Jonas E. Salk

Experimental allergic encephalomyelitis: An encephalitogenic basic protein from bovine myelin

Abstract A homogeneous encephalitogenic protein was prepared in high yield (35–40%) from bovine spinal cord and myelin. This protein, referred to as A1 protein, was found to be susceptible to proteolysis in situ at pH 6–7; this effect was arrested by prompt freezing of tissue upon removal from the animal. The purification procedure consisted of (1) defatting with chloroform-methanol (2:1); (2) acid extraction at pH 1.7; (3) precipitation of contaminating protein and dialysis at pH 7; (4) DEAE-cellulose chromatography; and finally, (5) gel filtration. CM-cellulose or Cellex-P chromatography was also used for final purification in place of gel filtration. The DEAE eluate contained the basic protein fraction of which the A1 protein comprised 80–95%. The data suggest that the A1 protein is very likely the only encephalitogenic basic protein of native myelin, constituting approximately 30% of the total myelin protein. It has been obtained in homogeneous form as demonstrated by a single sharp band on polyacrylamide gel electrophoresis at pH 4.5 and 8.6, and by immunoelectrophoresis and immuno-double diffusion. As little as 0.1 μg induced histologic lesions in the CNS and 10–100 μg induced clinical disease in 80–100% of guinea pigs. In addition to its encephalitogenic activity the A1 protein was found to induce in rabbits and guinea pigs the formation of precipitating antibody which was detected by immuno-double diffusion and assayed in a passive hemagglutination test. Antibody-combining activity of the A1 protein was also demonstrated by immuno-double diffusion and was measured by a passive hemagglutination-inhibition test. The delayed hypersensitive response developed in guinea pigs after injection of 20 μg of the A1 protein and was demonstrated using 5 μg as skin test antigen. The positive skin test and histologic lesions characterizing EAE both appeared on days 4–5 after sensitization and developed simultaneously. The principal findings here reported, on the in situ proteolysis of the encephalitogenic basic protein, provide a partial explanation for the reports from different laboratories of encephalitogens possessing widely differing properties.

Formaldehyde treatment and safety testing of experimental poliomyelitis vaccines.

an experimental vaccine, and for safety testing, as yet have not been published. It is the purpose of this communication to discuss the principles underlying the procedures being followed in preparing material for more extensive studies than have been carried out thus far; essentially, this represents a discussion and an elaboration of the specifications which have been prepared for processing the vaccine for this purpose. A fuller presentation and documentation of details here referred to will be covered in several reports to be made in the appropriate technical journals.

Initial studies on active immunization of HIV-infected subjects using a gp120-depleted HIV-1 Immunogen: long-term follow-up.

In 1987, exploratory clinical studies were initiated to determine whether the development of AIDS in HIV-infected individuals might be delayed or prevented by immunization with an inactivated HIV preparation. Preclinical studies had shown the preparation to be safe and immunogenic. Twenty-three patients with biopsy-confirmed persistent generalized lymphadenopathy (CDC III) and two with asymptomatic HIV infection and CD4 lymphocyte counts between 135 and 769/mm3 were studied, of whom eight (32%) had additional HIV-related symptoms. Over a 3-year period, they received a median of eight open-label inoculations of 100 micrograms of inactivated gp 120-depleted HIV-1 Immunogen in incomplete Freunds adjuvant (IFA). Clinical, general laboratory, immunologic, and virologic parameters were followed for up to 6 years. No serious treatment-related adverse experiences were reported, nor was accelerated HIV disease progression seen. Twelve patients developed a delayed-type hypersensitivity response (HIV-DTH) to the immunogen and nine showed fourfold or greater increases in anti-p24 antibody titers. In the follow-up period, 10 of the 25 patients developed AIDS and one with Kaposis sarcoma (KS) at baseline progressed. Of the 12 patients who became HIV-DTH-responsive, one developed an opportunistic infection (OI), occurring approximately 5 years from study onset, and subsequently died. One additional HIV-DTH responder developed KS. Of the 13 patients who remained HIV-DTH-nonresponsive, nine (69%) progressed to AIDS and seven of these have died. Differences were also observed in terms of HIV-DNA copy number, CD4 percentages, and anti-p24 antibody patterns between the HIV-DTH-responsive and -nonresponsive groups, suggesting a more favorable clinical course in the former. HIV-1 Immunogen in IFA appears to be safe and immunogenic. Further studies are indicated to determine clinical efficacy of the HIV Immunogen as well as the significance of the apparent correlation between HIV-DTH responsivity and a more favorable clinical course.

