Personal care formulations containing alkyl ketal esters having the structure

Personal care formulations are available in a wide range of formulation forms and are used in a wide variety of specialized applications. Because these various types of formulations differ enormously, as do the conditions under which they are used, the individual formulations tend to be formulated specifically for the end-use application for which they are intended. Further, the use of materials based on renewable resources is becoming of increasing interest as formulators redesign their formulations to meet environmental sensitivities of their consumer bases.

Personal care formulations containing alkyl ketal esters and methods of manufacture

Abstract: wherein a is 0 or an integer of 1 to 12; b is 0 or 1; R2 is a divalent C1-8 group optionally substituted with up to 5 hydroxyl groups; and R1 is C1-6 alkyl.


A metallic mold for tire curing is disclosed, which comprises a plurality of pieces for forming a tread pattern of a tire by sections, the pieces being fixed to a back block and arranged in an annular form. In this case, each piece is composed of a plurality of piece block layers laminated by performing a plurality of shots of die casting for metallic materials, and a very small gap is formed in a joined portion between the piece blocks by means of coagulation and contraction of the metallic materials. Because of this constitution, formation of a gap having necessary exhaust performance in an optional position of a molding surface can be facilitated. Accordingly, an appearance of a produced tire can be improved without preventing spew formation, and the production of defectives can be reduced during curing and molding.

The present invention relates to a metallic mold for tire curing, which includes a plurality of pieces used to form a tire tread pattern by sections, and a process for producing the same. More specifically, the invention relates to a metallic mold for tire curing, which is capable of improving an appearance of a produced tire by preventing spew formation, and also capable of reducing the production of defectives during curing and molding, and a process for producing the same.
Conventionally, regarding a sector mold used as one of metallic molds for tire curing, a sector is formed by arranging a plurality of pieces in an adjacent relation to each other, the pieces having concave parts and convex parts corresponding to a profile of a tread section of a produced tire, and then fixing these pieces to a back block. Then, an annular sector mold is made by interconnecting a plurality of such sectors.
It has been known that during tire curing and molding, air is left or gas is generated between an outer peripheral surface of the tire and a molding surface of the metallic mold to form very small concaves in the surface of the produced tire and, consequently, a defective product is produced. To deal with this problem, an air vent through-hole called a vent hole or an exhaust groove called a vent groove has conventionally been formed in the molding surface of the metallic mold.
However, a rubber material flows into the vent hole or the vent groove during tire curing and molding to form spews (or flashes: whisker-like protruding portions) in the surface of the produced tire. This necessitated finishing work to be carried out for removing the spews after molding. But even the removal of the spews didn""t erase traces thereof. Thus, an appearance of the produced tire was damaged, and tire noises were increased.
Under these circumstances, in recent years, in order to improve an appearance of the tire and reduce the production of defectives during tire curing and molding, a spewless mold including a slit vent composed of a very small gap has been made available without forming any vent holes or vent grooves in the molding surface.
Regarding such a spewless mold, for example, the followings have been presented: {circle around (1)} Japanese Patent Laid-Open Hei 4 (1992)-223108 (Title of the Invention: METALLIC MOLD FOR CURING UNCURED TIRE CARCASS, PROCESS FOR PRODUCING THE SAME, AND TIRE CURING PROCESS USING THE SAME); and {circle around (2)} Japanese Patent Laid-Open Hei 10 (1998)-24423 (Title of the Invention: METALLIC MOLD FOR TIRE CURING, AND PROCESS FOR PRODUCING THE SAME).
The process disclosed in the former invention {circle around (1)} is for assembling back blocks for forming a tire tread pattern by sections divided in a pitch, and then sucking air existing between the tire and a metallic mold through a gap provided between back blocks. The process disclosed in the latter invention {circle around (2)} is for casting a matrix metal (aluminum) to form a pattern piece around a core piece of a different kind of metal (SS400), and then discharging air through a gap formed at a tire curing temperature (160xc2x0) based on a difference in a linear thermal expansion coefficient between the metals. In addition, for a slit vent mold, there is available a method of venting air by providing a level difference between pieces by means of machine work or inserting a spacer between the pieces.
However, in the case of the invention {circle around (1)}, it is difficult to secure a proper air vent in an optional position, and it is always necessary to install a vacuum device or the like as exhaust means. In the case of the invention {circle around (2)}, a sufficient volume must be provided for forming a gap based on a difference in a linear thermal expansion coefficient between the metals, and it is difficult to secure a necessary gap quantity in an optional position. Thus, it is difficult to substantially vent air from the metallic mold.
It is an object of the present invention to provide a metallic mold for tire curing, which is capable of facilitating formation of a gap having necessary exhaust performance in an optional position in a molding surface, thus preventing spew formation to improve an appearance of a produced tire, and also capable of reducing production of defectives during curing and molding. It is another object of the invention to provide a process for producing the metallic mold.
In order to achieve the above object, in accordance with the present invention, a metallic mold for tire curing comprises a plurality of pieces for forming a tread pattern of a tire by sections, the pieces being fixed to a back block and arranged in an annular form. In this case, each of the pieces is composed of a plurality of piece block layers laminated by performing a plurality of shots of die casting for metallic materials, and a very small gap is formed in a joined portion between the piece blocks by means of coagulation and contraction of the metallic materials.
In accordance with the present invention, a process for producing the tire curing metallic mold comprises the steps of:
performing a first shot of die casting to cast a molten metallic material in a first metallic mold, and forming a first piece block having a partial molding surface corresponding to a tread pattern of a tire; performing a second shot of die casting to cast the same kind of a molten metallic material in a second metallic mold while the first piece block is arranged in the second metallic mold, and forming a second piece block on the first piece block so as to complement the molding surface; and forming a very small gap in a joined portion between the first and second piece blocks by means of coagulation and contraction of the metallic materials.
With the invention, during casting and laminating the plurality of piece block layers constituting each piece by using the same kind of metallic materials, a very small gap is formed in a joined portion between the piece blocks by means of coagulation and contraction of the metallic materials. This very small gap formed by coagulation and contraction of the metallic materials exhibits exhaust performance for discharging only air or gas remaining in the metallic mold well without allowing a rubber material to flow out. Accordingly, a gap having necessary exhaust performance for a spewless mold can be easily formed in an optional position of the molding surface based on the arrangement of the joined portion, and the production of defectives can be reduced during curing and molding while preventing spew formation.
Moreover, a tire appearance can be improved without performing spew removal after tire curing or without leaving any spew traces on a tire surface, the spew traces causing deteriorated tire noises.
In accordance with the present invention, a pneumatic tire comprises a tread pattern having no spew traces, which is formed on a tire surface by using the foregoing metallic mold for tire curing.
Furthermore, in accordance with the present invention, a process for producing a pneumatic tire comprises the step of forming a tread pattern having no spew traces on a tire surface by using the foregoing metallic mold for tire curing.

Metallic mold for tire curing and process for producing the same


A polyaxial bone screw for attaching a rod to a vertebra comprises a fastener having a threaded shank and a head. A yoke has a rod receiving channel for receiving the rod. A screw support rotatably attached to the yoke has a cavity articulatingly and rotatably supporting the fastener head, a hole receiving the threaded fastener shank therethrough, and a plurality of slots for maximum screw angulation. A crown has an upper rod receiving surface, a lower surface in contact with the fastener head and a projecting surface in contact with the yoke. A biasing element between the screw support and the yoke applies biasing forces among the fastener, the crown, and the yoke to provide sufficient friction to retain the yoke in a manipulable position relative to the fastener. A set screw is supported by the yoke to overcome the bias and to rigidly secure the components.

Several techniques and systems have been developed for correcting and stabilizing injuries to or malformation of the spine. In one type of system, an elongated member such as an elongate bendable rod is disposed longitudinally along a length of the spine, spanning two or more vertebral levels. In certain applications, the rod is bent to correspond to the normal curvature of the spine in the particular region being instrumented, such as the normal kyphotic curvature of the thoracic region or the lordotic curvature of the lumbar region. In accordance with such a system, the rod is engaged to various vertebrae along a length of the spinal column by way of a number of anchor devices that utilize a variety of fixation elements configured to engage specific portions of the vertebra and other bones. For instance, one such fixation element is a hook that is configured to engage the laminae of the vertebra. Another very prevalent fixation element is a bone screw that can be threaded into various parts of the vertebrae, particularly the pedicle.
As these systems have evolved, various degrees of freedom of relative orientation were integrated into the systems in order to accommodate misaligned spinal curvature as well as to minimize rod bending and stress risers in the anchor. Presently, the standard in rod-based spinal systems is a polyaxial pedicle screw which includes a yoke having a slot for receiving a bendable elongate connecting rod that is coupled to a threaded bone engaging shank for polyaxial swiveling movement with respect thereto. One example of such a polyaxial screw is shown and described in commonly assigned U.S. Pat. No. 8,491,639, entitled "Multi-axial Spinal Fixation System", issued on Apr. 24, 2012 to Anthony James et al (the '639 Patent).
One particular use of polyaxial pedicle screws is for posterior application in the cervico-thoracic spine. In such application, due in part to the relatively small size of the vertebrae and access area for surgery, low profile and high angulation of the polyaxial screws are desirable. While considering these desirable features, some have recognized that pull out strength of the polyaxial screw may be affected in an effort to provide a structure to achieve such results. This concern is described, for example, in U.S. Pat. No. 8,636,778, issued to Gephart et al. on Jan. 28, 2014. Other patents describing polyaxial screws that may be used in cervico-thoracic applications include U.S. Pat. No. 8,337,530, issued to Hestad et al. on Dec. 25, 2012, U.S. Pat. No. 8,100,946, issued to Strausbaugh et al. on Jan. 24, 2012 and U.S. Pat. No. 8,021,397, issued to Farris et al. on Sep. 20, 2011.
In addition, after placement of the polyaxial screw during such spinal surgery, it is also desirable that the yoke of the polyaxial screw remain upright. Screw yokes are often manipulated with a positioner instrument for later rod placement and for a preview of any rod contouring that may be necessary. Yokes that stay in position negate the need for repositioning and therefore facilitate a more efficient operation. The '639 Patent noted hereinabove describes use of a wave spring to provide friction between the yoke and the bone screw to releasably retain the yoke in position after screw placement.
Nevertheless, improvements in polyaxial screws that provide relatively high angulation and low profile, as well as retention of the screw yoke in a releasable manipulable position once introduced into a vertebra, particularly for application in the posterior cervico-thoracic spine, are desirable.

