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We investigate the scattering off three nonoverlapping disks equidistantly spaced along a line in the two-dimensional plane with the radii of the outer disks equal and the radius of the inner disk varied. This system is a two-dimensional scattering analog to the double-well-potential (bound state) problem in one dimension. In both systems the symmetry splittings between symmetric and antisymmetric states or resonances, respectively, have to be traced back to tunneling effects, as semiclassically the geometrical periodic orbits have no contact with the vertical symmetry axis. We construct the leading semiclassical ``creeping'' orbits that are responsible for the symmetry splitting of the resonances in this system. The collinear three-disk-system is not only one of the simplest but also one of the most effective systems for detecting creeping phenomena. While in symmetrically placed n-disk systems creeping corrections affect the subleading resonances, they here alone determine the symmetry splitting of the 3-disk resonances in the semiclassical calculation. It should therefore be considered as a paradigm for the study of creeping effects. PACS numbers: 03.65.Sq, 03.20.+i, 05.45.+b, replaced with published version (minor misprints corrected and references updated); 23 pages, LaTeX plus 8 Postscript figures, uses epsfig.sty, espf.sty, and epsf.tex

A quantum generalization of the semiclassical theory of Gutzwiller is given. The new formulation leads to systematic orbit-by-orbit inclusion of higher $\ensuremath{\Elzxh}$ contributions to the spectral determinant. We apply the theory to billiard systems, and compare the periodic orbit quantization including the first $\ensuremath{\Elzxh}$ contribution to the exact quantum mechanical results.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

The bacterium Neisseria meningitidis of serogroups A and W-135 has in the recent decade caused most of the cases of meningococcal meningitis in the African meningitis belt, and there is currently no efficient and affordable vaccine available demonstrated to protect against both these serogroups. Previously, deoxycholate-extracted outer membrane vesicle (OMV) vaccines against serogroup B meningococci have been shown to be safe and induce protection in humans in clonal outbreaks. The serogroup A and W-135 strains isolated from meningitis belt epidemics demonstrate strikingly limited variation in major surface-exposed protein structures. We have here investigated whether the OMV vaccine strategy also can be applied to prevent both serogroups A and W-135 meningococcal disease. A novel vaccine combining OMV extracted from recent African serogroup A and W-135 strains and adsorbed to aluminium hydroxide was developed and its antigenic characteristics and immunogenicity were studied in mice. The specificity of the antibody responses was analysed by immunoblotting and serum bactericidal activity (SBA) assays. Moreover, the bivalent A+W-135 vaccine was compared with monovalent A and W-135 OMV vaccines. The bivalent OMV vaccine was able to induce similar SBA titres as the monovalent A or W-135 OMV towards both serogroups. High SBA titres were also observed against a meningococcal serogroup C strain. These results show that subcapsular antigens may be of importance when developing broadly protective and affordable vaccines for the meningitis belt.

A previous report of a large, double blind, efficacy trial of an experimental Group B meningococcal outer membrane protein vaccine carried out in Norwegian Teenagers, showed a protection rate of 57%. Previous studies had demonstrated the occurrence of mutations in the class-1 outer membrane protein which alter its immunological properties. The occurrence of new mutations might compromise the efficacy of a vaccine and explain the occurrence of any vaccine failures. The porA gene, which encodes expression of the class 1 protein, was sequenced in all isolates from vaccine failures and compared to that of the vaccinating strain H44/76 (B:15:P1.7,16). The porA DNA and deduced amino acid sequences were all identical to that of the vaccinating strain except for that of one isolate which had a sequence identical to strains previously reported in Norway and England with a 'masked P1.7' epitope. The absence of new mutations in the trial was encouraging for the further development of outer membrane protein vaccines.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Alternative strategies exist for prevention of group B Neisseria meningitidis (meningococcal) disease through vaccination (see Chapters 5 , 8 , 13 , 14 in this volume). However, the most promising approach to date has been the use of outer-membrane vesicle (OMV) vaccines for induction of bactericidal antibodies against cell-surface outer-membrane proteins (OMPs).