Development and evaluation of a vaccine for human immunodeficiency virus (HIV) infection

The development of a safe and effective vaccine for infection with human immunodeficiency virus (HIV) is complicated by several unique scientific, logistic, and ethical issues. These issues include a lack of understanding of protective immunity to HIV and disease development, the absence of an adequate and convenient animal model for studying HIV infection, and difficulties in phase III evaluation of candidate vaccines. Because HIV can be transmitted as either a cell-free or cell-associated virus, a protective immune response against HIV infection will likely require both humoral and cell-mediated immunity. A neutralizing antibody against HIV and an antibody involved in antibody-dependent cellular cytotoxicity have been shown in HIV-infected persons, but their precise relation to protection is unclear. Cytotoxic lymphocytes from HIV-infected persons have been shown to lyse target cells expressing HIV or its proteins. Cloned T cells have been developed that manifest HIV-specific, major histocompatibility-complex class I-restricted cytotoxic capabilities that are broadly specific. Thus far, all attempts to protect chimpanzees, currently the only suitable animal model, from HIV infection have failed. Ongoing vaccine studies in humans include phase I trials of recombinant proteins of the HIV envelope in uninfected persons as well as the administration of whole killed virus to persons already infected with HIV. Rapid progress is being made in the development of new animal models for HIV infection. The establishment of alternative animal models, both primate and small animal models, will greatly facilitate the development of a vaccine for HIV infection.

INDUCTION OF LONG-TERM IMMUNITY TO PARALYTIC POLIOMYELITIS BY USE OF NON-INFECTIOUS VACCINE

Naturally acquired immunity to paralytic poliomyelitis, which is of long duration, is associated with serum antibody, immunological memory, or both. Persistence of circulating antibody is reassuring but not essential for long-term protection. Immunological memory induced by killed poliovirus vaccine is similar to that induced by infection, which is not a prerequisite for the induction of long-term immunity. Animal studies of experimental vaccines indicate that some antigenic components of poliovirus induce immunological memory without producing detectable antibody; killed poliovirus vaccine has the same effect in man. Killed vaccine containing 40, 8, and 32 D-antigen units of poliovirus types 1, 2, and 3, respectively, protects all recipients in both one-dose and two-dose schedules. This means that either schedule can be chosen to fit in with local immunisation programmes and thus reduce costs and increase population coverage.

Vaccinology of poliomyelitis

Vaccinology requires not only an understanding of the fundamental properties of the immune system and specific immunogens, but also the application of basic knowledge to develop effective immunization programmes for particular social, political, and environmental conditions. Two problems exist in the use of oral, live poliovirus vaccine: the occurrence of vaccine-associated paralytic poliomyelitis and the uncertain antibody response in tropical climates. These difficulties can be overcome by the use of killed poliovirus vaccine formulated to be effective with a single dose. Application of basic principles can bring poliomyelitis fully under control in all parts of the world.

Immunologic memory induced at birth by immunization with inactivated polio vaccine in a reduced schedule.

Abstract of inactivated polio vaccine (IPV) of enhanced potency. Following the administration of a second vaccine dose six months later, a considerable proportion of babies responded with neutralizing antibody (NA) to the three poliovirus types. The very rapid occurrence and high antibody titer were indicative of an anamnestic response. Twenty-one infants who still had NA < 1:4 to one-more poliovirus types after the second vaccine dose responded with very high NA values 7–10 days after a supplementary dose of IPV. It appears that IPV of enhanced potency administered at birth is apt to induce immunologic memory, which should provide the basis for protection against paralytic poliomyelitis in case of exposure to wild poliovirus later in life.