This patent is subject to a terminal disclaimer.


A dry processing apparatus for plasma etching or deposition includes a chamber for plasma processing having an external wall for housing a work piece with a surface to be plasma processed. A source of an induction field is located outside the chamber on its opposite side from the work piece. A radio frequency induction field applied to the chamber generates a plasma. The plasma is confined within the external wall in the chamber by magnetic dipoles providing a surface magnetic field for confining the plasma. The surface magnetic field is confined to the space adjacent to the external wall. An R.F. generator provides an R.F. generated bias to the work piece. The chamber is lined with a material inert to a plasma or noncontaminating to the work piece, and the induction source in the form of a spiral or involute shaped induction coil is located on the exterior of the liner material on the opposite side of the chamber from the work piece. Distribution of gas to the chamber is uniform because a manifold located about the periphery of the chamber and an orifice formed by the surface of the chamber and the manifold admits gas from the manifold into the chamber at a uniform pressure about the periphery of the cover of the chamber.

1. Field of the Invention
This invention relates to apparatus for plasma processing of substrates, and more particularly to subtractive (etching) and additive (deposition) processing of electronic circuit chips and packaging materials.
2. Technical Problem
The problem with capacitive coupling of R.F. energy to a plasma employed for etching or depositing films is that to increase power to the level required to generate the plasma required, the voltage will be so high that the charged particles in the plasma will be accelerated to an excessive level of kinetic energy and will tend to sputter the work piece and to etch or sputter away any masks. The effect will be to chamfer the mask opening, i.e. increase the size of openings in masks by etching the edges of the masks. The effect also leads to ion damage and loss of selectivity. This is unacceptable as the requirements in the art are to decrease the size of openings as dimensions are decreasing in microelectronics. Instead one would like the flexibility of varying the ion energy according to the desired process.
2. Related Art
U.S. Pat. No. 3,705,091 of Jacob for "Gas Discharge Apparatus" shows a cylindrical glass reaction chamber coaxially wound with a helical R.F. coil energized by high frequency (13.5 MHz) R.F. to generate a plasma in a vacuum for etching of a tray of semiconductor slices The system operates in the 1 Torr pressure range and produces mainly reactive radicals. The Jacob system does not operate in the desired reactive ion etching, RIE mode of this invention. In the pressure range desired for the present invention of 1 to 50 mTorr, the Jacob system would produce very non-uniform and very slow etching No means for confining the plasma is shown
M. C. Vella, K. W. Ehlers, D. Kippenhan, P. A. Pincosy, and R. V. Pyle "Development of R.F. Plasma Generators for Neutral Beams", J. Vac. Sci. Technol. A 3(3), (May/Jun 1985) pp 1218-1221 describes an R.F. plasma source used to generate a high power neutral beam to heat a large fusion plasma to reaction temperatures. The pressure is from 1 to 20 mTorr. A plurality of magnetic dipoles surround the rf bucket chamber to create a magnetic shield for the plasma There is no reference to additive or subtractive treatment of a substrate. Vella et al teaches the use of R.F. induction for plasma production, but it does not teach the use of R.F. induction for etching or deposition where the plasma will etch the coating on an R F. coil and coat the insulators.
R. Limpaecher and K. R. MacKenzie, "Magnetic Multipole Containment of Large Uniform Collisionless Quiescent Plasmas", Rev. Sci. Instrum., Vol 44, No. 6, (June 1973) 726-731 discusses the use of magnetic multipoles for confinement of a plasma of argon at a pressure of 0.002 Torr. This reference is one of the original papers on multipole confinement of the primary electrons in plasma production from electron emission from a hot filament.
U.S. Pat. No. No 4,483,737 of Mantei, "Method and Apparatus for Plasma Etching a Substrate" uses an electrically heated filament to emit electrons, but states at Col. 5, lines 53.65, that a hollow cathode or ion cyclotron resonance can be used to generate electrons. Later, it states that R.F. power sources are not used for the discharge current or for generation of the surface magnetic field confining the plasma. At Col. 6, lines 52-58, it states "The plasma is produced by impact from fast ionizing electrons drawn from a set of heated tungsten filaments, rather than by an applied rf voltage".
See also T. D. Mantei and T. Wicker, "Plasma Etching with Surface Magnetic Field Confinement" Appl. Phys. Lett. 43(1), (1 Jul. 1983) pp. 84-86, and T. D. Mantei and T. Wicker, "Low Pressure Plasma Etching with Magnetic Confinement", Solid State Technology (April 1985) pp. 263-265.
I. Lin, D. C. Hinson, W. H. Class, R. L. Sandstrom, "Low-Energy High Flux Reactive Ion Etching by R.F. Magnetron Plasma", Appl Phys. Lett. Vol. 44 (Jan. 15 1984) pp. 185-187 describes magnetic confinement of a plasma and R.F. power being used for plasma production. The R.F. power is capacitively coupled to a copper prism used as an electrode. This is one of many magnetron reactive ion etching systems. Most of them involve an effort to achieve uniformity from a system in which the electron density increases in the direction of E.times.B drift of secondary electrons from the cathode. These systems also provide limited adjustability of the energy of the ions striking a wafer.
U.S. Pat. No. No. 4,632,719 of Chow et al for "Semiconductor Etching Apparatus with Magnetic Array and Vertical Shield" describes etching a semiconductor wafer in an R.F. field in argon gas A pair of rings of concentric dipoles above the wafer create a pair of rings in the plasma above the wafer. This leads to the kind of lack of uniformity of the plasma which would be avoided in systems required to provide uniform etching or deposition. Thus the Chow et al patent would lead one in the opposite direction from the purpose to which this invention is directed.
U.S. Pat. No. 4,384,938 "Reactive Ion Etching Chamber" of B. Desilets et al describes a reactive ion etching tool having a cylindrical reactive ion etching chamber acting as an anode and a plate arrangement acting as a cathode and wherein an R.F. signal applied between cathode and anode acts to produce an active glow region within the chamber with a dark space existing over the internal surfaces thereof. A reactive ion etching chamber structure has an internal top surface and sidewall surfaces forming a physically symmetrical arrangement with respect to the cathode plate positioned between the sidewall surfaces below the top surface, the top surface and surfaces being uniform except for gas input and exhaust ports with the gas exhaust ports having an opening dimension less than the thickness of the dark space existing over the internal surface.
See also Keller et al U.S. Pat. No. 4,383,177 for "Multipole Implantation Isotope Separation Ion Beam Source".
Objects of this invention are:
a) a uniform plasma, PA1 b) plasma density which does not saturate with power, PA1 c) control of ion energy, PA1 d) high etch rates for a given power level, and PA1 e) relative simplicity.
In accordance with this invention, dry processing apparatus for plasma etching or deposition includes a chamber for plasma processing having an external wall for housing a work piece with a surface to be plasma processed in a gas. A source of an induction field is located outside the chamber on its opposite side from the work piece. A radio frequency (R.F.) induction field applied to the chamber generates a plasma in the gas. The plasma is confined within the external wall in the chamber by magnetic dipoles providing a surface magnetic field for confining the plasma. The surface magnetic field is confined to the space adjacent to the external wall. An R.F generator provides an R.F. generated bias to the work piece. The chamber is lined with a material inert to a plasma or noncontaminating to the work piece, and the induction source in the form of a planar involute or spiral induction coil is located on the exterior of the liner material on the opposite side of the chamber from the work piece. Delivery of and distribution of the gas to the chamber is uniform about the periphery of the top cover because a manifold is located about the periphery of the chamber. An orifice for controlling the gas pressure of the gas being admitted to the chamber is formed by the surface of the chamber and the manifold admits gas from the manifold into the chamber at a uniform pressure about the periphery of the cover of the chamber Preferably a surface magnetic field is positioned adjacent to the induction coil to confine the field at the top of the chamber It is further preferred that a capacitive or inductive reactance be connected in series with the induction coil to adjust the R.F. generated bias.

Radio frequency induction/multipole plasma processing tool

International Business Machines Corporation


Disclosed is a manufacturing method of a keypad for a mobile phone and the keypad manufactured thereby, in which a vacuum deposition layer and a cellophane paper are attached to a lower surface of a key in the keypad. The keypad for a mobile phone includes a plurality of keys, each key includes a key body of a transparent resin material and a high-hardness transparent coating layer coated onto a surface of the key body in order to increase hardness, and each key comprises a key background color paint layer for color presentation, coated onto a lower surface of the key body; an opaque paint layer coated onto a lower surface of the key background color paint layer; a transparent marking portion formed by performing a laser processing according to a shape of a numeral, a character, or a symbol with respect to the lower surface of the key body so as to remove the color paint layer and the opaque paint layer; a metallic vacuum deposition layer formed on the lower surface of the key body and a lower surface of the opaque paint layer; and a transparent color-printed layer or a cellophane paper attached to a lower surface of the metallic vacuum deposition layer so as to present various colors.

