IFN-gamma and IL-12 plasmid DNAs as vaccine adjuvant in a murine model of grass allergy.

BACKGROUND: Plasmids encoding cytokines such as IFN-gamma and IL-12 are potential genetic adjuvants that might increase the effectiveness of allergen vaccines. OBJECTIVE: The role of plasmids expressing the cytokines IFN-gamma (pIFN-gamma) and/or IL-12 (pIL-12) as adjuvants in modulating allergic immune responses, inflammation, and asthma was investigated in a murine model of Kentucky blue grass (KBG) allergy. METHODS: Groups of naive B6D2F1 mice were vaccinated subcutaneously with KBG allergens and administered intramuscularly with pIFN-gamma, pIL-12, pIFN-gamma plus pIL-12, or a vector control. Mice were then sensitized with KBG allergens in alum (intraperitoneally) and later challenged intranasally. Mice were examined for modulation of specific immunity, prevention of the development of airway hyperresponsiveness, and inflammation. RESULTS: Mice vaccinated with cytokine plasmid adjuvants had relatively lower levels of total serum IgE and higher levels of grass allergen-specific IgG2a in comparison with control mice. The lowest IgE and highest IgG2a levels were found in mice vaccinated with the combination of pIFN-gamma and pIL-12 as an adjuvant. The vaccination of mice with both pIFN-gamma and pIL-12 as an adjuvant induced the highest level of T(H)1 cytokines, IFN-gamma, and IL-2 in comparison with mice given either of the plasmids alone. The most profound decrease in airway hyperresponsiveness and pulmonary inflammation was observed in mice receiving both pIFN-gamma and pIL-12 as an adjuvant. CONCLUSION: These results demonstrate that pIFN-gamma and pIL-12 together provide an effective adjuvant to parenteral grass allergen vaccines and show that this adjuvant can significantly enhance the effectiveness of allergen immunotherapy in human beings.

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection.

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.

Differential cytokine mRNA expression in Dermatophagoides farinae allergen-sensitized and respiratory syncytial virus-infected mice.

The interaction between mite allergen sensitization and respiratory syncytial virus (RSV) infection at the level of cytokine mRNA expression was examined in a murine model in the present study. Primary RSV infection enhances expression of inflammatory cytokines such as IL-6, IFN-gamma, and eotaxin in the lung and upregulates the expression of Th2-like cytokines IL-10 and IL-13 in the spleen in BALB/c mice. Mite antigen-sensitized and RSV-infected (ASRSV) mice show enhanced (P < 0.05) total serum IgE compared to antigen-sensitized mice. However, the levels of viral mRNA in the lung tissues are comparable between RSV-infected and ASRSV mice. It is concluded that compartmentalization of cytokine expression following RSV infection plays a role in the augmentation of Th2-like and IgE antibody response to RSV.

Recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness.

Respiratory syncytial virus (RSV) infection is considered a risk factor for bronchial asthma; however, the synergy between allergen sensitization and RSV infection in the development of pulmonary inflammation and asthma has been controversial. In this study the effects of primary and recurrent RSV infection on allergic asthma were examined in a group of control, RSV-infected, Dermatophagoides farinae (Df) allergen-sensitized, and Df allergen-sensitized plus RSV-infected BALB/c mice. Primary RSV infection in Df-sensitized mice transiently increases airway responsiveness, which is accompanied by increases in eosinophilic infiltration, the expression of ICAM-1, and macrophage inflammatory protein-1alpha (MIP-1alpha) in the lung tissue. A secondary RSV infection persistently enhances airway responsiveness in Df-sensitized mice, with a concomitant increase in MIP-1alpha and RSV Ag load in lung tissues. Bulk cultures of thoracic lymph node mononuclear cells demonstrate that acute RSV infection augments both Th1- and Th2-like cytokines, whereas secondary and tertiary infections shift the cytokine profile in favor of the Th2-like cytokine response in Df-sensitized mice. The elevated total serum IgE level in the Df-sensitized mice persists following only RSV reinfection. Thus, recurrent RSV infections in Df-sensitized mice augment the synthesis of Th2-like cytokines, total serum IgE Abs, and MIP-1alpha, which are responsible for persistent airway inflammation and hyperresponsiveness, both of which are characteristics of asthma.

