Efficient assembly of a large fragment of monkeypox virus genome & recombinant poxvirus based vaccine against COVID-19 -- What could go wrong?

Efficient assembly of a large fragment of monkeypox virus genome & recombinant poxvirus based vaccine against COVID-19 -- What could go wrong?

Greg Krasovsky
May 26, 2022

Earlier this week I came across the following scientific paper written by nine (9) leading scientists at the infamous Wuhan Institute of Virology in China

"Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination."

That's right, folks, monkeypox! Wuhan Institute of Virology!
 
Ever hear of them -- recently?
    
In their research article, these highly-trained and experienced professionals proudly reported that

"A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR."

    ["Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs."]
 
In plain English -- they artificially created (assembled) a "genomic fragment of monkeypox virus" by using DNA recombination (also known as playing God with animal & viral DNA).
 
What could possibly go wrong? In Wuhan?
 
For some ideas, please read "The Infamous Wuhan Lab Recently Assembled Monkeypox Strains Using Methods Flagged For Creating ‘Contagious Pathogens’." (see below).
 
Then today I came across U.S. Patent Application No. 20210260182, dated Feb 26, 2021 for...

"RECOMBINANT POXVIRUS BASED VACCINE AGAINST SARS-CoV-2 VIRUS
The invention relates in various aspects to a recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses. The recombinant poxviruses are well suited, among others, as protective virus vaccines against SARS-CoV-2 virus." (See below)

What? Pox virus based COVID-19 vaccine?

Does that sound like a great idea?
 
Hmmm, does the pox virus family just happen to include Monkeypox? Yep!

After all, what could possibly go wrong?
 

 

      
So I started to get a little philosophical and hypothetical. Just a little...
 
Do you believe in Coincidences?

Do you have Doubts about the the origins, potential man-made objectives, handling, consequences and potential sequels to the recent pandemic?
 
Do you believe in the usefulness & safety or extreme bat-shit level craziness & danger of "Gain of Function" research on lethal & very contagious viruses?

FYI, Gain of Function research  means (mad) scientists artificially make a pathogen more lethal and contagious just so that they can see  

   - how deadly & contagious the pathogen can get, including in military and terrorist biological weapon applications,
   - how an epidemic would develop, its extent & damage and how it could/should be handled "properly,
   - if current vaccines & treatments will work, and
   - if new and effective vaccines & treatments can be invented and quickly manufactured & distributed.
 
Do you believe that lab leaks in dangerous Gain of Function research are inevitable or that they can be prevented entirely or properly handled with no danger to the population at large?
 
Do you believe that the danger of lab leaks, the public outrage over such dangerous research and its political repercussions and the potential civil & criminal liability of those responsible could convince the private & public sector to outsource and perform such dangerous research in developing countries where the likelihood of independent press coverage, legal, political and financial fall-out in the event of a deadly lab leak are minimal and/or can be quashed & "contained" with minimal or acceptable damage & losses?
   
Do you believe that if a virus leaks from a lab -- through infected workers, animals or improperly disposed lab materials & trash -- the research facility (along with its sponsors, investors, creditors, customers, contractors, suppliers and etc.) and government regulators would tell you the truth and the extent of the danger at all times, especially when individuals, organizations and even governments could face severe civil and criminal liability and sanctions?

Do you believe in a remote possibility that a nefarious pharmaceutical conglomerate or the people behind it (private and/or public sector stakeholders) could ever hope for, facilitate or even cause intentionally the surreptitious release of a pathogen just to make absurd amounts of money (billions!) from expensive patented treatments and/or vaccines?
 
Do you believe that just "theoretical" threat & response assessment exercises, conferences, forums and war games could be organized and conducted as a test run to evaluate not just "highly likely" scenarios but plans in progress or in the reserve for the use of weapons of mass destruction ("WMDs") or what they call Black Flag Special Operations?

  FYI, a Black Flag Op is where you commit secretly (either yourself or through proxies) some horrible crime (e.g. mass terrorist act) and then falsely blame some enemy group to justify taking action against them (persecution, prosecution, torture, incarceration, death) or their country (sanctions, blockade, embargo, invasion, war, occupation)?
 
Do you believe that there could be evil, immoral, irresponsible, greedy and/or power-hungry people in medicine, pharmacology, pharmaceuticals, the public & private military-industrial complex, that could accept, advocate and even conspire to use biological weapons, including

    - lethal & contagious natural or man-made/modified pathogens (viruses, bacteria and etc.),
        and/or
    - supposedly safe treatments & vaccines for such very lethal and highly contagious pathogens,
    
to achieve military, political, economic, cultural, anthropological, demographic and even spiritual/occult goals?
   