In the absence of an affordable conjugate meningococcal vaccine, mass vaccination campaigns with polysaccharide vaccines are the means to control meningitis epidemics in sub-Saharan Africa. Facing global vaccine shortage, the use of reduced doses, which have been shown to be protective by serum bactericidal activity, can save many lives. In this study, we investigated the antibody responses and avidity of IgG antibodies evoked against the serogroup A capsule of Neisseria meningitidis by different doses of an A/C/Y/W135 polysaccharide vaccine. Volunteers in Uganda were vaccinated with 1/10, 1/5 or a full dose (50 μg) and revaccinated with a full dose after 1 year. Specific IgG geometric mean concentrations and geometric mean avidity indices (GMAI) were determined by a modified enzyme-linked immunosorbent assay (ELISA) using thiocyanate as a chaotropic agent. After vaccination with 1/10 or 1/5 doses, the GMAI increased from 1 month to 1 year. One year following the initial dose, the GMAI levels were higher in the arm receiving reduced doses than for the arm receiving a full dose. Following the second full dose, avidity indices equalized at approximately the same level in the three arms. Although there are practical challenges to the use of reduced doses in the field, our findings suggest that reduced doses of polysaccharide vaccine are able to elicit antibodies of as good avidity against serogroup A polysaccharide as a full dose.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

Chrysanthemum inducum-hybrid 'Coral Charm', Hibiscus rosa-sinensis L. 'Cairo Red' and Spathiphyllum wallisii Regel 'Petit' were grown in natural light in a greenhouse at three levels of irradiance using permanent shade screens. Light acclimation of photosynthesis was characterized using modulated chlorophyll a fluorescence of intact leaves. A close correlation was found between the degree of reduction of the primary electron acceptor Q(A) of Photosystem II (PS II) approximated as the fluorescence parameter 1-q(P), and light acclimation. The action range of 1-q(P) was 0-0.4 from darkness to full irradiance around noon, within the respective light treatments in the greenhouse, indicating that most PS II reaction centres were kept open. In general, the index for electron transport (ETR) measured by chlorophyll fluorescence was higher for high-light (HL) than intermediate-(IL) and low-light (LL) grown plants. However, HL Chrysanthemum showed 40% higher ETR than HL Hibiscus at light saturation, despite identical redox states of Q(A). The light acclimation of the non-radiative dissipation of excess energy in the antenna, NPQ, varied considerably between the species. However, when normalized against q(P), a strong negative correlation was found between thermal dissipation and ETR measured by chlorophyll fluorescence. To be able to accommodate a high flux of electrons through PS II, the plants with the highest light-saturated ETR had the lowest NPQ/q(P). The possibility of using chlorophyll fluorescence for quantification of the energy balance between energy input and utilization in PS II in intact leaves is discussed.

Chlorophyll fluorescence research began in the early 1930s with Kautsky (Kautsky and Hirsch, 1931) who was trained as a physical chemist and stumbled upon the phenomenon by accident. The main paradigm in those days was that the chlorophyll molecules alone bound CO2, absorbed photon energy and transferred that energy to the carbon atom, releasing oxygen to produce carbohydrates. When Kautsky presented his work in 1931 and stated that there was hardly any change in chlorophyll fluorescence yield when going from a CO2-containing atmosphere to a CO2-free atmosphere, it was believed that his equipment had failed. It was thought impossible, if he really was measuring chlorophyll fluorescence, that it would be unperturbed by the presence or absence of CO2. Although he was not a plant physiologist, Kautsky suggested that the light-dependent reactions and carbon fixation could be spatially separated processes in photosynthesis. It was not until the 1950s with the work of Rabinowitch (1951), Rabinowitch (1956) and Duysens (Duysens et al., 1961; Duysens and Sweers, 1963) that the idea of separate light and dark reactions was accepted.

The infant rat infection model previously developed to evaluate protective ability of passively administered murine antibodies to group B meningococcal (MenB) surface antigens was adapted for human sera. Several challenge doses were tested, aiming at sensitive detection of protection with little interassay variability. Doses of 10(5) and 10(6) colony forming units of strain IH5341 (MenB:15:P1.7,16) injected intraperitoneally gave consistently high levels of bacteremia and meningitis developed in 6 h in 50-100% of the pups. A monoclonal antibody mAb735 to the MenB capsule, injected 1-2 h before bacterial challenge, gave full protection at a dose of 2 microg/pup. Sera from adult volunteers immunized with a MenB outer membrane vesicle vaccine reproducibly reduced bacterial counts in the blood and cerebrospinal fluid, whereas a normal human serum, lacking bactericidal and opsonophagocidal activity, was unprotective.

