The end objective is an operational system that detects new viral ourbreaks as early as is technically feasible, regardless of whether the infectious agents is known or new, performs the analytical studies requires as expeditiously as is technically possible, and provides primers, vaccine candidates, convalescent antibodies, and vaccines within the time frame of a viral outbreak. The stated end, (RD/RR), may be readhed either by original research and development, collaborative efforts, license, or purchase.
A major hurdle in such integrative work are intellectual property rights. From the not-for-profit base of the Viral Defense Foundation, we believe these problems can be solved, especially in view of the overshadowing fact that national concerns ultimately trump private ones. Conflicting interests - our own and others - must be resolved in the quests for efficiency, reliability and speed.
Note that a major contribution of the VDF may be to define important projects that can be done, regardless of whether we have any part in the eventual outcome or not.
Project 1. Global Screen
Project Global Screen has been outlined in detail in a curet article in Emerging Infectious Diseases included in this web site. The elements of this project are:
1.1 Development of a database of the physical properties of known viruses.
1.2 Organization of collection of pooled human serum samples on a timely basis. This includes the development of arrangements, shipping conditions, and storage methods applicable to this problem. As the project progresses, the problem of sub-pool organization will be addressed to allow the geographical source of new infectious agents to be determined.
1.4 Experimental evaluation of alternative methods of virus isolation from pooled sera. This will required minor modifications to either filtration systems or centrifugal system. Safety is a major concern, and automated cleaning and sterilization in place systems will be required.
1.5 Organization of an operational facility for the routine preparation of viral concentrates.
1.6 Development or choice of means for converting viral concentrates into genomic libraries for sequencing.
1.7 Organization of a network for sequencing viral genomic libraries rapidly and routinely.
1.8 Organization of the data analysis and reduction facilities required to rapidly turn analytical data into useful epidemiological information.
1.9 Development of an interface with Projects 2 and 3, to take newly discovered virion sequence data into clinical use in the form of primers and antigens.
1.10 Organization of meetings and symposia to promote information and technology transfer in this project.
Project 2. Rapid Response: Convalescent Antibodies
The basic methods for the commercial fractionation of human plasma were developed during WWII, and have not been modified in a major way since. The methods are largely frozen in regulations, and it is generally agreed that these cannot be modified in major ways under existing regulations except at high cost. In this project we attempt to examine what could be accomplished now with current technology, with the assumption that in times of a really major crises, the same conditions that so effectively stimulate innovation during wartime will return.
2.1 Development of single-blood-unit devices and methods that allow convalescent immunoglobulins to be recovered, free of infectious particles, to the limits technically feasible, so that experimental studies on the effectiveness of such preparations may be undertaken early in an epidemic. In a really serious outbreak it is essential that there be some hope based on reality, and this project could provide it. Obviously the technology involved would be developed to the point where there was minimal risk to the technicians involved, or of transfusing virus with antibodies
2.2 Development of intermediate scales immunoglobulin isolation and purification systems for use with intermediate-sized batches of plasma or serum.
It is evident that evaluation of the use of convalescent antibodies requires some rapid objective test for efficacy. These are described in Project 3.
Note that one of the major health care problems associated with a new lethal outbreak is the retention of patient-contact staff. Therefore some immediate means for protecting a relatively small number of individuals is required, and for positively identifying and rewarding those who are immune and not at risk. Hence one must return to Pasteur and initiate on-the-spot experiments that are preplanned and also pre-approved.
Certified convalescent patient-contact staff need analytical assurance of safe passage, and should be identified by a special ring or badge. Individuals willing to risk care delivery should be transfused with convalescent antibodies as soon as available, and carefully monitored for possible infection. As a group, if the infection rate is lower than in controls, the process of producing and using such antibodies may be continued.
The efficacy of convalescent antibodies during an infection can obviously be best tested during an outbreak, and would start with the most severely ill.
It is essential that the procedures and technologies associated with rapid antibody production be available at the outset of an outbreak.
Project 3. Rapid Physical Isolation of Viruses Non-Plasma Sources
If intact virions can be isolated in a high state of purity early in the course of an outbreak, these can be used to develop a variety of immunologically based assays, even before PCR primers can be developed based on sequence data.
The viral load in affected tissues is generally much higher than in serum or plasma. Large quantities of virus are made during febrile stages, but are rapidly removed into lymphocytes, i.e., turn over rapidly. Further, the infected tissues are generally the sources of the virus in the first place and would be expected to contain the largest amount. In any serious viral outbreak, especially with high mortality, relatively large amounts of infected tissue will be available. While relatively little hard information is available on tissue viral loads, it is suggested that, using S-? technology and large zonal rotors, milligram quantities of virus will be available and can be isolated. (Estimates of tissue viral loads, tissue average mass, and hence more precise estimates are presented elsewhere).
With such material it is feasible to assay convalescent antibody preparations for activity, and to develop simple assays to identify individuals with circulating antibodies against a new virus.
Project 4. Rapid Assay Development
Many individuals, groups and companies can rapidly produce sensitive assays given antigens and antibodies, or even antigens alone. Hence the important thing is to provide these as quickly as possible.
If well-developed quantitative PCR methods are available, then, given the known average serum viral load for SARS, for example, it will be possible to detect the virus contributed by one individual in a 100 liter batch of pooled 0.5 ml samples (from 200,000 individuals using the technology described for Project Global Screen). Hence in any new outbreak, the development of sensitive, specific detection methods will be essential to follow the course of an outbreak. It is important that some centralized laboratory exist to cross evaluate new tests, and to rapidly apply them.
