From: Billi Goldberg <bigoldberg@igc.org>
Subject: Polio Vaccine & HeLa Cells
Note: non-commercial reproduction.
Archive-Number: 4843
Organization: UCLA, Computer Science Department
Date: Fri, 10 Apr 92 18:24:11 PDT

Reference is made to "Crossing the species - viruses and the origins of 
AIDS in perspective."  J. Seale, (1989) Journal of the Royal Society of 
Medicine, v 182, 519-523.

No precursor genetically nearly identical to HIV-1 has been found in any 
other species.  The nucleotide sequence of HIV-1 is little closer to 
HIV-2 than it is to the naturally occurring primate lentiviruses of 
green monkeys, mangabeys and mandrils.  It is possible that a naturally 
occurring lentivirus much closer genetically to HIV-1 may yet be 
discovered in some other species.  On the other hand, HIV-1 may have 
evolved rapidly from known animal lentiviruses replicating in the highly 
artificial, selective conditions, of serial passage in human cell 
cultures.  It is highly relevant that all lentiviruses so far identified 
in non-human primates (green monkeys, macaques, mangabeys and mandrils) 
have been obtained by culturing them, not in cells from their natural 
host species, but in human cells.*  As all the primate lentiviruses 
readily grown in human cells, it is possible that any of them may be 
adapted to infect intact humans.

In humans highly virulent viruses with short incubation periods and poor 
transmissibility, like the haemorrhagic fever viruses, are easily 
controlled by quarantine, but the greatest threat is posed by slow virus 
diseases, like AIDS and BSE (Bovine spongiform encephalopathy), which 
can spread silently far and wide before sounding any alarms.  It is of 
interest that one of the co-discoverers [sole discoverer] of HIV-1, 
warned of even more virulent infectious agents than HIV-1 which now 
threatened mankind.  'The greatest danger lies', he said, 'in non-
conventional viruses that produce no immune reaction' (Montagnier L. 
Interview: Luc Montagnier. Omni December 1988: 102-34).

*References: Fultz PN, McLure HM, Anderson DC, et al.  Isolation of a T-
lymphotropic retrovirus from naturally infected sooty mangabey monkeys 
(Cercoebus atys). Proc Natl Acad Sci USA 1986;83:5286-90.

Murphey-Corb M, Martin LN, Rangan SRS, et al. Isolation of a HTLV-III-
related retrovirus from macaques with simian Aids and its possible 
origin in asymptomatic mangabeys. Nature 1986;321:435-7.

Fukasawa M, Miura T, Hasegawa A, et al. Sequence of simian 
immunodeficiency virus from African green monkey, a new member of the 
HIV/SIV group. Nature 1988;333:457-61.

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Reference is made to "A Conspiracy of Cells."  Michael Gold (1986) State 
University of New York Press, Albany.  

p. 24
Cancer researchers craved HeLa most of all because it was their long-
sought tumor in a test tube.  They watched the cells react to a battery 
of toxic chemicals and photographed the weirdly shaped chromosomes.  
Scientists interested in the general workings of human cells clocked the 
rate at which HeLa cells multiplied and studied their production of 
proteins.  virologists, who found that polio viruses multiplied a 
million fold just two to three days after infecting a HeLa culture, made 
HeLa their major new tool for studying viruses.  A year later [1953?] 
they knew enough about polio to produce the first successful vaccine.

pp. 25-26
A few of the old guard saw the calamity coming.  With so many new cells 
in circulation, and with no means of positively identifying them, they 
knew there would be mix-ups.  So they tried to head of the problem.  
Some learned to tell cells apart by the shapes of the chromosomes, 
others by how antibodies reacted to particular cultures.  the methods 
were crude, but they worked well enough to show the scientists they had 
been right to worry.

One of the first cultures they checked was a human line that one day, 
for no apparent reason, lost its susceptibility to polio.  Their 
identification tests suggested the cells were no longer human but had 
somehow been replaced by mouse cells.  Then the same thing happened to a 
monkey cell line.  Soon they found human cells growing in what should 
have been pig, duck, and mouse cultures.  And there were mouse cells 
growing in rabbit cultures and rabbit cells where monkey cells should 
have been.

Most cell culturists were still celebrating the renaissance.  But by the 
late 1950s many samples of the useful cell lines--both the old, 
established animal cultures and some of the new human ones--had lost 
their identities.  An investigator who need a normal monkey cell for his 
experiments could no longer be sure he wasn't working on a tumor cell 
from a human larynx.  How could he interpret results when he couldn't 
even say what he had been experimenting on?

