Long-term immunogenicity of an inactivated virosome hepatitis A vaccine.

The aim of this study was to predict the long-term protection induced after immunisation with inactivated, aluminium-free virosome hepatitis A vaccine. The study population consisted of adult volunteers enrolled in four different clinical trials. Lower 95% confidence interval limits and seroconversion rate were calculated by using a linear mixed model to estimate the persistence of serum antibodies over time. To assess the robustness of the mathematical model, several sensitivity analyses were performed with more conservative protective threshold (20 mIU/ml vs. 10 mIU/ml), higher yearly decline rate, and exclusion of volunteers who had increasing titres over time. Based on 190 volunteers with at least two valid assessments of titres from year 3 onward, the median duration of protection was 55.5 years, with a lower limit of the 95% CI of 48.7 years. Duration below 25.3 years was predicted for only 5% of the subjects. Women tended to have higher titres to start with, but their rate of decline was higher, resulting in similar duration of protection overall. The use of a more conservative threshold, higher yearly decline rate, and exclusion of volunteers with increasing titres over time did not affect these results. According to this model, 95% of the volunteers should have anti-HAV titres above the minimum protective threshold for 20 years or more following immunisation with two doses of this aluminium-free vaccine.

Perspectives: towards a peptide-based vaccine against hepatitis C virus.

Hepatitis C virus (HCV) is a widespread infectious disease in humans with the negative implication of becoming chronic in most persons. Patients infected with HCV are at risk of liver cirrhosis or hepatocellular carcinoma at later stages. In contrast to hepatitis A and hepatitis B, there is no immunization yet available, neither prophylactic nor therapeutic. Thus, there is an urgent need to develop a safe, protective vaccine against this fatal disease. Developing countries are even more at risk for HCV. There are currently a number of scientific approaches aimed towards solving this problem. Taking both risks and costs of immunization into consideration, a peptide-based vaccine may be a reasonable prophylactic protection. Also, it might be of therapeutic use in already infected patients by increasing a specific CTL response against HCV. In our lab, we are focusing on immunopotentiating reconstituted influenza virosomes (IRIVs) as carriers for immunogenic HLA-A2-restricted core epitopes to induce peptide-specific cytotoxic T lymphocytes (CTLs). The IRIVs are similar to liposomes, but in addition contain influenza-derived hemagglutinin and neuraminidase on their outer surface which makes them fusogenic, thus, permitting antigen delivery to host cells. So far, virosomes have been successfully used for vaccine development and as a result a virosomal vaccine against both influenza virus (Inflexal) BERNA) and hepatitis A virus (HAV) (Epaxal) BERNA) already exist on the market. This paper focuses on the importance of development of a successful vaccine against HCV and, more specifically, we discuss the use, advantages and disadvantages of a peptide-based vaccine. A brief report of our latest findings will be included.

Pre-clinical and clinical investigation of the safety of a novel adjuvant for intranasal immunization.

Nasalflu Berna is a trivalent influenza virus vaccine for active immunization against influenza by the nasal route. It consists of influenza virosomes which are formulated from inactivated influenza strains and heat-labile toxin from aseptic Escherichia coli bacteria strain, as an adjuvant (HLT). The results of preclinical studies in ferrets, baboons, minipigs, mice and rabbits are presented here, and issues concerning route of administration, mechanism of action (preventing the disease and halting further spread of the disease), and the specific safety issues of the adjuvant itself (possible neurological activity of HLT) are examined. No clinical signs were detected in the animals, and hematological values were in the normal range. In particular, there was no evidence of any systemic adverse reaction, including sensitization to the test substances, and no evidence of possible neurological activity of the HLT. Further clinical studies in humans conducted over five influenza seasons using this virosome-formulated intranasal vaccine, elicited high levels of influenza-specific hemagglutination inhibition IgG antibody titers to the strains incorporated in the administered vaccine. In addition, IgA antibodies were also elicited in the nasal mucosa, and in the saliva. In addition to the systemic IgG antibody titers, the nasal mucosal IgA antibody response may provide additional local protection by the inhibition of viral replication and further spread in the respiratory tract. Nasalflu was well tolerated by most of the vaccinated subjects, both in terms of nasal symptoms and possible vaccination-mediated systemic symptoms. Both local and systemic symptoms were primarily mild, with only an occasional subject reporting moderate intensity. Out of four serious adverse events seen during the clinical development, only one was thought to be remotely related to the test vaccine.Nasalflu, developed by the Swiss Serum and Vaccine Institute, is a novel, highly immunogenic and safe influenza subunit vaccine which is easily administered as a nasal spray. This new route of administration is likely to increase compliance to vaccination, and could become an important tool to promote vaccination in population groups which show high resistance to vaccination.