Three different formulations of an outer membrane vesicle (OMV) vaccine against group B meningococcal disease have been prepared and tested for immunogenicity and reactogenicity in adult volunteers. The vaccines were prepared with or without aluminium hydroxide and serogroup C-polysaccharide (C-ps). Doses from 12.5 to 100 micrograms protein were given twice at a six weeks' interval. All three formulations were well tolerated and highly immunogenic, inducing bactericidal and opsonizing antibodies in humans. Adsorption of OMVs to aluminium hydroxide reduced the pyrogenicity in rabbits. The differences in immunogenicity between the formulations were relatively small, but after the second dose a stronger booster response was observed when the vaccines were adsorbed. Thus, a formulation with OMVs and C-ps represents a safe and highly immunogenic vaccine, even without aluminium hydroxide.

The diffraction trace formula ({\em Phys. Rev. Lett.} {\bf 73}, 2304 (1994)) and spectral determinant are tested on the open three disk scattering system. The system contains a generic and exponentially growing number of diffraction periodic orbits. In spite of this it is shown that even the scattering resonances with large imaginary part can be reproduced semiclassicaly. The non-trivial interplay of the diffraction periodic orbits with the usual geometrical orbits produces the fine structure of the complicated spectrum of scattering resonances, which are beyond the resolution of the conventional periodic orbit theory., Comment: Latex article + 3 ps figures

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

An extension of the Gutzwiller trace formula is given that includes diffraction effects due to hard wall scatterers or other singularities. The new trace formula involves periodic orbits which have arcs on the surface of singularity and which correspond to creping waves. A new family of resonances in the two disk scattering system can be well described which is completely missing if only the traditional periodic orbits are used., Comment: 4 pages, latex/REVTEX, 2 PS figures

Based on differences in reaction pattern with monoclonal antibodies against the P1.16 epitope, a new variant of the class 1 protein in Neisseria meningitidis serogroup B was identified in Norway. A single amino acid deletion was revealed when the part of the gene region encoding the second variable region of the protein was sequenced. This new variant was designated P1.16c. About 5% of the B:15:P1.7,16 strains in Norway from the time period 1987-1991 were P1.16c. In a localized area in Southern Norway, 5/8 (62%) of the P1.7,16 strains were P1.16c. The P1.16b mutant, recently described in England, was not found among the Norwegian meningococcal isolates. Strains carrying the P1.16c mutation showed increased resistance to bactericidal killing, not only by P1.16-specific monoclonal antibodies, but also by the sera from individuals immunized with a vaccine based on outer membranes from a B:15:P1.7,16 strain.

We investigate a new type of approximation to quantum determinants, the ``\qFd", and test numerically the conjecture that for Axiom A hyperbolic flows such determinants have a larger domain of analyticity and better convergence than the \qS s derived from the \Gt. The conjecture is supported by numerical investigations of the 3-disk repeller, a normal-form model of a flow, and a model 2-$d$ map., Revtex, Ask for figures from vattay@nbivax.nbi.dk

Results from the serum bactericidal assay (BA) after immunization of human volunteers with the Norwegian serogroup B meningococcal outer membrane vesicle vaccine are surveyed. In the phase II trials with adults we found very high seroconversion rates (greater than 98%) against the vaccine strain in the BA. Details in the antigenic composition of the inoculum used in the BA seem very important as shown here by finding lower bactericidal titres with teenager sera when tested with a variant of the standard inoculum. The present preliminary report corresponds to the presentation given at the Report Meeting on the Norwegian Meningococcal Vaccine Trial, Oslo, 12 September, 1991.

A survey is given of the efforts made to inform the general public, the potential vaccinees and their parents, and the health care personnel about meningococcal disease in general and the vaccination trial in particular, as a preparation for the meningococcal outer membrane vesicle serogroup B vaccine (MenB-vaccine "Folkehelsa") trials in secondary school students and military conscripts in Norway. Our case reporting system, supplementing the official notification, concerning even vaguely suspected cases in the age cohorts involved, is described. The efforts made to collect clinical material as well as laboratory and clinical data from 221 registered suspected cases are delineated. We also briefly summarize our cerebrospinal fluid antigen detection methods and diagnostic meningococcal serology work on these suspected cases. The compiled information on findings done at the admitting hospital of the possible cases and the additional diagnostic data provided at the National Institute of Public Health were put at the disposal of the independent Diagnosis Review Committee (DRC) as a basis for their diagnostic decisions before code opening for the meningococcal serogroup B outer membrane vesicle vaccine protection trial 3 June 1991.