Project 5. Rapid Vaccine Development
To prevent the chaos of a new lethal epidemic, of whatever origin, it is essential that some credible hope of protection and treatment exist.
It is important to state clearly here that if a new (or modified existing) agent suddenly appeared, spread naturally or artificially with the efficiency of smallpox, produced the same mortality, and were unchecked to the end, the course of human civilization would be dramatically and permanently altered. The two contributing factors are the death rate itself, and chaotic behavior in the almost total absence of credible hope. Small groups of individuals can and have borne up under mortalities of 30% or higher, but not without some glimmer of hope.
And that means an effective antiviral or vaccine very rapidly produced.
George Washington sometimes had green troops that were not immune to smallpox. When there was little action he arranged for them to be variolated - i.e., to have the exudates from the skin of individuals ill with smallpox placed under a flap of skin. The result was a mortality rate of about 1% (compared to ~30% for natural infections) probably due to the difference between the response of the immune systems to a large antigen dose, and the relatively long incubation period for the virus. The result was that the troops were immune to smallpox.
Pasteur depended on a a somewhat analogous process when he prepared serial injections of the spinal cords of rabies infected rabbits dried for decreasing periods of time. The original Merck HeptaVax was prepared by isolating active antigenic particles from the serum of individuals chronically infected with hepatitis B virus. K-II centrifuges, originally developed at Oak Ridge, were used in the process. This vaccine has been replaced by a recombinant vaccine produced in yeast. Thus there is a history of using vaccines prepared from human and animal sources to immunize human patients.
Given a totally new virus, and sufficient masses of the virus isolated from tissues, it should be feasible to prepare an inactivation vaccine series and, starting with the most inactivated (for example with formalin), to produce effective and measurable immunity. Essential to this is a technology for virus isolation, and tests for antibody production.
The objective would be initially to protect contact health care workers, and then to vaccinate a larger circle of individuals around outbreaks. Vaccinated individuals are also a source of more antibodies.
It is difficult to estimate the number of virions actually required to make a killed vaccine, since doses are usually determined with reference to a standard that is not a physical one. However, the dose is generally in microgram to nanogram range, and 108 polio virus particles weigh about 40 ng. In some viremias 108 particles are present per ml, and often more per gram of infected tissue. It appears technically feasible, therefore, to make vaccine in extremis from human sources.
While direct physical isolation of virus particles may provide a very rapid means for vaccine production, it must ultimately be possible to develop recombinant vaccines rapidly. Once a virus is sequenced, the genes for many of the virion capsid proteins may be identified, and candidate sequences for antigenic sites identified. A rapid means for expressing these is in tobacco plants using methods developed to make vaccines for non-Hodgkins lympoma. These vaccines can be effective, but, when made using conventional methods, often require more time to make than the remaining life of the patient. Hence attempts, some partially successful, to produce the vaccines in tobacco in only a few weeks. The production of vaccine-producing plants can be rapidly scaled up in large fields and conventional agricultural methods. Other means for rapid expression of viral genes also exist, and all should be explored and intercompared.
If any response is to work, or can be made to work, it is essential to time-shrink every step in the process maximally, since thousands of lives may be lost at the end due to time lost at the beginning.
None of these systems can be stitched together after an outbreak occurs. All must be available under "Hot Start" conditions, as will be the case with antiballistic missiles. They must be proven out, and must be on constant operation to demonstrate reliability, and to develop new vaccines against existing threats. It may require one major lethal epidemic before such systems are set in place.
Project 6. Safe Automation
As noted in the attached article in Emerging Infectious Diseases, biological containment originated in nuclear energy laboratories and was then transferred to biological warfare and other biological laboratories. Level 4 biocontainment now puts the operator in a full "space suit" for protection, and uses largely standard laboratory equipment for experimental work. In the nuclear energy field, in contrast, the cells or safe compartments are "hot", and are remotely controlled and operated. The operator is never exposed to the conditions in the cell. The same concepts of remote operation are employed in unmanned spacecraft where the physical operation is often separated from control and data reduction. In clinical chemistry, what were once manual operations are now almost universally mechanized, robotic and automated.
In this project, where many operations must ultimately be standardized, repeatable, and rapidly performed, we proposed that a new level of safety (Level 6) be adopted in which all operations were performed in a contained, sterilizable, remotely controlled environment, and have proposed some of the details of its construction.
A widespread assumption in bioterrorism has been that many individuals, groups, and levels of government and industry would separately develop, control, and manufacture numerous independed systems and laboratories, possibly all under separate grants. The approaches described here have the singular defect of moving contrary to this approach, following rather the approaches ultimately found to be successful in space, military weapons development, and in the nuclear weapons field. These involve definition of objectives, trolling for the best technology, integrated development and evaluation, and ultimately, production and emplacement of systems proven to work.
New viral diseases, or natural or human origin, constitute the largest credible threat to the health and welfare of the United States. There is at present no corresponding rapid detection and response system or program commensurate to the danger.
Project 7. Virion Database
While several databases concerned with viral nucleic acid sequences and capsid protein composition exist, there is little integrated literature on the biophysical properties of viruses of interest here, and on viral and tissue loads as a function of time after infection. A database covering these subjects is under construction.