Review of intranasal influenza vaccine.

Influenza viruses cause costly recurrent annual epidemics. Current efforts to control the morbidity and mortality associated with influenza outbreaks are based on the use of annual intramuscularly administered inactivated vaccines which are not only painful to administer, but are suboptimal in efficacy. This paper reviews the composition, safety analysis, rates of adverse events, immune response and protective efficacy of a new inactivated intranasal influenza vaccine.

Tolerance and immunogenicity of the simultaneous administration of virosome hepatitis A and yellow fever vaccines.

BACKGROUND: The purpose of this study was to evaluate the tolerance and immunogenicity of a hepatitis A vaccine using immunopotentiating reconstituted influenza virosomes (IRIV) as adjuvant when administered simultaneously with a yellow fever vaccine (YFV). METHOD: An open prospective trial with two parallel groups was conducted with 105 volunteers to study the effect of these vaccinations on the anti-hepatitis A virus (HAV) antibody response. Half of the volunteers (53) received one dose of IRIV-HAV vaccine (Epaxal) and one dose of live attenuated YFV (Stamaril) on the same day at two different sites. Fifty-six volunteers were given a single injection of IRIV-HAV as a control group. Anti-HAV titers were measured at days 14, 28, months 3, 12, 13, and 24 using a standardized test (Enzymun test Anti-HAV). Neutralizing yellow fever antibodies were measured at days 14 and 28 for the YFV recipients. Regarding vaccine tolerance, the volunteers were asked to record all their adverse reactions on a standard report sheet for the 6 days following the immunization. RESULTS: Seroconversion rates for HAV were 88% after 14 days and 100% after 4 weeks. There was no statistically significant difference between the two groups every time the titers were checked (IRIV-HAV vs HAV only: D14: 81 vs 101; D28: 275 vs 368; M3: 153 vs 169; M12: 117 vs 226; geometrical mean titers (GMT) in mIU/mL). However, lower titers were found among male volunteers, and were not attributable to YFV administration. The seroconversion rates for YFV were 90% after 14 days and 96% after 4 weeks. No serious general side-effects and only mild local reactions were reported. The administration of a booster of IRIV-HAV at 12 months resulted in a 24-fold increase in GMT. CONCLUSION: When needed, the simultaneous administration of the IRIV-HAV and YFV is immunogenic, safe and well-tolerated, as volunteers seroconverted to both antigens, with no cross-interference.

Efficacy study of a new albumin-free human diploid cell rabies vaccine (Lyssavac-HDC, Berna) in 100 severely rabies-exposed Thai patients.

A newly developed human diploid cell rabies vaccine (Lyssavac-HDC), produced without added serum albumin and with an effort to remove the virus-inactivating beta-propriolactone prior to addition of the gelatin, L-cysteine and potassium phosphate stabilizer, was tested for safety immunogenicity, adverse reactions and efficacy in 100 severely rabies-exposed Thais. All patients also received human rabies immune globulin and vaccine was administered using the conventional 5-dose intramuscular schedule of one dose on days 0, 3, 7, 14 and 28. One hundred percent of a subgroup of 40 subjects, where blood had been collected, had neutralizing antibodies greater than 0.5 IU ml-1 on days 28 and 90 and all had detectable titers on days 7, 14, 28, 90, 180 and 360. All patients could be followed for at least 1 year and remained well. No significant side-effects from this vaccine were noted.

Immunogenicity of purified duck embryo rabies vaccine (Lyssavac-N) with use of the WHO-approved intradermal postexposure regimen.

The World Health Organization (WHO) has recommended the following regimen for administration of intradermal postexposure rabies vaccines (tissue or avian cultures): 0.1 mL of the vaccine given intradermally at two sites on days 0, 3, and 7 and at one site on days 28 and 90. WHO did not specify which types of vaccines should be used when following this regimen. We evaluated the efficacy of purified duck embryo rabies vaccine (Lyssavac-N) under the above regimen conditions; although purified duck embryo rabies vaccine is highly immunogenic when given intramuscularly, it differs significantly from the other rabies vaccination products in that it is not a solution, but rather, a suspension with an added preservative. Lyssavac-N was found to produce lower mean neutralizing antibody titers with the WHO-recommended intradermal regimen. However, when the volume of each dose was increased from 0.1 mL to 0.2 mL and the same schedule of administration was followed, satisfactory titers were obtained. We concluded that the WHO intradermal postexposure rabies vaccine regimen should only be used with vaccines that have been subjected to immunogenicity studies with satisfactory results.

