Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity.

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Generation and characterization of reassortant influenza A viruses propagated in serum-free cultured MDCK-SF1 cells.

The replacement of embryonated chicken eggs by tissue culture cells for the production of influenza vaccines is likely to take place in the near future. Vaccines have already been produced in Madin Darby Canine Kidney (MDCK) cells (Brands et al, in this issue) and extensively tested in phase III trials in humans (Palache et al, in this issue) and it seems a matter of time before such vaccines will become available. For this reason, the generation of high-growth reassortants of influenza A virus strains in MDCK cells has been examined. Influenza A virus reassortants of the field strains A/Taiwan/1/86, A/Johannesburg/82/96 and A/Shenzhen/227/95 (all H1N1) were generated in serum-free cultured MDCK-SF1 cells by dual infection with A/Hong Kong/2/68 (H3N2), a strain selected for its high-growth phenotype. These reassortant viruses all contained at least the matrix gene of A/Hong Kong/2/68 which apparently correlates with an improvement of the viral yield.

Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine.

Influenza vaccine production technology based on large scale cell culture technology has been developed. From the characterization of the continuous cell line MDCK as well as drug safety studies we conclude that this cell line and the cell culture system are suitable for biological production. The Down Stream Process (DSP) of the virus-containing harvest fluids guarantees sufficient inactivation of influenza viruses and adequate removal or inactivation of putative adventitious or endogenous viruses, mycoplasma or bacteria. Our data indicate that the tissue culture-based production technology is feasible.

Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium.

In the present study reassortant influenza A viruses of both the H1N1 and H3N2 type were generated in Madin Darby Canine Kidney cells grown in the absence of fetal bovine serum (MDCK-SF1 cells). To this end, MDCK-SF1 cells were simultaneously infected with one of the high-growth laboratory strains A/Puerto Rico/8/34 (H1N1) or A/Hong Kong/2/68 (H3N2) and recent H3N2 and H1N1 vaccine strains, respectively. Reassortant viruses obtained from these mixed infections were genetically characterized by RT-PCR and restriction enzyme analysis and their growth properties were compared to those of the corresponding field strains. Reassortant H3N2 viruses inherited the matrix and polymerase pa gene whilst H1N1 reassortant viruses inherited the matrix and polymerase pbl gene of the high-growth parent. Reassortant viruses generally gave higher viral yields, as measured by a haemagglutination assay, than their wild type counterparts. The procedure followed results in the generation of high-growth reassortant viruses in weeks. The use of MDCK-SF1 cells together with these reassortants for generating influenza virus antigens can significantly speed up the vaccine production procedure.

Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen.

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines. However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity, and a pentamer of identical B subunits, which mediates binding to ganglioside GM1, the cellular receptor for the toxin. In this paper, we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself, retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). Immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However, supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT, rLTB in particular, represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases.

Immunogenicity and reactogenicity of influenza subunit vaccines produced in MDCK cells or fertilized chicken eggs.

A tissue culture method using MDCK cells grown under serum-free conditions was developed to produce an inactivated influenza subunit vaccine. The first clinical data suggest it to be equal to the conventional egg-derived influenza subunit vaccine. In a double-blind controlled trial, 2 groups (n = 57 each) of adult volunteers were immunized with experimental bivalent influenza subunit vaccine derived from either MDCK cells or hens' eggs. Each vaccine contained 15 microg of hemagglutinin of influenza A/Taiwan/1/186 (H1N1) and 15 microg of hemagglutinin of B/Panama/45/90. No clinically relevant adverse reactions were observed in either vaccine group, and the incidence of systemic and local vaccine reactions was comparable in both groups. Standard hemagglutination inhibition antibody titers were determined using both MDCK- and egg-derived test antigens. The data reveal that both vaccines are safe and well-tolerated and meet the criteria for immunogenicity as stated in the European Community's "Harmonisation of Requirements for Influenza Vaccines."

Mucosal immunoadjuvant activity of liposomes: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with an influenza subunit vaccine and coadministered liposomes.

