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HIV "Inserts" Thread
This Thread will examine in tedious detail:
1. HIV based Virus Like particles (VLPs) & Lentiviruses used mainly for vaccine development
2. Important papers & analyses published on HIV "inserts"
3. Claims & Arguments made
4. Counter Arguments
5. Conclusions
This Thread will examine in tedious detail:
1. HIV based Virus Like particles (VLPs) & Lentiviruses used mainly for vaccine development
2. Important papers & analyses published on HIV "inserts"
3. Claims & Arguments made
4. Counter Arguments
5. Conclusions
1. Examine Thesis A
A. Some argue that these putative HIV "inserts" appeared not by deliberate insertion, but are mere artefacts from VLPs/Lentivirus used as S protein vehicles, possibly used in pancoronavirus vaccine experiments
B. Others claim they were inserted deliberately
A. Some argue that these putative HIV "inserts" appeared not by deliberate insertion, but are mere artefacts from VLPs/Lentivirus used as S protein vehicles, possibly used in pancoronavirus vaccine experiments
B. Others claim they were inserted deliberately
2. Evidence for Thesis A
Are these "inserts" feasible via serial passage & selection of mutations?
Virus-like particles-universal molecular toolboxes
https://europepmc.org/article/pmc/pmc7126091
Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform (using HIV)
https://jvi.asm.org/content/89/17/9044
Are these "inserts" feasible via serial passage & selection of mutations?
Virus-like particles-universal molecular toolboxes
https://europepmc.org/article/pmc/pmc7126091
Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform (using HIV)
https://jvi.asm.org/content/89/17/9044
3. Several generations of HIV-1-based lentivirus vectors (deleting HIV viral envelope & some regulatory genes not required during vector production) were developed.
However, virulence of lentiviral-derived backbones, HIV-1, caused serious safety concerns
https://genemedi.net/i/vaccines-review
However, virulence of lentiviral-derived backbones, HIV-1, caused serious safety concerns
https://genemedi.net/i/vaccines-review
4. What is possible?
HIV-1 Conserved Mosaics Delivered by Regimens With Integration-Deficient DC-Targeting Lentiviral Vector Induce Robust T Cells
https://ncbi.nlm.nih.gov/pmc/articles/PMC5368423/
HIV-1 Conserved Mosaics Delivered by Regimens With Integration-Deficient DC-Targeting Lentiviral Vector Induce Robust T Cells
https://ncbi.nlm.nih.gov/pmc/articles/PMC5368423/
5. HIV Vaccines using Coronaviruses
Towards a Coronavirus-Based HIV Multigene Vaccine
https://researchgate.net/publication/6638139_Towards_a_Coronavirus-Based_HIV_Multigene_Vaccine
Recent Advances in Lentiviral Vaccines for HIV-1 Infection
https://researchgate.net/publication/304990427_Recent_Advances_in_Lentiviral_Vaccines_for_HIV-1_Infection
Towards a Coronavirus-Based HIV Multigene Vaccine
https://researchgate.net/publication/6638139_Towards_a_Coronavirus-Based_HIV_Multigene_Vaccine
Recent Advances in Lentiviral Vaccines for HIV-1 Infection
https://researchgate.net/publication/304990427_Recent_Advances_in_Lentiviral_Vaccines_for_HIV-1_Infection
6. Currently, HIV-1 derived vectors are considered the most efficient ones for gene transduction in mammalian tissues.
Here, they carry out vector genome insertion into host cell chromatin but integration in transcribed genes raises several safety issues
https://researchgate.net/publication/269099221_Comparison_Between_Several_Integrase-defective_Lentiviral_Vectors_Reveals_Increased_Integration_of_an_HIV_Vector_Bearing_a_D167H_Mutant
Here, they carry out vector genome insertion into host cell chromatin but integration in transcribed genes raises several safety issues
https://researchgate.net/publication/269099221_Comparison_Between_Several_Integrase-defective_Lentiviral_Vectors_Reveals_Increased_Integration_of_an_HIV_Vector_Bearing_a_D167H_Mutant
7. HIV-derived VLPs containing cGAMP
HIV-derived viral vectors & VLPs are routinely produced in cell line HEK293T by transfection of plasmids encoding viral components.
