William F. Friedewald

Factors Influencing the Inactivation of the Rabbit Papilloma Virus by X-Rays

Lacassagne found several years ago that the rabbit-papilloma virus is notably resistant to X-rays, 1 and Syverton has more recently reported that a dose of 14,000,000 r was required to abolish the infectivity of a cell-free suspension of the papilloma virus 2 though 1,000,000 r or less sufficed to inactivate certain other viruses and bacteria. In experiments undertaken lately for other purposes we have noted that the amount of irradiation required to inactivate the papilloma-virus is influenced by the virus concentration, as also by extraneous material present along with it in the irradiated suspensions. Berkefeld filtrates, which contained the virus in large amount, were rendered completely non-infectious upon exposure to 2 to 4 million r of irradiation, while only 400,000 to 800,000 r was required when the virus had been partially purified by repeated differential centrifugations. Virus was obtained by grinding infectious cottontail rabbit papil-loma-tissue and extracting it in 10 or 20 volumes of isotonic saline and centrifuging at about 4,400 rpm for 20 minutes in an angle centrifuge, with filtration of the supernatant fluid through Berkefeld V candles. Partially purified suspensions of the virus were prepared by centrifuging the filtrate at 30,000 rpm for 1 hour in an air-driven centrifuge, after which the supernatant fluid—which contained no detectable virus but much extraneous protein, as indicated by nitrogen-determinations—was removed. The small pellet of sediment was then resuspended in isotonic saline to the original volume and spun at about 4,400 rpm for 20 minutes. The highspeed centrifugation was then repeated, the pellet of sediment re-suspended in saline, and another low-speed centrifugation done to remove gross particles. The final virus suspensions were very faintly opalescent and contained less than 1% as much nitrogen as the whole filtrate.

Distinct Types of Antibodies in the Blood of Rabbits Carrying the Transplanted V2 Carcinoma

Rabbits carrying the transplanted V2 carcinoma—a squamous-cell cancer derived originally from a virus-induced papilloma—regularly develop in their blood an antibody capable of neutralizing in vitro the papilloma virus (Shope) and capable also of fixing complement in mixture with it. 1 Further studies have now shown that the virus-neutralizing and complement-fixing titers of sera procured from rabbits carrying the V2 carcinoma invariably parallel one another, and that the antibody responsible for both reactions can be readily absorbed from the sera upon admixture with the virus. From the findings it appears certain that the antibody is directed against the papilloma virus per se, and that it is identical with the antiviral antibody present in the blood of rabbits carrying benign virus-induced papillomas, which is in turn identical with that present in the blood of rabbits injected with purified papilloma virus. 2 In further serum tests, carried out by the methods used in the serological study of the Brown-Pearce tumor, 3 we have recently encountered in the blood of rabbits with the transplanted V2 carcinoma two other types of antibodies, wholly distinct from one another and from the antiviral antibody just described. When the sera of normal adult rabbits and of rabbits carrying the V2 carcinoma are heated at 56°C for 30 minutes and tested for capacity to fix complement in mixture with unheated 1:20 or 1:40 saline extracts of normal and neoplastic tissues (e. g., normal rabbit liver or kidney, V2 carcinoma, Brown-Pearce carcinoma) complement-fixation takes place in nearly all of the mixtures.∗ In general the normal sera react in dilutions as high as 1:16 to 1:32 with the normal tissue extracts, and only about half as well with the neoplastic-tissue antigens. On the other hand, sera from rabbits that have carried progressively enlarging V2 carcinomas for some weeks not only react with normal tissue extracts but are vastly more potent in mixture with the homologous antigen, generally fixing 2 units of complement completely in dilutions as high as 1:128 or 1:256 or more in mixture with extracts of the V2 carcinoma.

A Natural Antibody Reacting with Sedimentable Constituents of Normal Tissues

While making serological studies of several transplanted rabbit cancers by the methods that disclosed a distinctive substance in the Brown-Pearce carcinoma, 1 we have noted that the blood serum of normal rabbits will fix complement in mixture with saline extracts of normal rabbit tissues in dilutions far beyond any anticomplementary or summative effects. This phenomenon appears to be due to a natural antibody that reacts with sedimentable constituents of normal tissues. The antibody can be detected by means of a standardized complement fixation test in which 2 units of complement are employed and 2 hours at room temperature allowed for fixation. Antigens are prepared by extracting normal rabbit tissues (liver, kidney, brain, etc.—either fresh or preserved frozen at −22°C) with sand in a mortar, suspending the ground paste in physiological saline (1:10 to 1:40 or more) and centrifuging at 4400 rpm for 20 minutes. The unheated supernatant liquids, opalescent but free from gross particles, have proved notably effective in the tests. When the sera of 22 normal adult rabbits were mixed with antigens consisting of 1:20 or 1:40 saline extracts of normal rabbit livers, fixation invariably occurred, and it was complete with many of the sera in dilutions as high as 1:16 to 1:64. No fixation was got when the sera of 13 rabbits less than 4 weeks old from 7 litters were tested concurrently with the same antigens. None of the sera proved anticomplementary in dilutions of 1:2 or more in concurrent tests though a few showed slight anticomplementary effect when a double volume of the 1:2 dilution was tested, and the antigens were not anticomplementary when 4 and 8 times the standard amount were employed in control tests. Experiments were next undertaken to ascertain some of the properties of the substance present in the serum of normal adult rabbits which is responsible for the fixation.

Identity of “Inhibitor” and Antibody in Extracts of Virus-Induced Rabbit Papillomas:

Saline extracts of the virus-induced papillomas of domestic rabbits often contain something that inhibits or neutralizes the virus in vitro, as Shope first noted. 1 Serological studies have shown that when mixed with it the blood of rabbits carrying these papillomas also has in most instances the power to neutralize the virus in vitro. 2 Experiments were undertaken to learn whether the “inhibitor” procured from the papillomas may not be specific antiviral antibody of the sort present in the blood. To test for “inhibitor”, 10% extracts of freshly procured papillomas were prepared by grinding in sand, suspending in saline, and centrifuging at about 4400 rpm for 20 minutes in an angle-head centrifuge. The clear supernatant fluids were then mixed in equal parts with a Berkefeld filtrate of the virus-induced growths of cottontail rabbits, containing virus of known titer, incubated 2 hours at 37°C, and rubbed into scarified areas on the skin of normal domestic rabbits according to a titration technic already described. 2 The papillomas of 13 domestic rabbits with high serum-antibody titers, as determined by virus-neutralization and complement-fixation tests, 2 , 3 all yielded large amounts of the “inhibitor”, extracts of the growths neutralizing completely or almost completely an amount of virus equal to 500 minimal infective doses. Similar growths of 6 domestic rabbits which had but little circulating antibody yielded little or none of the “inhibitor”, the extracts of these, prepared in the same way, having practically no capacity to neutralize virus. To study further the relation between the “inhibitor” and antibody, pieces of the papillomas of 4 domestic rabbits, all produced by the same inoculum, were repeatedly removed and tested for yield of “inhibitor”, the amount of antibody in the serum of the rabbits being determined concurrently.