IMMUNOLOGICAL PARADOXES: THEORETICAL CONSIDERATIONS IN THE REJECTION OR RETENTION OF GRAFTS, TUMORS, AND NORMAL TISSUE

I am very pleased to have been exposed to such a wide variety of viewpoints here. It seems to me that the conference has been extremely successful in bringing together individuals who, although from such diverse points of view, have a common interest. The remarks that I have to make are of two kinds: First, to comment upon what I heard this morning, regretfully, without time for sufficient quiet thought such as one would like to have in pondering the information presented this morning, and, second, to present a few thoughts of my own that have developed over a number of years and that I think will fit into the context of this morning’s discussion. Dr. Bennette’s remarks were rather poetic, and I believe describe a process that in fact does go on in nature. The problem with which the experimental scientist is confronted is to try to relate structure and function in biological systems, to try to define the actual mechanism that is involved, and especially in ways that can be manipulated. When Dr. Weiss pointed out that there really is no fundamental distinction between a spontaneous neoplasm and one that is induced experimentally, he could very well have been clarifying the use of the term “spontaneous” as applied to mammary tumors of mice, as if they were different from the tumors induced by the milk factor or by the mammary tumor virus. The very brilliant analysis by Dr. Eva Klein should have revealed to you what can be done with a very heterogenous system, all lumped together under the same term “cancer” or “neoplasia,” when carefully analyzed. And, therefore, it does illustrate what-not to use the word disparagingly-a reductionist approach can do in an attempt to isolate and identify the particular factors that are crucial even in systems in which a multiplicity of factors are involved. I hope to illustrate this a bit further myself. What she was able to demonstrate is that certain laws do govern the relationships between structure and function. She showed very nicely how differently tumors induced by viruses behave from those that are chemically induced. She showed experimentally and quantitatively that 100% effects do not always occur. This tempts us further to try to look more precisely into the events that are involved. It says that we do not as yet have all the answers, and it is this that challenges the experimentalist who tries to find out much more precisely what variables are involved and tries thereby to reveal a means for controlling this natural phenomenon. Fundamentally, this is what we are all attempting to understand, and to be able to do, whether it be through the use of the tools of the immunologist, the virologist, or the psychiatrist. The problem with which this conference seems to be confronted is that of developing an understanding of the relative roles played by each of the multiplicity of factors that we presume, or that we know, are operative in neoplasia. Dr. Bahnson’s interests are broad, his bias is clear. At the same time, he does demonstrate an open mind in regard to the many factors that undoubtedly play a role in a disease process, the exact or precise nature of which is not yet clear

Antigenic Activity of Poliomyelitis Vaccines undergoing Field Test.

are of the opinion that lifelong immunity can best be provided through the use of a living attenuated virus for each of the three immunologic types and administered, preferably, by a natural route. If this is not a precise expression of the opinions of all whose predominant orientation is one way or the other, we believe it does convey the essential features of the two points of view and there are reasonable reasons for believing that either may lead to the solution of the practical problem of immunization of man. We have accepted the assumption that immunity is mediated principally, -or entirely, through the action of antibody. It might then be expected, in accordance with well established immunologic principles, that the presence of antibody either in the circulating blood, or within fluids bathing neural tissue-or the existence of a hyperreactive state of the antibody forming system, resulting from either natural or artificial immunization-might provide the modus operandi for effective immunity. The question then becomes whether or not a noninfectious vaccine can produce these effects, or do these effects result exclusively from contact with living virus? This question has been answered in part. It is now amply evident that the injection of noninfectious virus can simulate at least some of the effects resulting from infection with the living virus. The question that now remains is whether or not the kind of immunity that accompanies the serologic response to infection with living virus is the same or different from that which results from the injection of a noninfectious antigen. While it is true that the immune response induced by the infectious process results from multiplication of the living virus, the immune response to a noninfectious virus requires the administration of a sufficient quantity of effective antigen, given in such a way that the

Viral load, CD4 percentage, and delayed-type hypersensitivity in subjects receiving the HIV-1 immunogen and antiviral drug therapy

Two trials of subjects inoculated with the inactivated, gp120-depleted HIV-1 Immunogen are reported. In one study, in which 19 subjects received ZDV and 8 subjects received ddI, treatment with the HIV-1 Immunogen did not affect the pharmacokinetic parameters of the antiviral drugs. In another study, 65 subjects who were previously immunized with the HIV-1 Immunogen over a mean period of 4.0 years (range, 1.2–5.4 years) received inoculations at 0 and 6 months. At some point during this 48-week study, 72% of the subjects (47/65) were receiving antiviral drug therapy. The HIV-1 DNA load in CD4 cells and CD4 percentage were found to be stable over the 48-week period. Delayed-type hypersensitivity to HIV-1 antigens increased after two inoculations with the HIV-1 Immunogen. In these two trials, no serious treatment-related adverse events were documented in the subjects. The two studies presented herein are the first to suggest that an immune-based therapy such as the HIV-1 Immunogen can be combined safely with antiviral drugs, supporting further study to evaluate the clinical utility of this approach.