Detection of remote myocardial infarction with quantitative real-time ultrasonic characterization.

We have previously shown that the intrinsic properties of myocardium can be characterized quantitatively by the assessment of ultrasonic integrated backscatter. In this study we utilized a novel, real-time, two-dimensional system capable of quantitative integrated backscatter imaging to determine whether zones of remote myocardial infarction in dogs could be delineated definitively by ultrasonic tissue characterization. Detection of such zones in patients is needed as a basis for management decisions related to thrombolysis, angioplasty, and coronary surgery. Integrated backscatter was measured through the closed chest from 25 myocardial sites. Zones of infarction exhibited time-averaged integrated backscatter values approximately 10 dB (9.5 +/- 0.5 dB, standard error of the mean) greater than those in normal regions (p less than 0.001). In addition, the physiologic cardiac cycle--dependent variation of integrated backscatter was blunted significantly in zones of infarction [0.8 dB +/- 0.3 vs. 3.8 +/- 0.6 (p less than 0.01) for normal regions]. Ultrasonic results matched the histopathologic features assessed directly. Thus quantitative ultrasonic tissue characterization can differentiate infarcted tissue from normal myocardium and offers promise for quantitative detection of histopathology in vivo.

Changes in ultrasonic attenuation and backscatter of muscle with state of contraction.

We have previously demonstrated that backscatter (uncompensated for attenuation) of canine myocardium varies systematically throughout the cardiac cycle and in relation to regional contractile performance. To elucidate these phenomena under conditions independent of blood flow and complex myofibrillar architecture, we measured attenuation with a phase-insensitive receiver and backscatter over a wide range of frequencies in an intermittently tetanized (10 stimulations), isolated frog gastrocnemius preparation (n = 12 muscles). Muscle contraction, as compared with relaxation, was associated with increased values of slope of attenuation (0.78 +/- 0.04 vs 0.58 +/- 0.03 dB/(cm MHz); p less than 0.001) and increased values of integrated backscatter (-29.5 +/- 0.9 vs -35.5 +/- 0.8 dB; p less than 0.005). Differences in attenuation and backscatter diminished with the number of muscle stimulations (as the muscle fatigued). Thus, quantitative ultrasonic indices of skeletal muscle vary systematically with the contractile performance of the tissue. Extrapolation of these results to cardiac muscle suggests that the sensitivity of these indices to contractile function of muscle may provide an approach for noninvasive assessment of intrinsic properties of myocardium that determines its performance.

Effects of coronary artery occlusion and reperfusion on cardiac cycle-dependent variation of myocardial ultrasonic backscatter.

We have recently reported a systematic variation in integrated ultrasonic backscatter throughout the cardiac cycle in canine hearts. This study was performed to determine whether the pattern of such variation is modified systematically by ischemia. Measurements of integrated ultrasonic backscatter in selected regions of normal, ischemic, and reperfused hearts were compared in view of known differences in systolic function of myocardium in each of these regions. Integrated ultrasonic backscatter (3-7 MHz) gated to the first derivative of left ventricular pressure was measured at the apex, midwall, and base in 10 dogs and at the apex before and during transient ischemia and reperfusion in four dogs. Quantitative integrated ultrasonic backscatter was referenced to a steel reflector. Cyclic variation of integrated ultrasonic backscatter was greatest at the apex [peak to trough variation 5.5 +/- 0.9 dB (mean +/- SE)] with the maximum near end diastole (-52.9 +/- 0.9 dB) and minimum near end systole (-58.4 +/- 1.0 dB). Variation at the apex (5.5 +/- 0.9 dB) and the midwall (4.3 +/- 0.8 dB) was greater than at the base (0.5 +/- 1.0 dB) (P less than 0.01 for either region compared with base). Left anterior descending coronary occlusion for 10 minutes in four of 10 dogs reduced variation at the apex to 0.4 +/- 1.5 dB (P less than 0.02 compared with preocclusion). Reperfusion for 2 hours restored apical cyclic variation to 3.9 +/- 1.7 dB, i.e., to values not significantly different from those before occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional differences in the cyclic variation of myocardial backscatter that parallel regional differences in contractile performance.