The reason for the new mess were obvious to the old guard.  In a word 
these new people were sloppy.  They were probably mislabeling cultures.  
Hell, they were probably using the same pipettes to feed different 
cultures, inadvertently picking up a few cells out of one dish and 
dropping them into another.  If the first cells were stronger, they 
would grown over the second culture and replace it.  Such faux pas 
slipped by unnoticed in many hectic labs where accurate record keeping 
had become a lost art.  In addition, cell swapping was rampant.  And 
when one researcher traded materials with another, he also traded 
mistakes.

p. 33-34.
HeLa was so tenacious, in fact, that it probably didn't need to wait for 
a lab worker to use the same pipette on different cultures.  A startling 
series of experiments reported in 1961 by Lewis Coriell, one of the old 
guard who had helped start the reference cell bank, had shown HeLa could 
literally appear out of thin air.  Coriell, working at the Institute for 
Medical Research in Camden, New Jersey, found that merely pulling a 
stopped from a test tube or dispensing liquid from a dropper could 
launch tiny airborne droplets containing a few HeLa cells.  When the 
drops landed on open petri dishes holding live cultures, the HeLa cells 
began growing so feverishly that in three weeks they overwhelmed the 
original cultures.

Cultures contaminated by HeLa cells were no longer bound by the normal 
cells' lifetime limit of fifty to sixty divisions.  The transformed 
cells [by HeLa contamination] ignored their biological clocks and 
continued doubling without end.

************************************************************************

The following is from "Persistent Infection of Human Carcinoma and 
Primary Check Embryo Cell Cultures with Simian Virus 40."  Bernice E. 
Eddy et al. (1962) Proceedings of Society for Exerimental Biology and Medicine, 
v111,718-722.

Before means for detecting the virus were known, SV-40 had been 
administered to man as a contaminant of poliomyelitis and adenovirus 
vaccines and as a contaminant of the respiratory syncytial virus tested 
experimentally in man.

HeLa and HEp-2 continuous line cultures.  HeLa (from Henrietta Lacks), 
originally derived from a carcinoma of the uterus and HEp-2 from an 
epidermoid carcinoma of the Larynx had been propagated in vitro in 
different laboratories for a period of approximately 9 years prior to 
receipts.  

Inoculation of test cell cultures with SV-40.  Two-oz. prescription 
bottles containing confluent sheets of HeLa, HEp-2 and CE cultures were 
inoculated serially with 1 ml each of 10-fold dilutions of SV-40 that 
had been propagated in CMK.

Results.  Multiplication of SV-40 in test cultures.  SV-40 was found to 
multiply in each of the cell lines under study as shown in Table I and 
the cells continued to shed virus into the fluid during periods of 
observation ranging from 178 to 241 days.  No specific cytopathic 
changes occurred in the cells that adhered to the glass by depending on 
the amount of virus in the inocula the HeLa, and to a lesser extent the 
HEp-2 cell cultures underwent a series of macroscopic changes.

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Billi's Comments:

The main criticisms to Elswood and Stricker's Origin of Aids theory, as 
propagated by Tom Curtis in Rolling Stone and elsewhere were twofold:
(1) How could one infected monkey infect other monkeys when they were 
"gangcaged? and (2) How could SIV become HIV-1 in such a short time--
1960 to the present since the genetic sequencing was only 40-50% 
comparable.  

The first criticism could be answered by researchers drawing blood 
samples from the "imprisoned" monkeys and using contaminated syringes 
and needles.  Even if obviously sick monkeys were disposed of, other 
monkeys contaminated from syringes would shown no sign of infection 
during the incubation period and would not be disposed of.  Their 
kidneys would have been taken and used to prepare the polio vaccines.  
African green monkeys would show signs of SV40 infection but not SIV 
infection.  Rhesus monkeys would shown symptoms of SIV infection but not 
SV40 infection.

Now for the second criticism.  If, for some reason, the culture of 
monkey kidney cells used by Koprowsky et al. to prepare the polio 
vaccine were contaminated by HeLa cell(s), there would be unlimited cell 
proliferation.  Considering the stated ability of SIV/HIV and SV40 to 
mutate upon replication, it would be quite feasible that genetically 
comparable SIV would soon be obtained.  The SIV/HIV species barrier was, 
in all probability, crossed thanks to HeLa cell contamination of polio 
kidney cultures.

New cells would not have to be added to these cultures thanks to the 
immortalization of the monkey cultures by HeLa cells.  In a very short 
time, not only the gap between genetic sequences of SIV and HIV would be 
closed, but unlimited supplies of polio vaccine was available for any 
mass inoculation and immunization programs being planned.  Large 
quantities of SIV/HIV and SV40 would also be available.
Newsgroups: sci.med.aids