1. Field of the Invention
The present invention relates to a manufacturing method of a keypad for a mobile phone and the keypad manufactured thereby, and more particularly to a method for manufacturing a keypad which can provide a transparent or translucent marking portion (such as a number, a character, or a symbol of the key) with an elegant metallic glossy image, and present various color images when light is emitted, by attaching a vacuum deposition layer and a cellophane paper to a lower surface of a key in the keypad of the mobile phone.
2. Description of the Related Art
In general, "portable communication devices" refers to devices that are portable and enable the users of the devices to perform wireless communications. Portable communication devices include an HHP (hand-held phone), a CT-2 (cordless telephone 2) cellular phone, a digital phone, a PCS (personal communication system) phone, and a PDA (personal digital assistant), and are classified into various types according to their external appearance. For example, mobile phones are classified into bar-type, flip-type, folder-type, and slide-type mobile phones according to their external appearance. The above-mentioned conventional mobile phones necessarily include an antenna unit, a data input/output unit, and a data transceiver. A keypad for enabling the user to input data through a pressing operation using his/her fingers is generally used as the data input unit, and includes a plurality of keys arranged thereon.
The structure of a mobile phone including a keypad 1 will now be described with reference to FIG. 1. A plurality of keys 4, each of which includes a transparent or translucent marking portion 3 for displaying a number, a character, or a symbol, are constructed on an elastic pad 2 so that the user may easily identify operational functions and touch a key to input a desired operational signal.
The key 4 secures a desired color and durability through the spraying, coating, and printing processes performed with respect to the upper surface of the key body 5 (see FIG. 3) of a transparent or translucent resin material (see FIG. 2). In addition, in order to increase marking resolution of numerals, characters, and symbols, a laser etching process is performed adaptively to the transparent or translucent marking portion 3, so that light generated by a light emitting unit 11 installed on a printed circuit board 10 can be easily transmitted through the transparent or translucent marking portion 3, thereby remarkably displaying the transparent or translucent marking portion 3.
The manufacturing method of the key 4 will now be described with reference to FIGS. 2 and 3.
First, a color paint for forming a transparent or translucent marking background color paint layer 6 for the transparent or translucent marking portion 3 is coated onto the key body 5 made of a transparent or translucent resin material, and then an opaque paint layer 7 is coated so as to prevent light from being transmitted through portions other than the transparent or translucent marking portion 3.
For the opaque paint layer 7, a black paint is generally used in consideration of a light absorbing characteristic and a laser workability characteristic. If a key background has a color of the black series or gray series, it is possible to omit the process of coating the opaque paint layer 7. Thereafter, a second color key background paint layer 8 for presentation of a key background color is coated. Then, in order to form the transparent or translucent marking portion 3, a laser etching process is performed adaptively to the shape of a numeral, a character, or a symbol, so that the second color paint layer 8 for a key background color and the opaque paint layer 7 may be removed, and only the first marking color paint layer 6 for forming the transparent or translucent marking portion will remain at the corresponding laser-etched portion, thereby enabling light, which has been generated by the light emitting unit 11 installed on the printed circuit board 10, to be transmitted through the corresponding portion. Finally, a high-hardness transparent coating layer 9 is coated onto the surface of the key 4 so as to secure hardness of the surface and improve abrasion resistance thereof.
Meanwhile, the construction of the coating layers 7 and 8 varies depending on the key background color. That is, when the key background color is a light color such as white, generally a silver paint layer (not shown) is formed between the opaque paint layer 7 and the key background color paint layer 8 so as to prevent the light background color from being dark due to the opaque paint layer 7 of a black color.
However, although a high-hardness transparent or translucent coating layer is formed as the last layer for a key in order to secure hardness of the key surface in the prior art, an abrasion phenomenon happens due to the use of the key after a predetermined period has passed, so that the high-hardness transparent coating layer, the key background color paint layer, and a transparent or translucent marking portion, which have been formed on the upper portion of the key, wear away.
In addition, according to the conventional key manufacturing method, since a color is presented by a method of spraying/coating each of the paint layers on the upper surface of the key, it is impossible to present an elegant metallic gloss. Also, according to the conventional key manufacturing method, since the transparent or translucent marking portion is constructed with only one color paint layer, such as green, red, white, etc., it is impossible to present various colors and effects when the keypad emits light.

Manufacturing method of keypad for mobile phone and keypad manufactured thereby


In a multitasking system executing real-time harmonic and dynamic tasks having various priority levels, slack is stolen from both timeline and reclaimed slack to enable the execution of high priority non-essential tasks on a best efforts basis. Counts of the amount of slack consumed, slack reclaimed, and periodic compute time consumed are maintained by individual priority level and dynamically updated at certain times. Idle time is calculated by priority level. Available slack is calculated, and slack is allocated and consumed by rate, with the highest rate first and the lowest rate last. Also described are a computer system and various methods that perform slack stealing in a multitasking system in which dynamic tasks can request activation or deactivation at any time.

Computer processes are often subdivided into a variety of functions which may be executed as tasks in serial and/or parallel fashion. These computer processes can be used to gather and act upon information, and to bring about some result in response to the information. These functional task systems find use in a variety of important environments. Examples may include monitor and control of an industrial process, such as a power generation and distribution system, or monitor and control of complex equipment, such as an aircraft or spacecraft.
In real-time systems, such as those mentioned above, execution of tasks can include both periodic tasks and aperiodic tasks. One known way of executing periodic tasks is to use rate monotonic scheduling (RMS). The classical RMS formulation is for strictly periodic task sets. To specify a periodic task set, each of n tasks, say τi, where 1≦i≦n, is associated with a period Ti and a worst-case compute time C1. Each task τi will be dispatched and executed at the rate 1/Ti, and in worst-case it will consume processor time equal to Ci at each dispatch. Each task is implicitly assigned a priority that is determined by its rate (or equivalently, the inverse of its period), with priority equal to the ranking of the rate.
The RMS scheduler schedules periodic tasks having hard deadlines. Viable real-time systems must also be capable of executing aperiodic tasks, which can have hard deadlines or soft deadlines. It is desirable for a task scheduling system to schedule a mixture of periodic and aperiodic tasks in such as way that all periodic task deadlines are met and the response times for the aperiodic tasks are as small as possible.
In "An Optimal Algorithm for Scheduling Soft-Aperiodic Tasks in Fixed-Priority Preemptive Systems", John P. Lehoczky and Sandra Ramos-Thuel, Real-Time Systems Symposium, IEEE Proceedings, December, 1992, there is described a slack-stealing algorithm. This algorithm creates a passive task which, when prompted for service, attempts to make time for servicing aperiodic tasks by "stealing" all the processing time it can from the periodic tasks without causing their deadlines to be missed. This is equivalent to "stealing slack" from the periodic tasks. The slack-stealing algorithm was demonstrated to provide substantial improvements in the response times for aperiodic tasks. In addition, the slack stealing algorithm was described as further improved by cooperating with a reclaiming algorithm that makes available for aperiodic service any processing time unused by the periodic tasks when they require less than their worst-case execution times.
The slack-stealing algorithm as described above was limited to only a static set of execution threads, i.e. a fixed set of recurring tasks without any new periodic tasks being activated and without any periodic tasks being deactivated. However, actual real-time processing environments typically contain a dynamic set of threads, as certain periodic tasks become active and others become inactive.
Thus there is a significant need in the art for a task scheduler that can provide slack-stealing in a real-time environment that encompasses periodic and dynamic tasks.

Method and apparatus for slack stealing with dynamic threads


This invention relates to a plasma-collecting device which comprises a blood filtering material comprising glass fiber filter and microporous membrane, a holder having a blood inlet and a plasma outlet accommodating the blood filtering material, a blood-collecting needle being connected to the blood inlet, and a plasma receiver being provided on the plasma outlet side. The plasma-collecting device can collect a necessary amount of plasma for analysis from blood vessel directly, easily and surely.

This invention relates to a device for collecting plasma from blood vessel directly by drawing and filtering blood successively.
Type or concentration of blood components, such as metabolites, proteins, lipids, electrolytes, enzymes, antigens, and antibodies, is measured, in general, using a plasma or serum sample obtained by centrifuging whole blood. However, centrifuging takes labor and time. Particularly, centrifuging is unsuitable for an urgent case of measuring a small number of samples promptly and on site inspection, because of requiring a centrifuge and electricity. Thereupon, it has been investigated to separate serum from whole blood by filtration.
Several filtration methods using glass fiber filtration have been known wherein whole blood is charged into the glans fiber in a column from one side of the columns and pressurized or sucked to obtain plasma or serum from the other side (Japanese Patent KOKOKU Nos. 44-14673, 5-52463, Japanese Patent KOKAI Nos. 2-208565, 4-208856).
However, drawing blood and plasma separation are carried out independently in every known method and this requires time and labor. Moreover, waiting time between drawing blood and plasma separation varies considerably, and coagulation and denaturation of blood therebetween are also a problem. On the other hand, techniques concerning successive drawing blood and plasma separation have been developed from old times, however, plasma separation was carried out by centrifugation.
A device for conducting both drawing blood and plasma separation is disclosed in Japanese Patent KOKAI 52-100783 wherein silica gel is used for plasma separation.
Although various means for obtaining blood plasma directly from blood vessel were developed, none of them has been put to practical use because of troublesome handling, insufficient separation of blood cell components, etc


The present invention is a flow-thru cosmetic dispenser and applicator that comprises a means for controlling the flow of product from a reservoir to an applicator tip. The flow control means comprises wedge structures attached to biased hinges. The wedge structures move on the hinges in response to the removal or placement of a closure. The movement of the wedges alternately opens and pinches-off a deformable conduit in which the product flows. The invention includes means for converting any ordinary cosmetic container into a flow-thru container.