Previous reports from our laboratory indicate that ultrasonic backscatter from myocardium exhibits a cyclic variation during the cardiac cycle that is reduced sharply by ischemia, a process which impairs both systolic contraction and diastolic relaxation. These results suggest that the cyclic variation of backscatter may be related to the cyclic variation of the contractile performance of the myocardium. Because contractile performance of the left ventricle is known to exhibit regional variability, the present study was undertaken to determine whether such regional differences in contractile performance are paralleled by differences in the magnitude of the cyclic variation of ultrasonic backscatter. Measurements obtained from representative zones of three regions of the hearts of ten open-chest dogs indicate that the magnitude of the cyclic pattern of variation of backscatter parallels the regional differences in contractile performance throughout the left ventricle with the maximum variation (5.5 +/- 0.9 dB peak-to-peak amplitude) occurring at the apex, intermediate values (4.3 +/- 0.8 dB) at the midwall, and minimum (0.5 +/- 1.0 dB) at the base. These results suggest that the ultrasonic backscatter may be sensitive to the regional myocardial contractile performance.

Applicability of ultrasonic tissue characterization for longitudinal assessment and differentiation of calcification and fibrosis in cardiomyopathy.

Progress in tissue characterization of myocardium with ultrasound suggests that quantitative recognition of ischemic or scarred tissue will be achieved. Despite the increasing recognition and importance of cardiomyopathy, its diagnosis generally requires invasive procedures such as cardiac catheterization and biopsy. To investigate methods that permit the characterization of longitudinal cardiomyopathic changes that might ultimately be extended for noninvasive studies in patients, quantitative ultrasonic methods were utilized for in vitro tissue characterization of hearts from Syrian hamsters of selected age of either 2 to 3 or 5 to 7 months. Normal hamsters were used as controls. Myocardial sites (n = 600) from the young Syrian hamsters exhibited values (+/- standard error) of integrated ultrasonic backscatter averaging -53.87 +/- 0.26 dB, which were significantly different from values (n = 500) in age-matched control hamsters (-58.07 +/- 0.08 dB; p less than 0.001). Cardiomyopathic hearts from older animals exhibited backscatter values (n = 500 sites) averaging -50.87 +/- 0.22 dB, again significantly different from values (n = 300 sites) in age-matched control hamsters (-55.91 +/- 0.11 dB; p less than 0.001). In addition, ultrasonic attenuation was significantly different for hearts from the control and cardiomyopathic hamsters of both age ranges. The results correlated with sequential calcification and fibrosis characteristics assessed histopathologically. This study indicates that quantitative characterization of myocardium with ultrasound may permit longitudinal assessment of cardiomyopathic changes in diverse disease entities and their response to therapy.

Hematin: unique effects of hemostasis.

Hematin is clinically useful in the treatment of acute intermittent porphyria. Recently, hematin-induced coagulopathy has been reported, and a patient we treated bled during hematin therapy. On 3 separate occasions, infusions of hematin (4 mg/kg) induced thrombocytopenia, prolongation of the prothrombin time, partial thromboplastin time. Reptilase time, and apparent decreases in fibrinogen and increases in fibrin(ogen) degradation products (FDP). However, fibrinogen assayed by heat precipitation was unchanged, the protamine paracoagulation test was negative, there was no red blood cell fragmentation, and plasminogen and antithrombin III remained normal, excluding the presence of disseminated intravascular coagulation. Furthermore, premedication with heparin, 5000 U i.v., failed to prevent the lengthening of the Reptilase time and exacerbated the thrombocytopenia. In vitro studies revealed that hematin, 0.1 mg/ml, aggregated platelets and induced the release of 14C-serotonin and adenosine triphosphate (ATP). Hematin also aggregated washed or gel-filtered platelets but had no effect on formalin-fixed platelets. Aggregation was inhibited by aspirin (0.12 mg/ml), adenosine triphosphate, and apyrase, suggesting that hematin aggregated platelets by inducing adenosine diphosphate (ADP) release. Hematin (0.07 mg/ml) progressively inactivated thrombin and 0.1 mg/ml prolonged the Reptilase time. Thus, hematin is unique in that it both induces platelet aggregation and inhibits coagulation.

Enhancement of formylmethionyl-leucyl-phenylalanine-induced directional locomotion of human neutrophils by serum low-density lipoproteins.

Human low-density lipoproteins (LDL) significantly increased neutrophil locomotion in a gradient of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), while LDL alone had no effect on either directional or random locomotion. The enhancing effect on directional locomotion was constantly observed at FMLP concentrations of 10(-8) M and 5 X 10(-8) M after addition of 50 microgram LDL/ml.

Modulation of concanavalin A-induced lymphocyte stimulation by human low-density lipoproteins.

Spleen lymphocytes and T cells were stimulated by concanavalin A in the presence of various concentrations of human low-density lipoproteins (LDL). The proliferative responses were measured by [14C]thymidine incorporation. Low LDL doses (5-50 microgram/ml) significantly enhanced the stimulation of splenic lymphocytes and T cells. Further, LDL had the capacity to partially relieve the suppression produced by supraoptimal doses of concanavalin A.