inactivated vaccine (INV), a modified live vaccine (MLV) or a combination of the two and the responses compared. In experiment 1 (E1), 21 piglets were distributed in three groups. Group A was vaccinated with a commercial INV at 2.5, 3.5 and 6.5months old; group B pigs received the INV at 1.5, 2.5, 5.5 and 6.5months old, while pigs in group C were kept as unvaccinated controls. At 7.5months of age all pigs were challenged with PRRSV and followed for 21days. In experiment 2 (E2), 32 piglets were distributed evenly in four groups. Groups A, B and C were vaccinated with a commercial MLV at 1.5months old, while group D pigs were kept as controls. At 4.5months old, groups A and C received the INV while B received a second MLV, 1month later group C pigs received a third INV. At 6.5months old all pigs were challenged as in E1. In both experiments, total antibodies, neutralizing antibodies (NA) and cell-mediated immunity (CMI) were evaluated, and viraemia was determined after challenge.In E1, immunization with an INV induced high interferon-γ responses after the second and subsequent vaccinations. Development of NA after challenge was faster in INV vaccinated pigs compared to unvaccinated controls. In E2, re-vaccination with INV induced NA responses similar to re-vaccination with MLV; however, a significant increase in NA titres after challenge was only detected in group C pigs. The use of combined protocols (MLV+INV) was superior to the use of MLV alone in inducing cell mediated immunity. In conclusion, the highest immune responses against PRRSV after a single shot were achieved with MLV; after that, INV re-vaccination should be considered as the best strategy to induce significant boosters.