Flexible, flow-thru applicator packages for holding and dispensing cosmetic products are known. Such applicator packages generally comprise a deformable reservoir for holding a cosmetic product. Suitable cosmetic products may be in fluid, semi-viscous or powder form. A typical flow-thru applicator package further comprises an applicator tip, such as a bristled brush, a powder puff, a foam sponge, a molded plastic base covered with absorbent material or any other substrate for transferring onto a surface. The applicator package also comprises a means for conveying product from the reservoir to a specific location on or in the applicator tip. In use, product reaching the specific location of the applicator tip moves further to a position where the product may be transferred from the applicator tip to another surface. For example, the cosmetic product may emerge onto an exterior surface of the applicator tip from where it can be applied to the skin of a user.
In order to prevent product from reaching the surface of the applicator tip when the flow-thru applicator package is not in use, some means may be employed to reduce or prevent the unwanted flow of cosmetic product to the applicator tip. Unwanted flow of product may occur as a result of the reservoir being inadvertently squeezed, or as a result of gravity or vibration. Also, unwanted flow may occur as a result of an adverse pressure differential that can arise between the inside of the reservoir and the ambient atmosphere. This may occur as a result of temperature and/or pressure changes in the ambient atmosphere. For example, if the package is located in an unpressurized compartment of an airplane, the temperature and pressure inside the reservoir are likely to be greater than the ambient temperature and pressure outside the applicator package. This adverse differential tends to force the flowable product in the reservoir out of the package and will do so, if effective seals are not provided. Following are some examples of known flexible, flow-thru cosmetic applicator packages.
U.S. Pat. No. 2,994,897 discloses a conventional plastic squeeze bottle with threadably mounted closure. The container orifice is fitted with a relatively simple snap-fit or friction-fit insert that carries a tuft of bristles. A flow passage through the insert leads to the base of the bristles. No means of preventing unwanted flow is disclosed which means that leakage problems previously discussed may occur. Furthermore, the product flow passage ends at the very base of the bristles, still inside the container insert and does not extend into the bristle tuft. Because of this, the product must force its way between a section of tightly crimped bristles, whereby an even distribution of the product on the bristles is difficult to achieve. In contrast, the present invention does provide means to control unwanted flow and the flow passage extends out of the package housing proper and into the applicator tip for better product distribution.
In U.S. Pat. No. 4,040,753, the flow passage between a squeezable reservoir and a brush a head is normally sealed by the abutment of two surfaces that are snap fitted or friction fitted together. The act of squeezing the reservoir distorts one or more of the surfaces so that the seal is broken and the product under pressure can flow to the bristles. When pressure is removed, the distorted surface return to its original shape and the seal is restored. This device is fairly simple, but suffers from the fact that the flow passage leads to the outside of the bristle envelope at a level that is still inside the package housing proper, rather than leading some distance into the bristle envelope outside of the package housing. In order to be useful, product must be squeezed out of the reservoir onto the brush until some of the product is pushed up toward the distal end of the brush. This requires a significantly large dose of product, all of which may not be needed by the user. The portion of dispensed product that remains near the base of the bristles will not be used and may dry out between uses. Furthermore, with this device the flow passage is only open when the bottle is being squeezed. Therefore, if a user prefers to maintain the flow of product to the brush, she must maintain a tight, forceful grip on the reservoir while manipulating the brush for precise make up application. In contrast, the present invention injects product into the interior of the brush, substantially above the base of the bristles and outside the package housing proper, from where the product will flow evenly throughout the brush. Furthermore, once the closure is removed from the container, the flow passage is open even without squeezing the reservoir.
U.S. Pat. No. 3,545,874 is a squeeze container with brush applicator having a valve member housing disposed in the container neck. The valve member housing defines a flow passage from the reservoir to the bristles and comprises an axially movable valve stem which is normally sealingly biased against an orifice in the flow passage. The sealing bias is accomplished with a springs or with a bellows integrally molded as the walls of the valve member housing. The valve may be opened only by pressing the brush applicator sufficiently hard against a surface to overcome the sealing bias. Then, if the container is squeezed and/or upside down, product can flow from the reservoir to the bristles. Because the orifice in the flow passage is normally sealed, unwanted flow is controlled. However, this device is relatively complicated and requires several custom designed components each of which adds to the cost of the package. Furthermore, the flow passage does not extend into the bristle tuft beyond the package housing proper. As discussed above, this makes even distribution of the product on the bristles difficult to achieve. Other disadvantages of this design include the need for metal springs which are subject to corrosion, and a specially designed cap to engage the valve member housing. Both the spring and special cap drive up the cost of the package. Also, as noted, the brush applicator must be pressed sufficiently hard to open the valve. This precludes this device from being used for delicate applications, such as nail polish or eye makeup. In contrast, the present invention does not use springs, nor a complicated-to-manufacture bellows nor a custom designed closure. Furthermore, this device and some that follow are inferior to the present invention in that the prior art devices utilize components that slide relative to one another while having to maintain fluid tight seals. In order to achieve fluid tight seals of this type, tight tolerances must be maintained in manufacturing. The sliding fit of the components must be tight enough to form an effective seal but loose enough to permit one component to slid passed the other. Effective seals of this type are substantially more difficult to obtain than the sealing means used in the present invention, which does not utilize components that slide past one another in a sealing relationship.
More complicated approaches have been taken to develop squeeze containers having flow-thru brush applicators as, for example, disclosed in U.S. Pat. No. 4,748,990. Here an orifice in a flow passage between a reservoir and an applicator brush may be fully or partially closed off by a valve stem that moves toward or away from the orifice. The relative position of the fixed valve stem and the movable orifice may be continuously adjusted by the relative rotation of a threaded lower section on the container. In this way, the amount of product that can pass through the orifice may be controlled. Disadvantageously, this device is relatively complicated in that the valve stem (one essential part of the sealing mechanism) must be fixedly mounted in the container neck via a specially designed "central sealing plug", which is quite complicated and requires special care to ensure a good seal between it and the container neck orifice. Furthermore, the product flow passage orifice (the other essential part of the sealing mechanism) is itself part of a complicated specially designed housing and the product flow passage does not extend outside of the package housing.
U.S. Pat. No. 5,397,195 is another rotation-type system, this one analogous to a lipstick riser system. From the closed position, the relative rotation of a sleeve and container, axially advances the applicator holder. When this happens, an orifice at the base of the applicator holder moves away from a needle that seals the orifice when in the closed position. In this way, a flow passage from the reservoir to the brush is opened. When the relative rotation is performed in the opposite direction, the orifice moves toward and contacts the needle and is sealed off, so that product cannot flow. This and other flow-thru devices that use relative rotation to achieve translational motion and flow control are relatively complicated, costly to produce and less reliable than the present invention. Also, here again, the product flow passage does not extend outside of the package housing.
The disclosures of U.S. Pat. No. 3,655,290, 4,368,746 and 5,904,433 are alike in that they describe flow-thru applicators that have a flow control means that is activated by a container closure. In U.S. Pat. No. 3,655,290 a brush applicator is mounted in a "nozzle" that is capable of axial movement. When a closure that is specially designed for contact with the nozzle is applied to the container, then the nozzle is pushed further into the container and a portion of a flow passage from the reservoir to the brush becomes blocked. When the closure is removed from the container, the nozzle may be extended slightly, enough to open the flow passage. The nozzle may be extended by a pressure at the back of the nozzle that is created when a user squeezes the container or by a spring optionally provided to push on the back of the nozzle. This device is inferior to the present invention in that this prior art device utilizes components that slide relative to one another while having to maintain fluid tight seals. The disadvantages of this were discussed above. Also, the flow passage of this prior art device terminates at the base of the bristles, still inside the housing and does not extend into the center of the bristle tuft beyond the housing. As discussed above, this makes even distribution of the product on the bristles difficult to achieve. The use of a spring also makes this design inferior to the present invention.
U.S. Pat. No. 4,368,746 discloses a lip-cream applicator comprising a squeezable tube of cream having a product exit orifice. The exit orifice emerges into a flow passage of an applicator tip construction, that leads to the base of an applicator pad. The applicator tip construction is axially movable relative to the exit orifice of the tube. Unopposed, a spring holds the applicator tip construction in its fully extended position and the exit orifice is open. However, when a screw cap is applied over the applicator tip construction and allowed to engage threads of the tube, the applicator tip construction is forced closer to the exit orifice, against the action of the spring, until a valve head within the tip construction seats in the exit orifice to cut off the flow of product from the tube. This device is inferior to the present invention in that this prior art device utilizes components that slide relative to one another while having to maintain fluid tight seals. The disadvantages of this were discussed above. Other disadvantages of this design include the need for a metal spring, which is subject to corrosion, and a specially designed cap to engage the applicator tip construction. Both the spring and special cap drive up the cost of the package. A specially designed cap is needed because, over the life of the package, the cap is repeatedly forced against the applicator tip. If precautions are not taken, this abrasive contact may damage the applicator tip. Also, as noted, the applicator tip construction is axially movable and this movement may occur when the applicator tip is pressed against the skin during use. This may be a distraction to a user trying to accurately apply the product. Also, if the user presses hard enough, the flow of product is reduced or cut off during use.
U.S. Pat. No. 5,904,433 describes a squeezable flow-thru applicator system having a flow passage that opens when a container closure is removed from a container and that closes when the closure is applied to the container. As the closure is being applied to the container, the closure bears down on radially projecting tabs which causes the applicator housing to deform at specially molded thin-walled portions. This deformation displaces sealing strips located near the thin walled portions, moving the sealing strips toward opposing surfaces until the sealing strips and opposing surfaces form a seal, closing off the flow passage. Removing the closure, releases the deformation and the flow passage opens. This device is quite complicated and relatively difficult to manufacture because it requires custom components having centimeter-sized portions where the wall thickness must be tightly controlled to impart specific flexural properties to the wall. Deforming one component (i.e. thin walled portion) in order to precisely move an adjacent component (i.e. sealing strip) into a sealing relationship with a third component (opposing surfaces) is a significantly complex design, especially in the cosmetics market where pressure to keep component costs low is acute. Furthermore, the product flow passage terminates at the base of the bristles, still within the housing rather than terminating within the applicator tip. In contrast, the present invention uses a deforming sealing means that is responsive to a closure, while having a significantly simpler design than this prior art device.
U.S. Pat. No. 1,067,596 describes a piston-activated shaving cream brush that has a rubber product flow passage that extends substantially beyond the package housing proper into the interior of the applicator tip. To reduce the uncomfortable feel of drawing a rigid member over the skin, the flow passage is flexible rubber However, the uncomfortable feel is not completely eliminated and the flow passage interferes with the operation of the brush. This is unlike the present invention where the flow passage does extend beyond the package housing proper into the applicator tip for maximum product distribution, but does not interfere with the function of the applicator tip nor cause an uncomfortable experience. The present invention does not utilize a relatively complex piston system.