Antibody responses after vaccination with three different formulations of a new meningococcal group B outer membrane vesicle (OMV) vaccine have been studied with the ELISA technique using four different antigens. Sera from about 1200 vaccinees participating in steps 1, 2, 3 and 6 of the phase II clinical trials in Norway were analysed. The effects of non-covalently complexing the OMV antigen to group C polysaccharide (C-PS) and of adsorbing OMV (with and without C-PS) to aluminium hydroxide (AH) were studied. All three vaccine formulations were highly immunogenic in humans. Adsorption of the vaccine to AH had a relatively small effect on the immune response, but the results indicated that the booster response was stronger with the adsorbed than with the unadsorbed vaccines. Some increase in the immune response against OMV was also observed by non-covalent complexing OMV with C-PS, particularly after the second dose. In most of the vaccinees the antibody levels were significantly reduced 6 to 12 months after vaccination. Adsorption of the vaccine to AH had no effect on the antibody response against C-PS. Comparison with bactericidal activity of the same sera was done. A highly significant correlation was observed between the bactericidal titres and the levels of IgG antibodies against OMV and class 5C protein, whereas the correlation between antibody levels against lipopolysaccharide and the bactericidal activity was poor.

For more than 15 years, Norway has had the highest incidence of meningococcal disease in northern Europe, with 80% of cases being due to serogroup B meningococci. The case-fatality has remained high, at about 10%. In this study, an outer membrane vaccine, which had previously been shown to induce an increase in bactericidal antibodies to the parent strain, was assessed in a large-scale, randomised, double-blind trial. From October, 1988, 171,800 students in secondary schools volunteered to take part in a double-blind, placebo-controlled, efficacy trial with school as the randomisation unit. Hospitals and clinics that routinely receive patients with infectious disease were asked to report urgently all cases of suspected meningitis and/or septicaemia in 13-21-year-old students in Norway. These cases were registered and further investigated according to a detailed protocol. 89 out of the 221 cases investigated by June 3, 1991, were shown to be severe systemic disease due to group B meningococci. 36 cases in 35 schools took part in the trial (11 schools with vaccinated students and 24 with students given placebo). The calculated rate of protection was thus 57.2% (p = 0.012, one-sided test). The findings suggest that, although the vaccine conferred protection against group B meningococcal disease, the effect was insufficient to justify a public vaccination programme.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

Quasielastic light scattering experiments have been performed to study the aggregation kinetics of human immunoglobulins IgG. Rapid aggregation and irreversible cluster growth results when IgG solutions (2–15 mg/ml) are heated above 50 °C. The measured scattering intensity I and effective hydrodynamic radius R can be described consistently by a Smoluchowski aggregation process. The number of clusters ni(t) containing i monomers at time t are computed. The radius of an i cluster is assumed to be Ri =R0iβ where β is the cluster exponent. This kinetic process results in the following characteristic power law behavior: R/R0={1+ΓR(T,C,c)⋅t}αR and I/I0={1+ΓI(T,C,c)⋅t}αI. Here R0=5.51 nm, is the monomer radius and the I0 the scattered intensity from the monomer solution at temperature T, and concentration C. A fraction c∼0.48 of the monomers are heat stable and do not participate in the aggregation process. The power law behavior of R/R0 and I/I0 indicates scaling, and indeed a very satisfactory data col...

The crystal and molecular structure of the title compound C-22H-30O-4, has been determined by X-ray methods using 1106 reflections above background level collected by counter methods. The crystals are orthorhombic, space group P2-12-12-1 with cell dimensions a equals 16.514(2) A; b equals 14.324(2) A; c equals 8.744(1) A; there are four molecules per unit cell. The structure was refined to an R of 0.084 (weighted R-w equals 0.068). The cyclohexene and the pyran part of the molecule occurs in the half-chair conformation. The bond distances and angles, and a slight twist of the benzene ring, indicate considerable stains in the aromatic system. Both the phenolic and carboxylic group are significantly out of the plane through the aromatig ring. The angle between this plane and a plane through the cyclohexene ring is 37.7 degrees. The pentyl sidechain occurs in an extended gauche conformation, and the thermal parameters of this part of the molecule are very high. The molecules are held together by van der Waals forces in the c-directions, and hydrogen bonds (2.688 A) from phenolic to carboxylic groups in the a-b plane. There is a short ultra-molecular hydrogen bond (2.490 A) from the carboxylic group to the pyran oxygen.