Intranasal IFN-gamma gene transfer protects BALB/c mice against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants, young children and the elderly and causes severe bronchiolitis and asthma. In an effort to develop a preventive IFN-gamma therapy against RSV infection, an intranasal gene transfer strategy was utilized. Intranasal administration of a plasmid expressing the IFN-gamma cDNA (pIFN-gamma) resulted in the expression of IFN-gamma in murine lungs and decreased RSV replication. The mice administered with pIFN-gamma and then infected with RSV exhibited a significant decrease in broncho-alveolar lavage lymphocyte and neutrophil counts. A significant reduction in epithelial cell damage, infiltration of mononuclear cells in the peribronchiolar and perivascular regions, and thickening of the septa was observed in the lungs of mice treated with pIFN-gamma when compared to controls. These results suggest that intranasal IFN-gamma gene transfer results in decreased RSV replication and pulmonary inflammation and may be useful against RSV infection.

Expression, purification and characterization of peanut (Arachis hypogaea) agglutinin (PNA) from baculovirus infected insect cells.

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Galbeta1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice.

The tubercle vaccine, bacille Calmette-Guérin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases. The effects of rBCG vaccination on allergic responses in a murine model were examined in this study. A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E. coli beta-galactosidase as the model antigen and allergen. This vector enabled the expression of the E. coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis. Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response. The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0. 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0. 01). Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls. These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response.

Respiratory syncytial virus induces the expression of 5-lipoxygenase and endothelin-1 in bronchial epithelial cells.

Respiratory syncytial virus (RSV) infection causes and exacerbates asthma, yet the mechanism by which RSV triggers asthma is poorly understood. Herein, an in vitro model of RSV infection was established using HEp-2 and BEAS-2B bronchial epithelial cell lines, and the expression of 5-lipoxygenase (5-LO), and endothelin-1 (ET-1) was examined. RSV infection increased the expression of 5-LO mRNA and protein in both cell lines, as detected by RT-PCR and western blot analysis, respectively. The levels of leukotrienes also increased in the supernatants of RSV infected cells. Furthermore, RSV infection increased the expression of ET-1 mRNA and protein following RSV infection in a time-dependent manner. It is concluded that RSV infection upregulates the expression of ET-1 and 5-LO in the epithelial cells leading to the production of leukotrienes, which may mediate the consequent exacerbation of asthma.

Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli.

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception. Studies on this potential use can be facilitated by the availability of recombinant proteins. A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe. Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1. The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1). An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction. The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector. Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009[pREP4] and ompT and Ion protease-deficient BL21 (plysS). SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments. Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA. Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus.

Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of S1NPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of beta-galactosidase (beta Gal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest beta Gal activity was obtained with S1AcNPV4.lacZ. Production of beta Gal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.

Characterization of the polyhedrin gene of Spodoptera litura nuclear polyhedrosis virus.

The polyhedrin gene (polh) of a characteristically distinct Spodoptera litura nuclear polyhedrosis virus isolate (SIMNPV) is identified in the HindIII-F fragment of the viral DNA. The nucleotide sequence of the 1057 base pair (bp) region of this fragment contains an open reading frame (ORF) without any intervening sequence for coding a polypeptide of 246 amino acids. Analysis of the nucleotide sequence and deduced amino acid sequence indicate that this has more than 70% sequence identity to known polyhedrins. The coding region is preceded by an AT rich region containing the conserved late promoter motif TAAG. The upstream promoter and coding regions of this polh gene are more similar to polh of the NPVs of Spodoptera frugiperda (Sf), Spodoptera exigua (Se) and Panolis flamea (Pf).

N-glycosylated polypeptides synthesized in the early phase of AcNPV infected insect cells.

Infected cell specific polypeptides (ICSP) in Autographa californica nuclear polyhedrosis virus (AcNPV) infected Spodoptera frugiperda cell line, Sf9, were radiolabelled with [14C] mannose and the labelled proteins were monitored at various time intervals. A major glycoprotein of molecular size of 64 kDa appears as early as 3h post infection (p.i.) which peaks at 12h p.i., but in presence of tunicamycin this protein is not labelled. Two other glycoproteins of molecular size 67 kDa and 63 kDa appear at 12h post infection. The glycosylation of these and eight other cellular proteins is also inhibited by tunicamycin. AcNPV propagated in tunicamycin treated Sf9 cells was found to cause delayed infectivity.

A 39,X/40,XY/41,XYY mosaic male mouse.

Cytogenetic analyses of bone marrow and gonadal cells in a male mouse, which appeared to be normal, revealed mosaicism in both tissues. Three chromosome complements, 39,X, 40,XY, and 41,XYY, were found in both bone marrow and spermatogonia, while only the last two complements were found in spermatocytes. In this mouse, unlike in the human, the XYY cells showed a proliferative advantage over the XY cells. In XYY cells at diakinesis/metaphase I the gonosomes showed all possible types of association, and a pairing advantage of the X chromosome was clearly demonstrated. The fertility of the mouse was not determined. However, since the epididymal sperm count was reduced by only 55% and the incidence of sperm head abnormality was near normal, it is not evident that the mouse was sterile.