An oil-filled transformer monitoring system is provided which calculates an oil temperature of an oil-filled transformer with high precision from a current of the transformer and an ambient temperature. The oil-filled transformer monitoring system of this invention has an oil temperature calculation device to calculate a transformer oil temperature using measurements from a transformer current measuring device and an ambient temperature measuring device. The transformer monitoring system also has an oil temperature measuring device and compares an output value from the oil temperature calculation device with an output value from the oil temperature measuring device to detect any anomaly.

The present invention relates to a monitoring of a state of oil-filled transformers using information such as transformer oil temperature, current and ambient temperature.
A stable supply of electricity is important and it is therefore necessary to periodically monitor transformers to forestall possible failures. For this purpose, monitoring a voltage, current and oil temperature of the transformers is effective. The monitoring of the oil temperature generally involves estimating an oil temperature from an average load factor, visually checking a temperature reading on a thermometer, comparing the estimated oil temperature with the temperature reading on the thermometer to decide whether the transformer in question is normal or not, and at the same time checking if the temperature reading on the thermometer has exceeded a predetermined value.
Another practice involves the use of a thermometer with an alarm contact which issues an alarm for detection of an anomaly when the oil temperature exceeds a set temperature.
Another method of monitoring is disclosed, for instance, in JP-A-5-227644 which, when determining a remaining service life from a maximum winding temperature, calculates an oil temperature rise and determines an oil temperature by adding a measured ambient temperature to the oil temperature rise.
In another method disclosed in JP-A-8-213246, a means is provided that calculates a transformer oil temperature from a reading on a thermometer attached to the outside of a case of the oil-filled transformer and from a measured load factor of the transformer.
In the above conventional method using a visual check, however, since an oil temperature change lags load factor and ambient temperature changes, checking the measured temperature after the load factor or ambient temperature has changed cannot easily determine if it is a proper temperature or not.
Further, JIS C 4304 specifies that a limit of an oil temperature rise is 50K at maximum during the rated load operation and that the transformer can be used in an ambient temperature range of between −20° C. and 40° C. Thus, to precisely estimate the oil temperature requires considering influences of ambient temperature.
When the oil temperature exceeds the set temperature of the thermometer with an alarm contact, an alarm is issued for detection of an anomaly. However, since the oil temperature is a sum of a temperature rise caused by a transformer loss and a temperature rise caused by the ambient temperature, if the ambient temperature is low, the oil temperature will not reach the set temperature even during an overload operation. Further, even when the transformer is used below the rated load, an alarm may be issued if the ambient temperature is high and the set temperature is exceeded. This method therefore could not decide whether the oil temperature is correct or not.
Nor can the conventional monitoring system determine if the oil temperature is correct or not.
Further, since the above conventional technique uses a measured value for the ambient temperature in determining the maximum winding temperature, the fact is not taken into account that an oil temperature change lags behind an ambient temperature change.
Further, since the conventional technique does not consider ambient temperature variations when indirectly measuring the oil temperature from the case outer surface temperature, an error occurs between the measured value and the converted value if the ambient temperature changes sharply. Moreover, when the converted value is based on a case measured value, which indirectly measures the oil temperature, since the measured value constitutes a reference, it is not possible to estimate accurately what the true oil temperature is.
Therefore, if an abnormal condition occurs in connected portions of circuit within coil and case as a result of progressive degradations over time or vibrations caused by earthquakes, oil is heated and its temperature rises. However, since the true oil temperature cannot be estimated accurately, it is not possible to decide that an abnormal condition has occurred inside the transformer.

Hitachi Industrial Equipment Systems Co., Ltd.


Recombinant human parainfluenza virus type 1 (HPIV1) compostions, formulations and methods are provided. The recombinant HPIV1 viruses and HPIV1 chimeric and chimeric vector viruses provided according to the invention are infectious and attenuated in permissive mammalian subjects, including humans, and are useful in immunogenic composition s for eliciting an immune responses against one or more PIVs, against one or more non-PIV pathogens, or against a PIV and a non-PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a recombinant HPIV1 genome or antigenome.

