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Mouse retrovirus present in cell lines used for production of biologicals

MEKoan

Senior Member
Messages
2,630
That's really interesting, George!

Leo Kanner, who coined the term Autism for that condition, said he began to see it in 1938 and published the first paper in 1943.

Does anyone know how early vaccinations were made? Mice? There was intense pressure as far back as the turn of the 20th century, to mass vaccination against widespread and dangerous diseases.

SMALLPOX AND VACCINATION

J. P. LEAKE, M.D.; JOHN N. FORCE, M.D.
J Am Med Assoc. 1923;81(13):1072-1076.

Twenty-five years ago, the surgeon general of the U. S. Marine Hospital Service, in his annual report, said:

Smallpox is a disease so easily prevented by vaccination that the smallpox patient of today is scarcely deserving of sympathy, the improvement in the preparation of pure vaccine lymph having been so great that there is now little cause for fear of untoward results from vaccination. The spread of the disease also is so easily prevented under proper management that it is a disgrace to the sanitary authorities of any state, municipality or locality whenever this disease is permitted to get beyond their control. If the inability to manage the disease is due to a want of funds, then this lack of necessary provision is a disgrace to the state or locality infected.

I've always been a fan of vaccination. All of us who work(ed) in international development were. The risk/benefit ratio seemed a no brainer. It may still be but it may be a more complex issue than we thought.

I just don't know anything any more.
 

MEKoan

Senior Member
Messages
2,630
From: The Wilbur A. Sawyer Papers

The Yellow Fever Laboratory: Rockefeller Foundation, 1928-1937

They then turned their attention to developing a vaccine. This was badly needed; by early 1929, ten researchers in various Berlin and London labs had reportedly contracted yellow fever from working with infected monkey blood and tissue. In April that year, Sawyer himself caught the disease, despite elaborate precautions. It was not a severe case, but he was ill for several weeks, and it was five weeks before he returned to the lab. Many of the lab staff contracted it over the following two years, though none died.

Meanwhile, Max Theiler, working in Sellards's Harvard lab, had begun developing an attenuated yellow fever virus using mouse brains. Mice, it turned out, did not develop classic yellow fever symptoms when injected abdominally, but did develop encephalitis (inflammation of the brain) if the virus was injected directly into the brain. When the virus was passed from mouse to mouse (by transferring infected brain tissue to successive mice), it produced increasingly severe encephalitis, but when each "pass" was injected into rhesus monkeys, the virus was progressively less virulent to the liver and other organs. That is, the power of the virus had been weakened or attenuated. To prove that the mice had yellow fever and not some other encephalitis, Theiler demonstrated that if the virus were mixed with serum from a yellow fever-immune person and injected into the mice, it would protect them from the encephalitis. If non-immune serum was used, the mice developed encephalitis. When Theiler published these findings in 1930, Sawyer and Russell visited him to learn more. Soon afterwards, Theiler joined the team at the RF Yellow Fever Laboratory.

Within several months, Sawyer and his colleagues had developed a vaccine using the attenuated virus. The vaccine had two parts: a ten-percent suspension of mouse-brain tissue with yellow fever virus in fresh sterile human serum, and human immune serum from people recently recovered from yellow fever (i.e., many of the lab staff, including Sawyer and Theiler). These were injected simultaneously. Dr. D. Bruce Wilson, recently back from Brazil, volunteered to be the first human test case. With no ill effects beyond soreness at the injection sites, Wilson had a good level of immunity within several days. Because the vaccine required human immune serum, it was not suitable for large-scale production. But the RF was able to vaccinate eighty-five lab workers and field staff during the next four years, and put an end the accidental and sometimes fatal infections.


karma
 

natasa778

Senior Member
Messages
1,774
Thanks for the lengthy explanation George, but few things...

You used meat slicer analogy to describe contamination of cell lines, when here we are talking about something that is INTRINSIC to those cell lines. NOT something that would jump from those cutting blades, or something airborne. That ‘something’ would already come packaged integrated in the very genome of those cell lines.

When you say radiation etc is used to decontaminate the cell lines, would those processes destroy/fragment any nucleic acid? Including cells’ own DNA? Wouldn’t that render those cells useless? Or if not, if cellular DNA is left mostly or at least partially intact, wouldn’t that mean that any/most retroviruses integrated in the genome would also 'survive'? A possibility?

Also you say in the final stage the mice are checked… checked for what? How?
 

natasa778

Senior Member
Messages
1,774
Limitations of the process to remove infectious viruses from cell lines

www.emea.europa.eu/pdfs/human/bwp/026895en.pdf


Note points 1.6 and 1.7 on page 3 – I could not copy and paste…

On page 5 they say, under 3.2:
"Results have shown that even small modifications in procedure or the particular laboratory strain of virus used can have a large effect on virus removal or inactivation"

also note 3.5 "occasional cases have been reported" (what about unreported ones?)

On page 6 they talk about rodent retroviruses. This guideline was written in 1994, what was the knowledge/awareness before then?

Interesting discussion on page 9 on "Limitations of Validation Studies", i.e. numerous factors that may lead to INCORRECT ESTIMATE OF THE ABILITY OF THE PROCESS TO REMOVE NATURALLY OCCURRING VIRUS INFECTIVITY



Exact science? Anything but, it seems ....
 

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starryeyes

Senior Member
Messages
1,558
Location
Bay Area, California
Rodent cell lines have for many years been used as substrates for production of biological therapeutics such as monoclonal antibodies, recombinant proteins, vaccines and gene therapy virus vectors. It has long been recognised that such cell lines contain retrovirus elements that may be expressed as particles detectable by electron microscopy. Such particles may be infectious, as in the case of Murine leukaemia virus (MLV), or defective and non-infectious, as in the case of the Chinese hamster ovary (CHO) cell retrovirus.

This is the same information we came across back in October when we were trying to determine if DeFreitas's retrovirus CAV was the same as XMRV.

I suspect that I got CFS from a vaccine as a kid but then EBV triggered it when I was 20.
 

natasa778

Senior Member
Messages
1,774
Some further info and questions:

FDA Letter to Viral Vaccine IND Sponsors - Use of PCR-based Reverse Transcriptase Assay (re detection of retroviruses in their products), dated 1998


http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ucm105911.htm


Whichever PBRT test you choose to employ, its performance should be validated, especially with regards to the lower limit of detection, the specificity (generality for retroviral RTs, negative for non-retroviral polymerases with RT activity), and the reproducibility of the assay. The lower limit of detection ("sensitivity") should be comparable with the published literature.

it is possible that the results will be positive, which may require further characterization of the source of the positive activity. In the latter case, IND sponsors are recommended to contact CBER for further guidance in this regard, prior to undertaking any additional testing. . If positive results are obtained, infectivity studies may be requested to demonstrate that the source of the positive activity is not an infectious retrovirus.


are recommended, but not legally required? What would infectivity studies be? Would those studies definitely rule out XMRV or any novel/still unknown exogenous infective retrovirus?


for products manufactured from an established cell bank and viral seed, it may be sufficient to test the cell bank(s) and viral seed(s); you may not be requested to test lot-by-lot

does this leave room for in-lab contamination of cell lots via other cell lines?


Viral vaccines manufactured in insect, yeast, or prokaryotic cells would not be expected to be contaminated with adventitious retroviruses and would not be subject to the testing described below, unless the culture methods (e.g., medium) or processing introduced animal-derived materials.

is it completely impossible that insect etc DNA could be a vector and contaminated?
 

flybro

Senior Member
Messages
706
Location
pluto
Is each vaccinated generation increasing potency of the and variety of virus'

Is it possible that generation upon generation being vaccinated using disfferent strains of virus..

increases the potency of the vaccine, and the variety of material available to the virus to mutate.
 

natasa778

Senior Member
Messages
1,774
ok I'm answering one of my own questions here, sort of:


http://jvi.asm.org/cgi/content/abstract/73/7/5843

Evidence of Avian Leukosis Virus Subgroup E and Endogenous Avian Virus in Measles and Mumps Vaccines Derived from Chicken Cells: Investigation of Transmission to Vaccine Recipients

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.


Re "infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV" - how reliable are those??

PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results

This does not inspire that much confidence, or does it? No details on PCR preparation of cells etc. Why not a culture study? Electron microscope? Why only 33?
 

Gemini

Senior Member
Messages
1,176
Location
East Coast USA
Koan,

You asked "Does anyone know how early vaccines were made?"

How about the 1950's polio vaccines? Byron Hyde's list of 63 suspected ME/CFS outbreaks by decade:
1930-39...... 5
1940-49...... 4
1950-59...... 29
1960-69...... 6
1970-79...... 6
1980-89...... 12
1990...... 1

shows a cluster in the 1950's.

Gemini
 
G

Gerwyn

Guest
ok I'm answering one of my own questions here, sort of:


http://jvi.asm.org/cgi/content/abstract/73/7/5843

Evidence of Avian Leukosis Virus Subgroup E and Endogenous Avian Virus in Measles and Mumps Vaccines Derived from Chicken Cells: Investigation of Transmission to Vaccine Recipients

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.


Re "infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV" - how reliable are those??



i can understand the presence of RT just but not tthe activity.In times of infection endos express protein via normal transcription so why RT activity.That would normally be linked to an infective RNA virus.

They didnt actually show RT activity in vivo.They constructed a clone and through various manipulations induced replication.There is no evidence of RT activity produced by this endovirus in vitro.

To cap it all they looked for this hypothetical scapegoat with a method which would not have detected it even if present sound familiar
 

MEKoan

Senior Member
Messages
2,630
To cap it all they looked for this hypothetical scapegoat with a method which would not have detected it even if present sound familiar

Yes! If there exists the possibility that retroviruses may be transmitted from any other species to humans via vaccines, it is a game changing, mind bending revelation and it won't be easily accepted.
 
G

Gerwyn

Guest
Yes! If there exists the possibility that retroviruses may be transmitted from any other species to humans via vaccines, it is a game changing, mind bending revelation and it won't be easily accepted.

it could even be that the vaccine reactivates a certain virus which might already be in the person "sleeping" in the DNA
 

natasa778

Senior Member
Messages
1,774
To cap it all they looked for this hypothetical scapegoat with a method which would not have detected it even if present sound familiar

btw that study was done by CDC ...
 

natasa778

Senior Member
Messages
1,774
There is more....

Re retroviral contamination of chicken embryo-grown products there were a few studies in the late 90s indicating that there may be a problem. Have a look at this one by the Swiss group

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC191429/?tool=pubmed.

... read the first and second paragraphs of discussion. They detected RT activity in all vaccines, but WHO experts concluded that it poses no danger to humans. Then in the next sentence we find out that they haven't a faintest clue what causes that RT activity. But they do suspect retroviruses are involved. No worries then.

Shortly (very shortly!) after that study there were 3 negative studies, 2done by CDC, and 1 by FDA, saying there is no problem with retro contamination, everything is hunky dory, nothing to see, go home, book is closed.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631681/?tool=pubmed

http://jcm.asm.org/cgi/content/full/39/2/675?view=long&pmid=11158127

http://jvi.asm.org/cgi/content/full/73/7/5843?view=long&pmid=10364336

case closed!



then this one comes along in 2008:

Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek's disease vaccines.

Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Marek's disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation. Barbosa T, et al Avian Dis. 2008 Jun;52(2):245-52.


wt* ???

Can we sleep soundly as WHO says we should. Or toss and turn and reopen the book?

Could these be the same lab/s that were involved with De Freitas follow up research, those that could not find anything: HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control ?

are these labs linked to the branch of CDC that is now in charge of CFS??
 
G

Gerwyn

Guest
Re retroviral contamination of chicken embryo-grown products there were a few studies in the late 90s indicating that there may be a problem. Have a look at this one by the Swiss group

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC191429/?tool=pubmed.

... read the first and second paragraphs of discussion. They detected RT activity in all vaccines, but WHO experts concluded that it poses no danger to humans. Then in the next sentence we find out that they haven't a faintest clue what causes that RT activity. But they do suspect retroviruses are involved. No worries then.

Shortly (very shortly!) after that study there were 3 negative studies, 2done by CDC, and 1 by FDA, saying there is no problem with retro contamination, everything is hunky dory, nothing to see, go home, book is closed.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631681/?tool=pubmed

http://jcm.asm.org/cgi/content/full/39/2/675?view=long&pmid=11158127

http://jvi.asm.org/cgi/content/full/73/7/5843?view=long&pmid=10364336

case closed!



then this one comes along in 2008:

Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek's disease vaccines.

Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Marek's disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation. Barbosa T, et al Avian Dis. 2008 Jun;52(2):245-52.


wt* ???

Can we sleep soundly as WHO says we should. Or toss and turn and reopen the book?

Could these be the same lab/s that were involved with De Freita’s follow up research, those that could not find anything: HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control ?

are these labs linked to the branch of CDC that is now in charge of CFS??

the other study did not have a clue what produced rt activity either they were making stuff up

as to your other vcomments in my view yes yes yes
 

Athene

ihateticks.me
Messages
1,143
Location
Italy
OMG this is HUGE! XMRV in vaccines. Natasa you are amazing.

I found this web page about the 1950s polio vaccine, which was made using monkey organs and was contaminated with "simian virus - 40" from Rhesus monkeys.
This virus causes various types of cancers. People infected with it via vaccines can transmit it to others sexually and through blood transfusions.

http://209.85.129.132/search?q=cach...accine+1950s+made+by&cd=1&hl=it&ct=clnk&gl=it

When they realised the mistake they started using Green Monkeys instead. Anyone heard of Green monkey disease?

And finally, a quote from the web page:
"Dr. Urnovitz revealed significant evidence that human immunodeficiency virus type 1 (HIV-1) is a monkey hybrid virus which was produced when 320,000 Africans were injected with polio virus contaminated with live simian immunodeficiency virus (SIV) in the late 1950's. Apparently, viral fragments combine easily with other viruses to produce these hybrids called "chimeras." Prior to this revelation, health officials were blaming AIDS on the habit of certain Africans to consume monkey flesh. What can be done now? "Make it in anything but animals," said Barbara Loe Fisher of the National Vaccine Information Center, which criticizes vaccine safety."

As far as I understand it, this would be an exact parallel/precedent for similar contamination of vaccines cultivated on rodent organs with XMRV. Could this be why some people with XMRV are well and others have CFS and others cancer? Perhaps the reaction depends on which other viruses you are infected with and thus which "hybrids" are produced in your body.

Who had heard of this polio vaccine contamination? How many people know that Guillain Barre Syndrome is caused by vaccine damage? They're very good at hushing these things up.