A modified and improved spin membrane immunoassay has been developed for detecting complement-activating antibodies to Neisseria meningitidis capsular polysaccharide antigens. The polysaccharides were incorporated in the membranes of large unilamellar vesicles prepared by the reverse-phase evaporation method and filled with the water-soluble spin label tempocholine chloride. Upon addition of group-specific antisera and complement, the lipid membrane was damaged and the spin label leaked out. This process was monitored by electron spin resonance spectroscopy. A satisfactory assay was developed for polysaccharides of group A and C, whereas in the case of the B system the assay was more labile. The method is rapid and has a sensitivity comparable to that of radioimmunoassay. When studying paired sera from five recruits vaccinated with an A + C polysaccharide vaccine, significant rises in titers to both A and C polysaccharides were observed in all the postvaccination sera.

The antibody response of a group of adult volunteers given a combined meningococcal group B polysaccharide and serotype 2 outer membrane protein vaccine, has been studied by the ELISA technique. The antigen was an outer membrane preparation from a non-capsular strain of Neisseria meningitidis (the vaccine strain). The vaccination was performed as a double-blind experiment where one group of 27 persons was given the vaccine and a similar group of 28 persons was given a placebo. In addition, five volunteers from the laboratory staff were given the vaccine. Two weeks after the primary vaccination, 31 of the 32 vaccinated persons demonstrated a significant increase of specific IgG antibodies. The number with significant IgA and IgM increase was 21 and 12, respectively. A booster effect after revaccination four weeks later was found in 18 persons for IgG, in 10 for IgA and in one for IgM. Twenty-five weeks after the primary vaccination the ELISA values were significantly reduced, mostly for IgM antibodies. The mean values for IgG, IgM and IgA were then 150%, 130% and 110%, respectively, of the values before vaccination. A new way of analysing the data has also been tried for IgG determination. Instead of comparing OD values, we calculate the expression: B = D/2 . In(1 + OD/A)/(1-OD/A), where A is an experimental constant and D is the serum dilution. B then becomes linearly proportional to the antibody concentration. This way of expressing the results shows the geometric mean IgG titer 25 weeks after vaccination to be three times higher for the vaccinated than for the placebo group.

The thermal stability of immunoglobins is a critical parameter both to experimental immunologists and clinicians responsible for administration of antibody preparations. In diagnostic immunology heating of serum at 56C for 30 minutes has become a well established method of inactivating complement in immunological assays. This procedure usually does not influence the antibody activity or the other main biological properties of the immunoglobins. In contrast, heating of IgG at 63°C for 30 minutes is a widely used method to produce soluble IgG aggregates. Such aggregates possess many biological properties which make them suitable for use as controls in studies concerning soluble antigen-antibody complexes. 1,2 The structure of the heat aggregates and the mechanism by which they are formed has been studied by several methods. For the analysis of the hydrodynamic properties, ultracentrifugation and &elfiltration have been the most used techniques,3,4,5 and for the study of the biological properties of the soluble aggregates, complement fixation methods has been shown to be very sensitive.6 By dynamic light scattering we measure the diffusion coefficients of the components in a solution of IgG. In this way we can follow the process of aggregation under different conditions.

A vaccine against serogroup B meningococcal disease has been prepared from a B:15:P1.7,16 meningococcal strain (44/76) by fermentor growth and extraction of the bacteria with the detergent deoxycholate. Outer membrane vesicles (OMV) were purified by ultracentrifugation and adsorbed to aluminium hydroxide adjuvant. OMV contained the major class 1, 3, 4 and 5 proteins and some minor high molecular weight protein components. Relative to protein, the vaccine also contained about 8% phospholipid, 7% lipopolysaccharide and 16% deoxycholate. The product was generally non-pyrogenic to humans in ordinary doses and was highly immunogenic in mice and humans. Production and control steps, physical, chemical and immunological data for the vaccine are described.