Human parainfluenza viruses (HPIVs) are important pathogens in human populations, causing severe lower respiratory tract infections in infants and young children. HPIV1 and HPIV2 are the principal etiologic agents of laryngotracheobronchitis (croup), and can also cause pneumonia and bronchiolitis (Collins et al., 3rd ed. In "Fields Virology," B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996). HPIV3 ranks second after respiratory syncytial virus (RSV) as a leading cause of hospitalization for viral lower respiratory tract disease in infants and young children (Collins et al., 3rd ed. In "Fields Virology," B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1205-1243. Lippincott-Raven Publishers, Philadelphia, 1996; Crowe et al., Vaccine 13:415-421, 1995; Marx et al., J. Infect. Dis. 176:1423-1427, 1997).
PIVs are also important causes of respiratory tract disease in adults. Collectively, HPIV1, HPIV2, and HPIV3 have been identified through a 20 year study as responsible etiologic agents for approximately 18% of hospitalizations for pediatric respiratory tract disease (Murphy et al., Virus Res. 11:1-15, 1988). HPIVs have also been implicated in a significant proportion of cases of virally-induced middle ear effusions in children with otitis media (Heikkinen et al., N. Engl. J. Med. 340:260-4, 1999).
Despite considerable efforts to develop effective immunogenic compositions against HPIVs, no vaccines have yet been approved for any HPIV serotype, nor for ameliorating HPIV related illnesses. The most promising prospects to date are live attenuated vaccine viruses since these have been shown to be efficacious in non-human primates even in the presence of passively transferred antibodies, an experimental situation that simulates that present in the very young infant who possesses maternally acquired antibodies (Crowe et al., Vaccine 13:847-855, 1995; Durbin et al., J. Infect. Dis. 179:1345-1351, 1999). Two live attenuated PIV3 vaccine candidates, a temperature-sensitive (ts) derivative of the wild-type PIV3 JS strain (designated PIV3 cp45) and a bovine PIV3 (BPIV3) strain, are undergoing clinical evaluation (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Karron et al., J. Infect. Dis. 171:1107-1114, 1995a; Karron et al., J. Infect. Dis. 172, 1445-1450, 1995b). The live attenuated PIV3 cp45 vaccine candidate was derived from the JS strain of HPIV3 via serial passage in cell culture at low temperature and has been found to be protective against HPIV3 challenge in experimental animals and to be satisfactorily attenuated, genetically stable, and immunogenic in seronegative human infants and children (Belshe et al, J. Med. Virol. 10:235-242, 1982; Belshe et al., Infect. Immun. 37:160-5, 1982; Clements et al., J. Clin. Microbiol. 29:1175-82, 1991; Crookshanks et al., J. Med. Virol. 13:243-9, 1984; Hall et al., Virus Res. 22:173-184, 1992; Karron et al., J. Infect. Dis. 172:1445-1450, 1995b). Because these PIV3 candidate viruses for use in vaccines are biologically derived, there are no proven methods for adjusting the level of attenuation should this be found necessary from ongoing clinical trials.
To facilitate development of PIV vaccines, recombinant DNA technology has recently made it possible to recover infectious negative-stranded RNA viruses from cDNA (for reviews, see Conzelmann, J. Gen. Virol. 77:381-89, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, 1996). In this context, recombinant rescue of infectious virus has been reported for respiratory syncytial virus (RSV), rabies virus (RaV), canine distemper virus, mumps virus, infectious hematopoietic necrosis virus, simian virus 5 (SV5), rinderpest virus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), measles virus (MeV), and Sendai virus (murine parainfluenza virus type 1 (MPIV1)) from cDNA-encoded genomic or antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-81, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell et al., EMBO J. 13:4195-203, 1994; Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-92, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997; Kato et al., Genes to Cells 1:569-579, 1996, Roberts et al., Virology 247:1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; International Publication No. WO 97/06270; Collins et al., Proc. Natl. Acad. Sci. U.S.A. 92:11563-11567, 1995; Clarke et al., J. Virol. 74:4831-4838, 2000; Biacchesi et al., J. Virol. 74:11247-11253, 2000; Gassen et al., J. Virol. 74:10737-10744, 2000; U.S. patent application Ser. No. 08/892,403, filed Jul. 15, 1997 (corresponding to published International Application No. WO 98/02530 and priority U.S. Provisional Application No. 60/047,634, filed May 23, 1997, 60/046,141, filed May 9, 1997, and 60/021,773, filed Jul. 15, 1996); U.S. patent application Ser. No. 09/291,894, filed on Apr. 13, 1999; International Application No. PCT/US00/09695, filed Apr. 12, 2000 (which claims priority to U.S. Provisional Patent Application Ser. No. 60/129,006, filed Apr. 13, 1999); International Application No. PCT/US00/17755, filed Jun. 23, 2000 (which claims priority to U.S. Provisional Patent Application Ser. No. 60/143,132, filed by Bucholz et al. on Jul. 9, 1999); Juhasz et al., J. Virol. 71:5814-5819, 1997; He et al. Virology 237:249-260, 1997; Peters et al. J. Virol. 73:5001-5009, 1999; Baron et al. J. Virol. 71:1265-1271, 1997; Whitehead et al., Virology 247:232-9, 1998a; Whitehead et al., J. Virol. 72:4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; Bucholz et al. J. Virol. 73:251-259, 1999; and Whitehead et al., J. Virol. 73:3438-3442, 1999, each incorporated herein by reference in its entirety for all purposes).
Additional publications in the field of the invention report successful recovery of recombinant parainflunza viruses (PIVs), specifically HPIV2, HPIV3, and BPIV3 (see, e.g., Durbin et al., Virology 235:323-332, 1997; Schmidt et al., J. Virol. 74:8922-8929, 2000; Kawano et al., Virology 284:99-112, 2001; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998 (corresponding to U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997); U.S. Provisional Application No. 60/412,053, filed Sep. 18, 2002; and U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), each incorporated herein by reference). These reports further address genetic manipulation of viral cDNA clones to determine the genetic basis of phenotypic changes in biological mutants, for example, which mutations in a biological mutant HPIV3 (JS cp45) virus specify its ts, ca and att phenotypes, and which gene(s) or genome segment(s) of BPIV specify its attenuation phenotype. Additionally, these and related publications discuss construction of novel PIV vaccine candidates having a wide range of different mutations, as well as methods for evaluating the level of attenuation, immunogenicity and phenotypic stability exhibited by such recombinant vaccine candidates (see also, U.S. application Ser. No. 09/586,479, filed Jun. 1, 2000 (corresponding to U.S. Provisional Patent Application Ser. No. 60/143,134, filed on Jul. 9, 1999); and U.S. patent application Ser. No. 09/350,821, filed by Durbin et al. on Jul. 9, 1999, each incorporated herein by reference).
Thus, infectious wild-type recombinant PIV3, (r)PIV3, as well as a number of ts and other modified derivatives, have now been recovered from cDNA. Reverse genetics systems have been used to generate infectious virus bearing defined mutations that specify attenuation and other desirable phenotypes, and to study the genetic basis of attenuation and other phenotypic changes in existing vaccine candidate viruses. For example, the three amino acid substitutions found in the L gene of cp45, singularly or in combination, have been found to specify the ts and attenuation phenotypes. Additional ts and other attenuating mutations can be introduced in other regions of the PIV3cp45 genome.
In addition, a chimeric PIV1 vaccine candidate has been generated using the PIV3 cDNA rescue system by replacing the PIV3 HN and F open reading frames (ORFs) with those of PIV1 in a PIV3 full-length cDNA, that optionally contains selected attenuating mutations. Exemplary recombinant chimeric viruses derived from these cDNA-based methods include a HPIV3-1 recombinant bearing all three identified mutations in the L gene, rPIV3-1.cp45L (Skiadopoulos et al., J. Virol. 72:1762-8, 1998; Tao et al., J. Virol. 72:2955-2961, 1998; Tao et al., Vaccine 17:1100-1108, 1999, incorporated herein by reference). rPIV3-1.cp45L was attenuated in hamsters and induced a high level of resistance to challenge with PIV. Yet another recombinant chimeric virus, designated rPIV3-1.cp45, has been produced that contains 12 of the 15 cp45 mutations, i.e., excluding the mutations that occur in HN and F. This recombinant vaccine candidate is highly attenuated in the upper and lower respiratory tract of hamsters and induces a high level of protection against HPIV1 infection (Skiadopoulos et al., Vaccine 18:503-510, 1999). However, for use against HPIV1, the immunogenicity of chimeric HPIV3-1 vaccine candidates against HPIV1 challenge is dampened in hosts that exhibit immune recognition of HPIV3.
Recently, a number of studies have focused on the possible use of viral vectors to express foreign antigens toward the goal of developing vaccines against a pathogen for which other vaccine alternatives are not proved successful. In this context, a number of reports suggest that foreign genes may be successfully inserted into a recombinant negative strand RNA virus genome or antigenome with varying effects (Bukreyev et al., J. Virol. 70:6634-41, 1996; Bukreyev et al., Proc. Natl. Acad. Sci. U.S.A. 96:2367-72, 1999; Finke et al. J. Virol. 71:7281-8, 1997; Hasan et al., J. Gen. Virol. 78:2813-20, 1997; He et al., Virology 237:249-60, 1997; Jin et al., Virology 251:206-14, 1998; Johnson et al., J. Virol. 71:5060-8, 1997; Kahn et al., Virology 254:81-91, 1999; Kretzschmar et al., J. Virol. 71:5982-9, 1997; Mebatsion et al., Proc. Natl. Acad. Sci. U.S.A. 93:7310-4, 1996; Moriya et al., FEBS Lett. 425:105-11, 1998; Roberts et al., J. Virol. 73:3723-32, 1999; Roberts et al., J. Virol. 72:4704-11, 1998; Roberts et al., Virology 247:1-6, 1998; Sakai et al., FEBS Lett. 456:221-226, 1999; Schnell et al., Proc. Natl. Acad. Sci. U.S.A. 93:11359-65, 1996a; Schnell et al., J. Virol. 70:2318-23, 1996b; Schnell et al., Cell 90:849-57, 1997; Singh et al., J. Gen. Virol. 80:101-6, 1999; Singh et al., J. Virol. 73:4823-8, 1999; Spielhofer et al., J. Virol. 72:2150-9, 1998; Yu et al., Genes to Cells 2:457-66 et al., 1999; Duprex et al., J. Virol. 74:7972-7979, 2000; Subash et al., J. Virol. 74:9039-9047, 2000; Krishnamurthy et al., Virology 278:168-182, 2000; Rose et al., J. Virol. 74:10903-10910, 2000; Tao et al., J. Virol. 74:6448-6458, 2000; McGettigan et al., J. Virol. 75:8724-8732, 2001; McGettigan et al., J. Virol. 75:4430-4434, 2001; Kahn et al., J. Virol. 75:11079-11087, 2001; Stope et al., J. Virol. 75:9367-9377, 2001; Huang et al., J. Gen. Virol. 82:1729-1736, 2001; Skiadopoulos et al., J. Virol. 75:10498-10504, 2001; Bukreyev et al., J. Virol. 75:12128-12140, 2001; U.S. patent application Ser. No. 09/614,285, filed Jul. 12, 2000 (corresponding to U.S. Provisional Patent Application Serial No. 60/143,425, filed on Jul. 13, 1999), each incorporated herein by reference). When inserted into the viral genome under the control of viral transcription gene-start and gene-end signals, the foreign gene may be transcribed as a separate mRNA and yield significant protein expression. Surprisingly, in some cases foreign sequence has been reported to be stable and capable of expressing functional protein during numerous passages in vitro.