Do you understand the fable of Pandora's Box and its application to WMDs (chemical, biological, nuclear, environmental and etc.) that can be used against humans, animals & plant life and the environment (earthquakes, floods, droughts, fires, lightning storms, hurricanes, tornadoes, holes in the ozone layer, various other climate-based calamities & etc.)?
 
Do you know, understand and support why the U.S., including through the Department of Defense,
 
    - funds and manages "purely scientific" research on very lethal & contagious pathogens that can be used as bioweapons
       and
    - builds and operates biolabs, in developing countries (like Ukraine, Georgia, Kazakhstan, etc.) where such dangerous research is conducted?
    
Regardless of your answers & position on the above, the first person who guesses the exact number of times "monkeypox" is mentioned in the "Recombinant poxvirus based vaccine" application (see below) gets a PRIZE!
 
Get ready, 1... 2.... 3... Go!

Afterwards, please try to remember or watch the movies Ronin (1998) and The Counselor (2013), ponder over some of the key quotes from them (including those listed below) and think about how you could apply them to our recent & upcoming (imminent?) epidemics, including COVID-19 and monkeypox!

Just food for thought. That's all!
 
But what do you think?
   

***
 
Ronin 1998
Director: John Frankenheimer    
Stars: Robert De Niro, Jean Reno, Natascha McElhone, Stellan Skarsgaard and Sean Bean
   
Sam:
Whenever there is any doubt, there is no doubt. That's the first thing they teach you.

Vincent:
Who taught you?

Sam:
I don't remember. That's the second thing they teach you.

Vincent:
No questions. No answers. That's the business we're in. You just accept it and move on. Maybe that's lesson number three.
 
https://www.quotes.net/movies/ronin_9722
 
***
 
The Counselor 2013
Director: Ridley Scott
Stars: Michael Fassbender, Penélope Cruz, Cameron Diaz, Javier Bardem and Brad Pitt.
 
Westray:
Well, I'm perfectly willing to believe you had nothing to do with this but I'm not the party you have to convince.

Counselor:
Convince of what, for Christ sake?

Westray:
That this is some sort of coincidence. Because they don't really believe in coincidences. They've heard of them. They've just never seen one.
 
https://www.quotes.net/movies/the_counselor_145064
 
***
 

 

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US Patent Application for RECOMBINANT POXVIRUS BASED VACCINE AGAINST SARS-CoV-2 VIRUS
(Application #20210260182)
 
Feb 26, 2021

The invention relates in various aspects to a recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses. The recombinant poxviruses are well suited, among others, as protective virus vaccines against SARS-CoV-2 virus.
 
Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority and benefit from U.S. Provisional Application No. 62/981,997, filed Feb. 26, 2020 and U.S. Provisional Application No. 63/114,514, filed Nov. 16, 2020, the contents of which are hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 26, 2021, is named 104545-0047-101-SL.txt and is 766,062 bytes in size.

BACKGROUND OF THE DISCLOSURE

On Dec. 31, 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases in the city of Wuhan, China.

The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus. As of Jan. 28, 2020, there are in excess of 4,500 laboratory-confirmed cases, with >100 known deaths.

The novel coronavirus is currently referred to as SARS-CoV-2 or 2019-nCoV and is related to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), although with only approximately 80% similarity at the nucleotide level.
Ralph et al. J Infect Dev Ctries. 2020 Jan. 31; 14(1):3-17.

Coronaviruses are enveloped single stranded RNA viruses with positive-sense RNA genomes ranging from 25.5 to ˜32 kb in length.

The spherical virus particles range from 70-120 nm in diameter with four structural proteins.

Despite the fact that a much effort is currently being invested into methods of providing vaccines and delivery vectors for SARS-CoV-2, there is still a need to provide additional and improved approaches against this coronavirus.
 
SUMMARY OF THE DISCLOSURE

An aspect of the present disclosure provides a recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses, for example, as immunogens, in immunogenic formulations against SARS-CoV-2 virus.

Another aspect of the present disclosure provides a recombinant synthetic poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, methods for producing such viruses and the use of such viruses, for example, as immunogens, in immunogenic formulations against SARS-CoV-2 virus.