Here, they generated VLPs via plasmids expressing HIV-1 capsid protein Gag-GFP & VSV-G
https://biorxiv.org/content/10.1101/2020.01.03.893586v1.full
HIV-derived viral vectors & VLPs are routinely produced in cell line HEK293T by transfection of plasmids encoding viral components.
Here, they generated VLPs via plasmids expressing HIV-1 capsid protein Gag-GFP & VSV-G
https://biorxiv.org/content/10.1101/2020.01.03.893586v1.full
8. A bit of a mouthful!
DNA VaccinesâHow Far From Clinical Use?
Qiao and co-workers demonstrated that vaccination of mice with a "mannosylated cationic liposome that complexed HIV protein-encoding plasmid DNA" resulted in improved immune responses in mice
https://mdpi.com/1422-0067/19/11/3605/htm
DNA VaccinesâHow Far From Clinical Use?
Qiao and co-workers demonstrated that vaccination of mice with a "mannosylated cationic liposome that complexed HIV protein-encoding plasmid DNA" resulted in improved immune responses in mice
https://mdpi.com/1422-0067/19/11/3605/htm
9. It is important to delineate in detail recent developments in HIV based lentivirals & VLPs used in vaccine research into different viruses, especially coronaviruses, in order to prepare groundwork for subsequent analysis of HIV inserts found in 4 viruses originating from bats
10. Bats can Wait!
HIV-1-based lentiviral vectors have advantages as DC vaccine vectors due to their ability to transduce non-dividing cells & integrate into the target cell genomic DNA, allowing for expression of encoded antigens during the cell's life https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914507/
HIV-1-based lentiviral vectors have advantages as DC vaccine vectors due to their ability to transduce non-dividing cells & integrate into the target cell genomic DNA, allowing for expression of encoded antigens during the cell's life https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914507/
11. (Continued from Above)
"A major safety concern with LV vectors is the risk for potential mutagenesis caused by insertion of the viral genome into the host genome.
The risk of insertional mutagenesis remains controversial to date."
"A major safety concern with LV vectors is the risk for potential mutagenesis caused by insertion of the viral genome into the host genome.
The risk of insertional mutagenesis remains controversial to date."
12. Live attenuated virus vaccines safety concerns:
1. live attenuated HIV may revert to a virulent pathogenic form
####2. virus genome may integrate into the recipientâs genome
3. bio-safety issues in production
4. risk of incomplete inactivation of HIV https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810054/
1. live attenuated HIV may revert to a virulent pathogenic form
####2. virus genome may integrate into the recipientâs genome
3. bio-safety issues in production
4. risk of incomplete inactivation of HIV https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810054/
13. A little Joke
Just realised I haven't eaten yet..
Bearing in mind this thread, I will cook up some
Lentils (with no viruses)
this meal will be VLP (Very Low Protein)
with many HIV inserts (High in Vegetables)
Designed to vaccinate myself against hunger for a few hours
Just realised I haven't eaten yet..
Bearing in mind this thread, I will cook up some
Lentils (with no viruses)
this meal will be VLP (Very Low Protein)
with many HIV inserts (High in Vegetables)
Designed to vaccinate myself against hunger for a few hours
14. Nice Meal but some Unpleasant News!
Fresh stool specimen
obtained from patient
Viral sequences detected!
Viral genome analysis show HIV sequences!!
(High in Vegetables)
Angry Debate in Lavatory:
Were they inserted deliberately?
OR, was it just a by product of "Cooking"?
Fresh stool specimen
obtained from patientViral sequences detected!
Viral genome analysis show HIV sequences!!
(High in Vegetables)
Angry Debate in Lavatory:
Were they inserted deliberately?
OR, was it just a by product of "Cooking"?
15. Antigens, Exosemes & LVLPs
A new method to incorporate protein antigens in exosomes relying on the unique properties of a mutant of HIV-1 Nef protein, Nef(mut).
This is a biologically inactive mutant we found incorporating into exosomes at high levels https://www.researchgate.net/publication/232704983_A_strategy_of_antigen_incorporation_into_exosomes_Comparing_cross-presentation_levels_of_antigens_delivered_by_engineered_exosomes_and_by_lentiviral_virus-like_particles
A new method to incorporate protein antigens in exosomes relying on the unique properties of a mutant of HIV-1 Nef protein, Nef(mut).
This is a biologically inactive mutant we found incorporating into exosomes at high levels https://www.researchgate.net/publication/232704983_A_strategy_of_antigen_incorporation_into_exosomes_Comparing_cross-presentation_levels_of_antigens_delivered_by_engineered_exosomes_and_by_lentiviral_virus-like_particles
16. Antigens, Exosemes & LVLPs (cont)
(also when fused at its C-terminus with foreign proteins) "The unique properties of HIV-1 Nef(mut) in terms of exosome incorporation efficiency & as a carrier of foreign antigens remained unaltered during incorporation into the exo-some"
(also when fused at its C-terminus with foreign proteins) "The unique properties of HIV-1 Nef(mut) in terms of exosome incorporation efficiency & as a carrier of foreign antigens remained unaltered during incorporation into the exo-some"
17/ Recent Sino-Japanese Research which sheds some light on HIV-1 Envelope Glycoprotein inserts:
"Insertional mutagenesis analysis showed that V4 and V5 variable loops of HIV-1 Envelope Glycoprotein were tolerant to insertion of Green Fluorescent Protein"
https://jbc.org/content/290/24/15279.full
"Insertional mutagenesis analysis showed that V4 and V5 variable loops of HIV-1 Envelope Glycoprotein were tolerant to insertion of Green Fluorescent Protein"
https://jbc.org/content/290/24/15279.full
18. So far, I have just examined possibilities which could explain how & why HIV like sequences could appear in SARS-COV2 & other viruses as artefacts of their use as lentiviruses or virus like particles in vaccine research
If someone wants to disagree with this, please jump in!
If someone wants to disagree with this, please jump in!
19. Next step will be to assess likelihood or even remote possibility that HIV based lentiviruses or VLPs could somehow "contaminate" a pre-existing virus in its host, the bat, this giving rise to detction of "HIV inserts" in the viral sequences of SARS-COV-2, RaTg13 & 2 others.
20. This is an extremely complex topic!
Maybe I have bitten off more than I can chew, but I will soldier on in search of illumination.
Wish me Luck!
Maybe I have bitten off more than I can chew, but I will soldier on in search of illumination.
Wish me Luck!
21. Worth pursuing this thread at a leisurely pace
1. Even if no conclusion is reached it is part of the learning process
2. Hidden gems often emerge when digging in strange territories
3. Research into a Pancoronavirus vaccine via HIV VLPs is more likely than HIV vaccine work
1. Even if no conclusion is reached it is part of the learning process
2. Hidden gems often emerge when digging in strange territories
3. Research into a Pancoronavirus vaccine via HIV VLPs is more likely than HIV vaccine work
22. Some input by @sanchak74 who should be followed
"I may be wrong, but the problem is these inserts are small (6aa (18nt) or lesser), and not contiguous. Inserts don't happen that way. Find a larger contiguous (nucleotide) insert matching any HIV or LVLPs constructs"
"I may be wrong, but the problem is these inserts are small (6aa (18nt) or lesser), and not contiguous. Inserts don't happen that way. Find a larger contiguous (nucleotide) insert matching any HIV or LVLPs constructs"
23. Jumping some background papers on VLPs and Lentivirus contamination in host cells and recombination of viruses, let's take a look at WIV Lab methodology and how they used HIV in their experiments.
24. WIV Pseudoviruses
1. WIV used HIV-1 derived plasmids, co-transfecting them along with SARS-like virus derived spike gene plasmids, into human embryonic kidney (HEK) cells.
2. They created pseudoviruses to perform experiments under BSL-2 instead of BSL-4 (required for SARS)
1. WIV used HIV-1 derived plasmids, co-transfecting them along with SARS-like virus derived spike gene plasmids, into human embryonic kidney (HEK) cells.
2. They created pseudoviruses to perform experiments under BSL-2 instead of BSL-4 (required for SARS)
25. A case in point (Thanks @franciscodeasis)
For MERS-CoV infection, HIV-pseudovirus was used during a WIV experiment on fatal swine acute diarrhoea syndrome caused by HKU2-related bat cov https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094983/
For MERS-CoV infection, HIV-pseudovirus was used during a WIV experiment on fatal swine acute diarrhoea syndrome caused by HKU2-related bat cov https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094983/
26. Let's look in more detail:
1. Codon-humanized S genes of SADS-CoV or MERS-CoV cloned into pcDNA3.1 were used for pseudovirus construction
2. pHIV-Luc plasmid (pNL4.3.Luc.R-E-Luc) & S-protein-expressing plasmid (empty vector control) were co-transfected into HEK293T cells
1. Codon-humanized S genes of SADS-CoV or MERS-CoV cloned into pcDNA3.1 were used for pseudovirus construction
2. pHIV-Luc plasmid (pNL4.3.Luc.R-E-Luc) & S-protein-expressing plasmid (empty vector control) were co-transfected into HEK293T cells
27. HIV virions & S proteins
"To evaluate the incorporation of S proteins into the core of HIV virions, pseudoviruses in supernatant were concentrated by ultracentrifugation"
"To evaluate the incorporation of S proteins into the core of HIV virions, pseudoviruses in supernatant were concentrated by ultracentrifugation"
28. MERS-CoV HIV Pseudovirus
1. SADS-CoV entry & infection was tested in Hela cells with coronavirus receptors
2. SADS-CoV grown in Vero cells was used to infect HeLa cells transiently expressing APN, ACE2 or DPP4.
1. SADS-CoV entry & infection was tested in Hela cells with coronavirus receptors
2. SADS-CoV grown in Vero cells was used to infect HeLa cells transiently expressing APN, ACE2 or DPP4.
29. MERS-HIV-WIV16
3. SARS-related-CoV WIV16 & MERS-CoV HIV-pseudovirus were used as positive control for human/swine ACE2 or DPP4, respectively.
4. SARS-related-CoV WIV16 replication was detected using rabbit antibody against SARS-related-CoV Rp3 N protein (made in house ;)
3. SARS-related-CoV WIV16 & MERS-CoV HIV-pseudovirus were used as positive control for human/swine ACE2 or DPP4, respectively.
4. SARS-related-CoV WIV16 replication was detected using rabbit antibody against SARS-related-CoV Rp3 N protein (made in house ;)
30. Contamination?
5. Infection of MERS-CoV HIV-pseudovirus was monitored by luciferase 48 h after infection.
6. The spike gene plasmid solution could become contaminated with SARS-like viruses due to inadequate purification, which infected HEK cells
( Thanks to @ArumughamVinu )
5. Infection of MERS-CoV HIV-pseudovirus was monitored by luciferase 48 h after infection.
6. The spike gene plasmid solution could become contaminated with SARS-like viruses due to inadequate purification, which infected HEK cells
( Thanks to @ArumughamVinu )
31. With HEK cells containing transfected HIV-1 genes, novel recombinant SARS-like viruses emerged with HIV-1 derived inserts. Such novel coronaviruses would have been exposed to lab workers & animals during handling, especially in a relaxed BSL-2 setting https://zenodo.org/record/3766463#.Xw6lAaEzbHY
32. Contamination (2)
1. HEK were grown using fetal calf serum
2. Experiments contaminated with genetic material from:
A. SARS-Like Coronaviruses (WIV16 et al)
B. HIV-1
C. Bovine viruses in fetal calf serum
D. Human viruses in HEK
thus resulting in SARS-CoV-2 with HIV Inserts?
1. HEK were grown using fetal calf serum
2. Experiments contaminated with genetic material from:
A. SARS-Like Coronaviruses (WIV16 et al)
B. HIV-1
C. Bovine viruses in fetal calf serum
D. Human viruses in HEK
thus resulting in SARS-CoV-2 with HIV Inserts?
33. A big thanks to @ArumughamVinu for his analysis which deserves reading:
https://zenodo.org/record/3766463#.Xw6lAaEzbHY
We have seen that when cells are:
1. infected with multiple viruses
2. contaminated with genetic material from multiple viruses
3. recombination can occur creating novel viruses.
https://zenodo.org/record/3766463#.Xw6lAaEzbHY
We have seen that when cells are:
1. infected with multiple viruses
2. contaminated with genetic material from multiple viruses
3. recombination can occur creating novel viruses.
34. @ArumughamVinu concluded:
"With millions of contaminated cell lines, WIV labs were incubators for novel viruses including coronaviruses such as SARS-CoV-2. So it was only a matter of time before a virus with h2h transmission capability would leak"
It did, in Oct/Nov 2019!
"With millions of contaminated cell lines, WIV labs were incubators for novel viruses including coronaviruses such as SARS-CoV-2. So it was only a matter of time before a virus with h2h transmission capability would leak"
It did, in Oct/Nov 2019!
35. RaTG13 & SARS-COV-2
Despite spurious & childish claims by US based Chinese Researchers that the HIV inserts were found in 2 other bat betacovs (ZC45 & ZXC21) it is clear thanks to @flavinkins & my own findings that only the viruses RaTG13 & SARS-CoV-2 contain these inserts
Despite spurious & childish claims by US based Chinese Researchers that the HIV inserts were found in 2 other bat betacovs (ZC45 & ZXC21) it is clear thanks to @flavinkins & my own findings that only the viruses RaTG13 & SARS-CoV-2 contain these inserts
36. Significantly, only RaTG13 & SARS-COV-2 viruses contain the specific sequences in question!
(individually they appear in many other organisms)
see @trvrb who "debunked' the HIV Inserts claim by pointing out these inserts are also found in RaTG13.
He shot himself in the foot!
(individually they appear in many other organisms)
see @trvrb who "debunked' the HIV Inserts claim by pointing out these inserts are also found in RaTG13.
He shot himself in the foot!
37. HIV Insert debunking Session by @trvrb is here:
https://twitter.com/trvrb/status/1223666856923291648
&
https://twitter.com/trvrb/status/1223337991168380928
Worth a read, yes, but we can debunk @trvrb now!
Why?
Because his argument is mainly based on the inserts appearing in RaTG13 which he then uses as "proof" of natural origin
https://twitter.com/trvrb/status/1223666856923291648
&
https://twitter.com/trvrb/status/1223337991168380928
Worth a read, yes, but we can debunk @trvrb now!
Why?
Because his argument is mainly based on the inserts appearing in RaTG13 which he then uses as "proof" of natural origin
38. RaTG13 is anything but a natural virus!
1. IF anything, ACE2 binding optimised to lab animals
2. Amplified from "disintegrated" bat fecal swab (4991)
3. Amplicon sequences reveal lab contamination
@nerdhaspower and @flavinkins have both proved this!
https://nerdhaspower.weebly.com/ratg13-is-fake.html
1. IF anything, ACE2 binding optimised to lab animals
2. Amplified from "disintegrated" bat fecal swab (4991)
3. Amplicon sequences reveal lab contamination
@nerdhaspower and @flavinkins have both proved this!
https://nerdhaspower.weebly.com/ratg13-is-fake.html
39. Horizontal Gene Transfer
The next set of tweets will examine the phenomenon of "horizontal gene transfer" between viruses and between hosts and viruses.
For an introduction, please watch the video here:
The next set of tweets will examine the phenomenon of "horizontal gene transfer" between viruses and between hosts and viruses.
For an introduction, please watch the video here:
40. Before we explore Horizontal Gene Transfer in more detail, let's examine the claims of @Ayjchan that the "HIV Insert Claim has been put to rest" by a flawed paper from Chinese Researchers:
https://twitter.com/Ayjchan/status/1282377662015602689
She bases her arguments on this paper:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033698/
https://twitter.com/Ayjchan/status/1282377662015602689
She bases her arguments on this paper:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033698/
41. I suspect that @Ayjchan did not bother to look closely at the sequence data because it is glaringly obvious that they presented the sequences erroneously to claim that the "HIV sequences" could also be found in ZC45 and ZXC21
See images via @trvrb side by side with their data
See images via @trvrb side by side with their data
42. Their claim is patently FALSE!
It can only be explained as an amateur propaganda attempt by Chinese AIDS researchers working in the US to promote a natural origin hypothesis to deflect criticism from their motherland & discredit claims of "HIV Inserts"
Perverse Science!
It can only be explained as an amateur propaganda attempt by Chinese AIDS researchers working in the US to promote a natural origin hypothesis to deflect criticism from their motherland & discredit claims of "HIV Inserts"
Perverse Science!
43. Do you still doubt what I say?
Read their drivel here:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033698/
Take a magnifying glass to inspect their diagrams with fake sequence data & their unsupported claims about ZC45 & ZXC21
Of course, their "research" has been WELL used by "natural origin" fanatics
Read their drivel here:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033698/
Take a magnifying glass to inspect their diagrams with fake sequence data & their unsupported claims about ZC45 & ZXC21
Of course, their "research" has been WELL used by "natural origin" fanatics
44. Their claims are TOXIC
@Ayjchan
"They didn't compare SARS2 to closest genomes RaTG13, ZC45, ZXC21..otherwise, they would've seen that 3 of 4 inserts already exist in these natural CoVs"
https://twitter.com/Ayjchan/status/1282373705394520065
"This paper puts the hypothesis to rest"
https://twitter.com/Ayjchan/status/1282130093784076289
@Ayjchan
"They didn't compare SARS2 to closest genomes RaTG13, ZC45, ZXC21..otherwise, they would've seen that 3 of 4 inserts already exist in these natural CoVs"
https://twitter.com/Ayjchan/status/1282373705394520065
"This paper puts the hypothesis to rest"
https://twitter.com/Ayjchan/status/1282130093784076289
45. There is now no doubt that:
1. RaTG13 is a lab construct and that only RaTg13 and SARS-COV-2 contain the specific set of HIV insert sequences.
2. ZC45 & ZXC21 do not contain these HIV sequences by any stretch of the imagination
https://twitter.com/flavinkins/status/1281540190725521408 https://twitter.com/flavinkins/status/1282593578749849600
1. RaTG13 is a lab construct and that only RaTg13 and SARS-COV-2 contain the specific set of HIV insert sequences.
2. ZC45 & ZXC21 do not contain these HIV sequences by any stretch of the imagination
https://twitter.com/flavinkins/status/1281540190725521408 https://twitter.com/flavinkins/status/1282593578749849600
46. Debunking Finished, Time for a well deserved Swim!
I will also hope to practice some "horizontal gene Transfer" with Sally Brown cos "she's a nice young lady, way, hay, roll and go" before returning to this thread to examine that topic in more detail
I will also hope to practice some "horizontal gene Transfer" with Sally Brown cos "she's a nice young lady, way, hay, roll and go" before returning to this thread to examine that topic in more detail