In order to successfully develop vectors for vaccine use, however, it is insufficient to simply demonstrate a high, stable level of protein expression. For example, this has been possible since the early-to-mid 1980s with recombinant vaccinia viruses and adenoviruses, and yet these vectors have proven to be disappointing tools for developing vaccines for human use. Similarly, most nonsegmented negative strand viruses that have been developed as vectors have not been shown to be amenable for human vaccine use. Examples in this context include vesicular stomatitis virus, an ungulate pathogen with no history of administration to humans except for a few laboratory accidents; Sendai virus, a mouse pathogen with no history of administration to humans; simian virus 5, a canine pathogen with no history of administration to humans; and an attenuated strain of measles virus which must be administered systemically and would be neutralized by measles-specific antibodies present in nearly all humans due to maternal antibodies and widespread use of a licensed measles vaccine. Furthermore, some of these prior vector candidates have adverse effects, such as immunosupression, which are directly inconsistent with their use as vectors. Thus, one must identify vectors whose growth characteristics, tropisms, and other biological properties make them appropriate as vectors for human use. It is further necessary to develop a viable immunization strategy, including efficacious timing and route of administration.
Proposed mononegaviruses for use as vaccine vectors include measles, mumps, VSV, and rabies viruses. Each of these virues have serious limitations relating to their potential use as vaccine vectors. For example, measles virus has been considered for use a vector for the protective antigen of hepatitis B virus (Singh et al., J. Virol. 73:4823-8, 1999). However, this combined measles virus-hepatitis B virus vaccine candidate could only be administered after nine months of age, on a schedule comparable to the indicated schedule for the licensed measles virus vaccine, whereas the current hepatitis B virus vaccine is recommended for use in early infancy. This is because the currently licensed measles vaccine is administered parenterally and is sensitive to neutralization and immunosuppression by maternal antibodies, and therefore is not effective if administered before 9-15 months of age. Thus, measles virus is a poor vector for antigens of pathogenic agents that cause disease in early infancy, such as RSV and the HPIVs.
The attenuated measles virus vaccine has been associated with altered immune responses and excess mortality when administered at increased dosages, which may be due at least in part to virus-induced immunosuppression and indicates that even an attenuated measles virus may not be suitable for vaccine vector use. Furthermore, the use of measles virus as a vector would be inconsistent with the global effort to eradicate this pathogen. Indeed, for these reasons it would be desirable to end the use of live measles virus and replace the present measles virus vaccine with a suitable non-measles vector that expresses measles virus protective antigens.
Rabies virus, a rare cause of infection of humans, has been considered for use as a vector (Mebatsion et al., Proc. Natl. Acad. Sci. USA 93:7310-4, 1996), but it is unlikely that a virus that is so highly fatal as rabies for humans could be developed for use as a live attenuated virus vector. Moreover, immunity to the rabies virus, which is not a ubiquitous human pathogen, is not needed for the general population, whereas more desirable vectors should be capable of eliciting a multi specific immune response against both the vector virus and the pathogen for which the vector is used as a carrier of antigenic determinants. While mumps and measles viruses are less pathogenic than the rabies virus, infection by either of these other vector candidates can yield undesirable results. Mumps virus infects the parotid gland and can spread to the testes, sometimes resulting in sterility. Measles virus establishes a viremia with widespread infection and associated rash. Mild encephalitis during mumps and measles infection is not uncommon. Measles virus is also associated with a rare progressive fatal neurological disease called subacute sclerosing encephalitis.
In contrast to such vector candidates as rabies, measles and mumps, PIV infection and disease is typically more limited, in part by confinement of infection to the respiratory tract. Viremia and spread to secondary sites can occur in severely immunocompromised subjects, but this is not a typical effect of PIV infection. Acute respiratory tract disease is the only disease associated with PIVs. Thus, the use of PIVs as vectors will, on the basis of their biological characteristics, avoid complications such as interaction of virus with peripheral lymphocytes, leading to immunosuppression, or infection of secondary organs such as the testes or central nervous system, leading to other complications. These characteristics also render PIV a better vector candidate for successful immunization, which can be achieved more easily and effectively via alternate routes, such as direct administration to the respiratory tract, compared to immunization with vectors that require parental administration.
Among a host of human pathogens for which a vector-based vaccine approach may be desirable is the measles virus. A live attenuated vaccine has been available for more than three decades and has been largely successful in eradicating measles disease in the United States. However, the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries, and the virus contributes to approximately one million deaths per year.
Measles virus is a member of the Morbillivirus genus of the Paramyxoviridae family (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). It is one of the most contagious infectious agents known to man and is transmitted from person to person via the respiratory route (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The measles virus has a complex pathogenesis, involving replication in both the respiratory tract and various systemic sites (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Measles virus is discussed here as an exemplary pathogen for which a live attenuated vector vaccine is particularly desired. For reasons discussed in further detail herein below, a measles vaccine based on a recombinant HPIV1 vector system would satisfy a long-felt need in the art and fulfill an urgent need for additional effective vector systems to generate vaccines against other pathogens as well.
Although both mucosal IgA and serum IgG measles virus-specific antibodies can participate in the control of measles virus, the absence of measles virus disease in very young infants possessing maternally-acquired measles virus-specific antibodies identifies serum antibodies as a major mediator of resistance to disease (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). The two measles virus glycoproteins, the hemagglutinin (HA) and fusion (F) proteins, are the major neutralization and protective antigens (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996).
The currently available live attenuated measles vaccine is administered by a parenteral route (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). Both the wild-type measles virus and the vaccine virus are very readily neutralized by antibodies, and the measles virus vaccine is rendered non-infectious by even very low levels of maternally-acquired measles virus-specific neutralizing antibodies (Halsey et al., N. Engl. J. Med. 313:544-9, 1985; Osterhaus et al., Vaccine 16:1479-81, 1998). Thus, the vaccine virus is not given until the passively-acquired maternal antibodies have decreased to undetectable levels. In the United States, measles virus vaccine is not given until 12 to 15 months of age, a time when almost all children are readily infected with the measles virus vaccine.
As noted above, measles virus continues to exact a heavy toll of mortality in developing countries, especially in children within the latter half of the first year of life (Gellin et al., J. Infect. Dis. 170:S3-14, 1994; Taylor et al., Am. J. Epidemiol. 127:788-94, 1988). This occurs because the measles virus, which is highly prevalent in these regions, is able to infect that subset of infants in whom maternally-acquired measles virus-specific antibody levels have decreased to a non-protective level. Therefore, there is a need for a measles virus vaccine that is able to induce a protective immune response even in the presence of measles virus neutralizing antibodies—with the goal of eliminating measles virus disease occurring within the first year of life as well as that which occurs thereafter. Given this need, there have been numerous attempts to develop an immunization strategy to protect infants in the latter half of the first year of life against measles virus, but none of these strategies has been effective to date.
The first strategy for developing an early measles vaccine involved administration of the licensed live attenuated measles virus vaccine to infants about six months of age by one of the following two methods (Cutts et al., Biologicals 25:323-38, 1997). In one general protocol, the live attenuated measles virus was administered intranasally by drops (Black et al., New Eng. J. Med. 263:165-169; 1960; Kok et al., Trans. R. Soc. Trop. Med. Hvg. 77:171-6, 1983; Simasathien et al., Vaccine 15:329-34, 1997) or into the lower respiratory tract by aerosol (Sabin et al., J. Infect. Dis. 152:1231-7, 1985), to initiate an infection of the respiratory tract. In a second protocol, the measles virus was given parenterally but at a higher dose than that employed for the current vaccine. The administration of vaccines that can replicate on mucosal surfaces has been successfully achieved in early infancy for both live attenuated poliovirus and rotavirus vaccines (Melnick et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 655-712. 2 vols. Lippencott-Raven Publishers, Philadelphia, 1996; Perez-Schael et al., N. Engl. J. Med. 337:1181-7, 1997), presumably because passively-acquired IgG antibodies have less access to mucosal surfaces than they do to systemic sites of viral replication. In this situation, the live attenuated poliovirus vaccine viruses are able to infect the mucosal surface of the gastrointestinal tract or the respiratory tract of young infants, including those with maternal antibodies, resulting in the induction of a protective immune response.
Therefore, a plausible method for measles immunization is to administer a live attenuated measles virus vaccine to the respiratory tract of the young infant, since this is the natural route of infection for the measles virus. However, the live attenuated measles virus that is infectious by the parenteral route was inconsistently infectious by the intranasal route (Black et al., New Eng. J. Med. 263:165-169, 1960; Cutts et al., Biologicals 25:323-38, 1997; Kok et al., Trans. R. Soc. Trop. Med. Hyg. 77:171-6, 1983; Simasathien et al., Vaccine 15:329-34, 1997), and this decreased infectivity was especially apparent for the Schwartz stain of measles virus vaccine which is the current vaccine strain. Presumably, during the attenuation of this virus by passage in tissue culture cells of avian origin, the virus lost a significant amount of infectivity for the upper respiratory tract of humans. Indeed, a hallmark of measles virus biology is that the virus undergoes rapid changes in biological properties when grown in vitro. Since this relatively simple route of immunization was not successful, a second approach was tried involving administration of the live virus vaccine by aerosol into the lower respiratory tract (Cutts et al., Biologicals 25:323-38, 1997; Sabin et al., J. Infect. Dis. 152:1231-7, 1985).
Infection of young infants by aerosol administration of measles virus vaccine was accomplished in highly controlled experimental studies, but it has not been possible to reproducibly deliver a live attenuated measles virus vaccine in field settings by aerosol to the young infant (Cutts et al., Biologicals 25:323-38, 1997). In another attempt to immunize six-month old infants, the measles vaccine virus was administered parenterally at a 10- to 100-fold increased dose (Markowitz et al., N. Engl. J. Med. 322:580-7, 1990). Although high-titer live measles vaccination improved seroconversion in infants 4-6 months of age, there was an associated increase in mortality in the high-titer vaccine recipients later in infancy (Gellin et al., J. Infect. Dis. 170:S3-14, 1994; Holt et al., J. Infect. Dis. 168:1087-96, 1993; Markowitz et al., N. Engl. J. Med. 322:580-7, 1990) and this approach to immunization has been abandoned.