In some embodiments, the synthetic poxviruses are assembled and replicated from chemically synthesized DNA which are safe, reproducible and free of contaminants.

Because chemical genome synthesis is not dependent on a natural template, a plethora of structural and functional modifications of the viral genome are possible.

Chemical genome synthesis is particularly useful when a natural template is not available for genetic replication or modification by conventional molecular biology methods.

In one aspect, the disclosure relates to recombinant poxviruses comprising a nucleic acid encoding a SARS-CoV-2 virus protein, wherein the SARS-CoV-2 protein is selected from the group consisting of the spike protein (S), the membrane protein (M) and the nucleocapsid protein (N), or combinations of two or more of said proteins.

In another aspect, the disclosure relates to pharmaceutical compositions comprising the recombinant poxviruses of the disclosure.

In another aspect, the disclosure relates to cells infected with the recombinant poxviruses of the disclosure.

In another aspect, the disclosure relates to methods for selecting a cell that expresses a SARS-CoV-2 virus protein, comprising infecting said cell with the recombinant poxvirus of the disclosure and selecting the infected cell expressing said SARS-CoV-2 virus protein.

In another aspect, the disclosure relates to methods of inducing an immune response against a SARS-CoV-2 virus in a subject in need or at risk therefor, comprising administering to said subject an immunologically effective amount of a recombinant poxvirus of the disclosure.

In another aspect, the disclosure relates to methods of generating the recombinant poxviruses of the disclosure, the methods comprising: (a) infecting a host cell with a poxvirus; (b) transfecting the infected cell of step (a) with a nucleic acid encoding a SARS-CoV-2 virus protein to generate a recombinant poxvirus; and (c) selecting a recombinant poxvirus, wherein the nucleic acid encoding a SARS-CoV-2 virus protein is located, upon transfection, in a region of the poxvirus that is not essential for the replication of the poxvirus.
 
Preparation of Poxviruses
 
In some embodiments, the Orthopoxvirus is selected from the group consisting of camelpox virus (CMLV), cowpox virus (CPXV), ectromelia virus (ECTV, “mousepox agent”), horsepox virus (HPXV), monkeypox virus (MPXV), rabbitpox virus (RPXV), raccoonpox virus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus, vaccinia virus (VACV), variola virus (VARV) and volepox virus (VPV).
 
Methods of the Disclosure

 In some embodiments, the recombinant poxvirus is useful towards the method of inducing T cell immunity against the SARS-CoV-2 virus and the poxvirus, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.

In some embodiments, the recombinant poxvirus is useful for a bivalent vaccine against a SARS-CoV-2 virus and a poxvirus comprising a recombinant virus or a pharmaceutical composition.

In some embodiments, the recombinant poxvirus is useful for a bivalent vaccine against a SARS-CoV-2 virus, wherein the poxvirus is a vaccinia virus, variola, horsepox virus or monkeypox.
 
Exemplary Embodiments / Claims

1. A recombinant poxvirus comprising a nucleic acid encoding a SARS-CoV-2 virus protein, wherein the SARS-CoV-2 protein is selected from the group consisting of the spike protein (S), the membrane protein (M) and the nucleocapsid protein (N), or combinations of two or more of said proteins.

2. The recombinant poxvirus according to claim 1, wherein the poxvirus is an orthopoxvirus.

3. The recombinant poxvirus according to claim 2, wherein the orthopoxvirus is selected from the group consisting of camelpox (CMLV) virus, cowpox virus (CPXV), ectromelia virus (ECTV), horsepox virus (HPXV), monkeypox virus (MPXV), vaccinia virus (VACV), variola virus (VARV), rabbitpox virus (RPXV), raccoon poxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus and volepox virus.
 
56. The method of inducing an immune response against a SARS-CoV-2 virus or a SARS-CoV-2 virus and a poxvirus according to claim 44, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.
 
65. The method of inducing T cell immunity against a SARS-CoV-2 virus or SARS-CoV-2 virus and a poxvirus according to claim 57, wherein the poxvirus is vaccinia virus, variola, horsepox virus or monkeypox virus.
 
69. The method according to embodiment 68, wherein the orthopoxvirus is selected from the group consisting of camelpox (CMLV) virus, cowpox virus (CPXV), ectromelia virus (ECTV), horsepox virus (HPXV), monkeypox virus (MPXV), vaccinia virus (VACV), variola virus (VARV), rabbitpox virus (RPXV), raccoon poxvirus, skunkpox virus, Taterapox virus, Uasin Gishu disease virus and volepox virus.
 
88. A bivalent vaccine against a SARS-CoV-2 virus and a poxvirus, wherein the poxvirus is a vaccinia virus, variola, horsepox virus or monkeypox.
 
https://patents.justia.com/patent/20210260182

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Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Virol Sin. 2022 Feb 28;  S1995-820X(22)00041-4.
doi: 10.1016/j.virs.2022.02.009.
Online ahead of print.

Lei Yang (1), Lingqian Tian (1), Leshan Li (1), Qiuhong Liu (1), Xiang Guo (1), Yuan Zhou (2), Rongjuan Pei (2), Xinwen Chen (3), Yun Wang (4)
 
Affiliations
    1    State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
    2    State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China.
    3    State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China. Electronic address: chenxw@wh.iov.cn.
    4    State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China. Electronic address: wangyun@wh.iov.cn.
 
PMID: 35393265 DOI: 10.1016/j.virs.2022.02.009
 
Abstract

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs.

One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products.

To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a PHIS3-HIS3 cassette as a positive selection marker.

This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.

To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.

A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.

The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

Keywords: Monkeypox virus; TAR assembly; Transformation-associated recombination (TAR).
 
https://pubmed.ncbi.nlm.nih.gov/35393265/

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EXC: The Infamous Wuhan Lab Recently Assembled Monkeypox Strains Using Methods Flagged For Creating ‘Contagious Pathogens’.

Are we here because of China’s experiments again?

by Natalie Winters
May 22, 2022

The Wuhan Institute of Virology assembled a monkeypox virus genome, allowing the virus to be identified through PCR tests, using a method researchers flagged for potentially creating a “contagious pathogen,” The National Pulse can reveal.

The study was first published in February 2022, just months before the latest international outbreak of monkeypox cases which appear to have now reached the United States.

The paper, which was authored by nine Wuhan Institute of Virology researchers and published in the lab’s quarterly scientific journal Virologica Sinica, also follows the wide-scale use of Polymerase Chain Reaction (PCR) tests to identify COVID-19-positive individuals.

Researchers appeared to identify a portion of the monkeypox virus genome, enabling PCR tests to identify the virus, in the paper: “Efficient Assembly of a Large Fragment of Monkeypox Virus Genome as a qPCR Template Using Dual-Selection Based Transformation-Associated Recombination.”

Monkey pox viruses – referred to as “MPXVs” in the paper – have strains that are “more pathogenic and [have] been reported to infect humans in various parts of the world.”

“Quantitative polymerase chain reaction (qPCR) is the gold standard for the detection of orthopoxvirus (including MPXV). For pan-orthopoxviruses detection, the E9L (DNA polymerase) gene has been shown to be an excellent target for qPCR assays. For MPXV detection, Li et al. reported that the C3L (complement-binding protein) gene could be used as the qPCR target for the MPXV Congo Basin strain,” explained the paper before noting that China lacked sufficient genetic information on the virus for PCR detection:

    “Since MPXV infection has never been associated with an outbreak in China, the viral genomic material required for qPCR detection is unavailable. In this report, we employed dual-selective TAR to assemble a 55-kb MPXV genomic fragment that encompasses E9L and C3L, two valuable qPCR targets for detecting MPXV or other orthopoxviruses.”

“The primary purpose of assembling a fragment of the MPXV genome is to provide a nucleotide template for MPXV detection,” reiterated the study, which relied on the process of transformation-associated recombination (TAR) to isolate a genomic fragment of the monkeypox virus.

“As an efficient tool for assembling large DNA fragments up to 592 kb in length, TAR assembly has become essential for preparing infectious clones of large DNA/RNA viruses,” explained researchers.

The paper acknowledged that TAR “applied in virological research could also raise potential security concerns, especially when the assembled product contains a full set of genetic material that can be recovered into a contagious pathogen.”

“In this study, although a full-length viral genome would be the ideal reference template for detecting MPXV by qPCR, we only sought to assemble a 55-kb viral fragment, less than one-third of the MPXV genome. This assembly product is fail-safe by virtually eliminating any risk of recovering into an infectious virus while providing multiple qPCR targets for detecting MPXV or other Orthopoxviruses,” posited researchers.

The unearthed study follows the Wuhan Institue of Virology conducting similar research into strains of bat coronaviruses that could infect humans while admitting its facilities lacked proper laboratory safety protocols.

https://thenationalpulse.com/2022/05/22/wuhan-assembled-monkeypox-strains/

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