This paper reports on a novel immunoadjuvant activity of liposomes. An influenza subunit preparation, containing the isolated viral surface antigens, was incorporated in a liposomal formulation. Administration of this vaccine to mice via the intranasal (i.n.) route resulted in a stimulated serum IgG response relative to the response to i.n. immunization with the antigen alone. In addition, the liposomal vaccine induced a secretory IgA (sIgA) response in the mucosa of the lungs and nasal cavity. Both serum IgG and sIgA responses persisted up to at least 21 weeks postimmunization. Immune stimulation was observed with negatively charged liposomes consisting of phosphatidylcholine (PC), cholesterol and dicetylphosphate (DCP), but not with zwitterionic liposomes, consisting of PC and cholesterol alone. Remarkably, for stimulation of serum IgG responses and induction of an sIgA response, liposomes could be simply mixed with the antigen. Moreover, i.n. administration of empty liposomes up to 48 h prior to i.n. immunization with the subunit antigen also resulted in immune stimulation, indicating that the liposomes did not exert their adjuvant effect by acting as a carrier for the antigen. The liposomal vaccine conferred protection against infection. It is concluded that liposomes, administered i.n., provide a promising adjuvant system for stimulation of antibody responses in general, and mucosal sIgA responses in particular.

Effect of selective chemical modification and CNBr-cleavage at methionine20 in catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met20. It was found that porcine pancreatic iso-phospholipase A2, possessing only Met8, was not affected by either modification. Reaction of porcine phospholipase A2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met20. The enhanced reactivity of Met20 compared to that of Met8 is in agreement with the known X-ray structure of phospholipase A2 which shows that Met8 is located in the interior of the protein, while Met20 is at the surface. Both methylation and carboxymethylation of Met20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met20 perturbs the active site despite the fact that the N-terminal fragment Ala1-Hse20 is still bound via the disulfide bridge Cys11-Cys77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala1-Hse20 and/or Asp21-Gly26 are important for the maintenance of the special microenvironment of the active site cleft.

Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE.

The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.

Interaction of pancreatic phospholipases A2 and semisynthetic mutants with anionic substrates and substrate analogues.

Recently it was shown that porcine pancreatic phospholipase A2 aggregates in the presence of submicellar concentrations of anionic substrates [van Oort, M.G., Dijkman, R., Hille, J.D.R., & de Haas, G.H. (1985) Biochemistry 24, 7993-7999]. In the resulting complexes the enzyme displays very high catalytic activity. In this study the interaction process was further investigated by using pancreatic phospholipases A2 of different origins and several semisynthetic mutants in which one particular amino acid residue was substituted. By use of directing binding studies with a nondegradable anionic substrate analogue and monomolecular surface film kinetics on 1,2-didecanoyl-sn-glycerol 3-sulfate, it is shown that the aggregation process is controlled by the ionization state of the side chains of the amino acid residues at positions 6 and 17.

The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains.

The amino acid sequence of the gene for the peplomer protein of the vaccine strain M41 and the Beaudette laboratory strain M42-Salk of avian infectious bronchitis virus (IBV) have been derived from cDNA sequences. As found with other coronaviruses, the peplomer protein carries the epitopes eliciting neutralizing antibodies. The gene encodes a primary translation product of 1162 amino acids with a molecular weight of 128,079. The use of a recent algorithm to predict membrane-protein interactions led to the unambiguous localization of the signal peptide and a transmembrane anchor alpha-helix at the C-terminus. At 50 positions amino acid differences were found between M41 and two Beaudette strains (M42-Salk and M42-Houghton). They are partly clustered in two regions of the protein. These two regions are candidates for neutralization epitopes of the protein.

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids.

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.

Side-chain dynamics of two aromatic amino acids in pancreatic phospholipase A2 as studied by deuterium nuclear magnetic resonance.

The flexibility of individual amino acid side chains of pancreatic phospholipase A2 in aqueous and micellar solutions was studied with deuterium nuclear magnetic resonance (2H NMR). Bovine pancreatic phospholipase A2 was selectively deuterated at the aromatic ring systems of Trp-3 and Phe-5 and porcine pancreatic phospholipase A2 at Trp-3 only. Solid-state 2H NMR spectra of the lyophilized enzymes exhibited quadrupole splittings on the order of 130 kHz, indicating almost complete immobilization of the aromatic ring systems. Exposure to a water-saturated atmosphere did not remove these steric constraints. However, side-chain mobility could be induced for the tryptophyl residue of the bovine enzyme by dissolving this enzyme in aqueous buffer or micellar solution whereas the phenyl ring always remained immobile and served as a probe for the protein's overall rotation. Typical correlation times for the tryptophyl and phenyl aromatic ring systems in aqueous solution were 7 ps and 13 ns (at 20 degrees C), respectively. The correlation time of the phenyl ring was longer than expected for the monomeric protein (approximately 6 ns), suggesting some aggregation of the protein at the high concentrations used for the NMR measurements. Addition of a micellar solution of oleoylphosphocholine had no influence on the motional freedom of the tryptophyl residue but approximately doubled the correlation time of the phenyl ring, indicating an increase of the effective volume of the tumbling particle due to lipid-protein interaction. A different behavior was observed for the Trp-3 residue of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study.

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.

Structural importance of the amino-terminal residue of pancreatic phospholipase A2.

To study the structural importance of the NH2-terminal Ala1 residue of pancreatic phospholipase A2, several mutants were prepared by a stepwise semisynthetic approach. 13C NMR spectroscopy of 90%-enriched [[3-13C]Ala1] phospholipases A2 shows the pK values of the alpha-NH3+ groups of porcine enzyme, porcine isoenzyme, bovine enzyme, and equine enzyme to be 8.4, 8.8, 8.9, and 8.8, respectively. A group titrating with a pK of approximately 6.3, present only in the porcine and equine phospholipases A2, presumably originating from Glu71, disappears in the presence of 0.2 M Ca2+, while the pK values of their alpha-NH3+ groups shift to 9.3 and 9.0, respectively. No such effects were observed for the porcine isoenzyme and bovine enzyme, which lack an acidic side chain in position 71. It can thus be concluded that the equine phospholipase A2, like the porcine enzyme, possesses in addition to the catalytic Ca2+ binding site also a second, low-affinity Ca2+ binding site, which is not present in the porcine isophospholipase A2 and bovine phospholipase A2. From the titration behavior of the alpha-NH3+ group of equine and porcine [[3-13C]-Ala1]phospholipases A2 in the presence of micelles of n-hexadecylphosphocholine, it seems very likely that at alkaline pH the equine phospholipase A2, like the porcine enzyme, requires the second Ca2+ ion for optimal binding to neutral lipid-water interfaces. Semisynthetic porcine phospholipase A2 analogues in which the position of the alpha-NH3+ group is varied have lost their affinity toward neutral lipid-water interfaces and consequently their catalytic activity on micellar substrates. Most of these phospholipase A2 analogues retain, however, some of their enzymatic activity on monomeric substrate. Substitution of the side chain of Ala1 in porcine phospholipase A2 by hydrophobic side chains abolishes almost all activity due to the loss of affinity for neutral lipid-water interfaces. In contrast, NH2-terminal residues having more polar side chains affect only slightly phospholipase A2 activity. Compared to the native enzyme, these latter phospholipase A2 analogues show an increased penetration capacity for monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Probably, the interaction of hydrophobic amino acid residues at the 1-position with other hydrophobic side chains present in their vicinity prevents the correct positioning of the alpha-NH3+ group, thereby leading to loss of catalytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Semisynthesis of phospholipase A2. The effect of substitution of amino-acid residues at positions 6 and 7 in bovine and porcine pancreatic phospholipases A2 on catalytic and substrate-binding properties.

The N-terminal alpha-helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg-Ser at positions 6 and 7, whereas the bovine enzyme has Asn-Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of epsilon-amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6-Gly7]AMPA and bovine [Ser7]AMPA and [Arg6-Ser7]AMPA. As we had previously found for the Asn6 leads to Arg bovine substitution, an Asn6-Gly7 leads to Arg5-Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid-water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7 leads to Ser produces almost no effect on these properties. Ser7 leads to Ala and Ser7 leads to Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6 leads to Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6 leads to Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid-water interfaces of Asn6 leads to Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6 leads to Asn substitution in porcine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7 leads to Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.