A second strategy previously explored for a measles virus vaccine was the use of a formalin inactivated whole measles virus or a subunit virus vaccine prepared from measles virus (Griffin et al., In "Fields Virology", B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus, Eds., Vol. 1, pp. 1267-1312. Lippincott-Raven Publishers, Philadelphia, 1996). However, the clinical use of these vaccines in the 1960's revealed a very serious complication, namely, that the inactivated virus vaccines potentiated disease rather than prevented it (Fulginiti et al., JAMA 202:1075-80, 1967). This was first observed with formalin-inactivated measles virus vaccine (Fulginiti et al., JAMA 202:1075-80, 1967). Initially, this vaccine prevented measles, but after several years vaccinees lost their resistance to infection. When subsequently infected with naturally circulating measles virus, the vaccinees developed an a typical illness with accentuated systemic symptoms and pneumonia (Fulginiti et al., JAMA 202:1075-80, 1967; Nader et al., J. Pediatr. 72:22-8, 1968; Rauh et al., Am. J. Dis. Child 109:232-7, 1965). Retrospective analysis showed that formalin inactivation destroyed the ability of the measles fusion (F) protein to induce hemolysis-inhibiting antibodies, but it did not destroy the ability of the HA (hemagglutinin or attachment) protein to induce neutralizing antibodies (Norrby et al., J. Infect. Dis. 132:262-9, 1975; Norrby et al., Infect. Immun. 11:231-9, 1975). When the immunity induced by the HA protein had waned sufficiently to permit extensive infection with wild-type measles virus, an altered and sometimes more severe disease was seen at the sites of measles virus replication (Bellanti, Pediatrics 48:715-29, 1971; Buser, N. Engl. J. Med. 277:250-1, 1967). This a typical disease is believed to be mediated in part by an altered cell-mediated immune response in which Th-2 cells were preferentially induced leading to heightened disease manifestations at the sites of viral replication (Polack et al., Nat. Med. 5:629-34, 1999). Because of this experience with nonliving measles virus vaccines and also because the immunogenicity of such parenterally-administered vaccines can be decreased by passively-transferred antibodies, there has been considerable reluctance to evaluate such vaccines in human infants. It should be noted that disease potentiation appears to be associated only with killed vaccines.
An alternative approach to development of a vaccine vector for measles employed a replication-competent vesicular stomatitis virus (VSV), a rhabdovirus which naturally infects cattle but not humans, expressing the measles virus HA protein. This vector candidate virus was shown to replicate in the respiratory tract of animal hosts (Roberts et al., J. Virol. 73:3723-32, 1999; Schnell et al., Proc. Natl. Acad. Sci. U.S.A. 93:11359-65, 1996a). However, since VSV is an animal virus that can cause disease in humans, development of this recombinant vector for use in humans will first require that a VSV backbone that is satisfactorily attenuated in human infants be first identified (Roberts et al., J. Virol. 73:3723-32, 1999).
Yet another strategy that has been explored for developing a vaccine against measles for use in young infants has been the use of viral vectors to express a protective antigen of the measles virus (Drillien et al., Proc. Natl. Acad. Sci. U.S.A. 85:1252-6, 1988; Fooks et al., J. Gen. Virol. 79:1027-31, 1998; Schnell et al., Proc. Natl. Acad. Sci. U.S.A. 93:11359-65, 1996a; Taylor et al., Virology 187:321-8, 1992; Wild et al., Vaccine 8:441-2, 1990; Wild et al., J. Gen. Virol. 73:359-67, 1992). A variety of vectors have been explored including pox viruses, such as the replication-competent vaccinia virus or the replication-defective modified vaccinia virus Ankara (MVA) stain. Replication-competent vaccinia recombinants expressing the F or HA glycoprotein of measles virus were efficacious in immunologically naive vaccinees. However, when they were administered parenterally in the presence of passive antibody against measles virus, their immunogenicity and protective efficacy was largely abrogated (Galletti et al., Vaccine 13:197-201, 1995; Osterhaus et al., Vaccine 16:1479-81, 1998; Siegrist et al., Vaccine 16:1409-14, 1998; Siegrist et al., Dev. Biol. Stand. 95:133-9, 1998).
Replication-competent vaccinia recombinants expressing the protective antigens of RSV have also been shown to be ineffective in inducing a protective immune response when they are administered parenterally in the presence of passive antibody (Murphy et al., J. Virol. 62:3907-10, 1988a), but they readily protected such hosts when administered intranasally. Unfortunately, replication-competent vaccinia virus recombinants are not sufficiently attenuated for use in immunocompromised hosts such as persons with human immunodeficiency virus (HIV) infection (Fenner et al., World Health Organization, Geneva, 1988; Redfield et al., N. Engl. J. Med. 316:673-676, 1987), and their administration by the intranasal route even to immunocompetent individuals would be problematic. Therefore they are not being pursued as vectors for use in human infants, some of whom could be infected with HIV.
The MVA vector, which was derived by more than 500 passages in chick embryo cells (Mayr et al., Infection 3:6-14, 1975; Meyer et al., J. Gen. Virol. 72:1031-1038, 1991), has also been evaluated as a potential vaccine vector for the protective antigens of several paramyxoviruses (Durbin et al., J. Infect. Dis. 179:1345-51, 1999a; Wyatt et al., Vaccine 14:1451-1458, 1996). MVA is a highly attenuated host range mutant that replicates well in avian cells but not in most mammalian cells, including those obtained from monkeys and humans (Blanchard et al., J. Gen. Virol. 79:1159-1167, 1998; Carroll et al., Virology 238:198-211, 1997; Drexler et al., J. Gen. Virol. 79:347-352, 1998; Sutter et al., Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851, 1992). Avipox vaccine vectors, which have a host range restriction similar to that of MVA, also have been constructed that express measles virus protective antigens (Taylor et al., Virology 187:321-8, 1992). MVA is non-pathogenic in immunocompromised hosts and has been administered to large numbers of humans without incident (Mayr et al., Zentralbl. Bakteriol. [B] 167:375-90, 1978; Stickle et al., Dtsch. Med. Wochenschr. 99:2386-92, 1974; Werner et al., Archives of Virology 64:247-256, 1980). Unfortunately, both the immunogenicity and efficacy of MVA expressing a paramyxovirus protective antigen were abrogated in passively-immunized rhesus monkeys whether delivered by a parenteral or a topical route (Durbin et al., Virology 235:323-332, 1999). The immunogenicity of DNA vaccines expressing measles virus protective antigens delivered parenterally was also decreased in passively-immunized hosts (Siegrist et al., Dev. Biol. Stand. 95:133-9, 1998). Replication-defective vectors expressing measles virus protective antigens are presently being evaluated, including adenovirus-measles virus HA recombinants (Fooks et al., J. Gen. Virol. 79:1027-31, 1998). In this context, MVA recombinants expressing parainfluenza virus antigens, unlike replication-competent vaccinia virus recombinants, lacked protective efficacy when given by a mucosal route to animals with passively-acquired antibodies, and it is unlikely that they, or the similar avipox vectors, can be used in infants with maternally-acquired measles virus antibodies. Based on these reports, it is not expected that poxvirus vectors or DNA vaccines expressing a measles virus protective antigens will be satisfactorily immunogenic or efficacious in infants that possess passively-acquired maternal measles virus-specific antibodies.
More recent developments in the field of negative stranded RNA viral vaccines have involved the use of HPIV3-based vaccine vectors to deliver antigenic determinants of heterologous pathogens, including heterologous PIVs. In particular, recombinant HPIV3 vaccine candidates have been disclosed that use a HPIV3 "vector" genome or antigenome combined with one or more heterologous genes of a different PIV, or of a non-PIV pathogen to form a chimeric, bivalent or multivalent, HPIV3 vaccine candidate (see, e.g., Durbin et al., Virology 235:323-332, 1997; Skiadopoulos et al., J. Virol. 72:1762-1768, 1998; Skiadopoulos et al., J. Virol. 73:1374-1381, 1999; Tao et al., Vaccine 19:3620-3631, 2001; Durbin et al., J. Virol. 74:6821-6831, 2000; U.S. patent application Ser. No. 09/083,793, filed May 22, 1998; U.S. patent application Ser. No. 09/458,813, filed Dec. 10, 1999; U.S. patent application Ser. No. 09/459,062, filed Dec. 10, 1999; U.S. Provisional Application No. 60/047,575, filed May 23, 1997 (corresponding to International Publication No. WO 98/53078), U.S. Provisional Application No. 60/059,385, filed Sep. 19, 1997; U.S. Provisional Application No. 60/170,195 filed Dec. 10, 1999; and U.S. patent application Ser. No. 09/733,692, filed Dec. 8, 2000 (corresponding to International Publication No. WO 01/42445A2), each incorporated herein by reference. The recombinant HPIV3 viruses are engineered to incorporate one or more heterologous donor sequences encoding one or more antigenic determinants of a different PIV or heterologous pathogen to produce an infectious, chimeric, bivalent or multivalent virus or subviral particle. In this manner, candidate HPIV3-based chimeric vaccine viruses can be made to elicit an immune response against one or more PIVs or a polyspecific response against a selected PIV and a non-PIV pathogen in a mammalian host susceptible to infection therefrom. Various modifications to chimeric HPIV3 vaccine candidates are reported to yield desired phenotypic effects, such as attenuation.
Although there have been numerous advances toward development of effective vaccine agents against PIV and other pathogens, including measles, there remains a clear need in the art for additional tools and methods to engineer safe and effective immunogenic compositions to alleviate the serious health problems attributable to these pathogens, particularly among young infants. Among the remaining challenges in this context is the need for additional tools to generate suitably attenuated, immunogenic and genetically HPIV1 candidates for use in diverse clinical settings against one or more pathogens. Additional challenges arise from the fact that HPIV1, HPIV2, and HPIV3 represent distinct viral serotypes, that do not elicit significant cross-protective immunity. Accordingly, there is an urgent need in the art for new immunogenic compositions and methods directed against multiple HPIV serotypes to treat, prevent, or alleviate the frequency or severity of the serious lower respiratory tract disease and the otitis media that accompanies different HPIV infections. To facilitate these goals, existing methods for identifying and incorporating attenuating mutations into recombinant viral strains and for developing vector-based immunogenic compositions and immunization methods must be expanded. In this context, it is particularly desirable to develop a method for recovery and genetic manipulation of HPIV1, to generate immunogenic compositions to elicit immune responses against this important human PIV, and to provide additional tools to generate novel vectors and immunization methods. Surprisingly, the present invention satisfies these needs and fulfills additional objects and advantages as described herein below.

Recovery of recombinant human parainfluenza virus type 1 (HPIV1) from cDNA and use of recombinant HPIV1 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens