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Sequencing of bivalent Moderna and Pfizer mRNA vaccines reveals nanogram to microgram quantities of expression vector dsDNA per dose

Authors:
  • Medicinal Genomics
  • Medicinal Genomics
  • Medicinal Genomics Corporation

Abstract

Several methods were deployed to assess the nucleic acid composition of four expired vials of the Moderna and Pfizer bivalent mRNA vaccines. Two vials from each vendor were evaluated with Illumina sequencing, qPCR, RT-qPCR, Qubit™ 3 fluorometry and Agilent Tape Station™ electrophoresis. Multiple assays support DNA contamination that exceeds the European Medicines Agency (EMA) 330ng/mg requirement and the FDAs 10ng/dose requirements. These data may impact the surveillance of vaccine mRNA in breast milk or plasma as RT-qPCR assays targeting the vaccine mRNA cannot discern DNA from RNA without RNase or DNase nuclease treatments. Likewise, studies evaluating the reverse transcriptase activity of LINE-1 and vaccine mRNA will need to account for the high levels of DNA contamination in the vaccines. The exact ratio of linear fragmented DNA versus intact circular plasmid DNA is still being investigated. Quantitative PCR assays used to track the DNA contamination are described.
Sequencing of bivalent Moderna and Pzer mRNA vaccines reveals nanogram to microgram
quanes of expression vector dsDNA per dose

  !"#$%&'
(%)**+*
,-./01(*(
(233, //#3, /4567%1)5
)%801)+9
1%:91;66<3=81<3
%*/01>/#3, /%
)/01801*/01(/080
>())%*209#
/01(**801)+
)**801801))
4)), /%>801)
2ntroducon
**/01>
:$??@???@ ??6;*
%*0#%:A;/01*0et al
/01%).801--E.coli
in-vitroB%%*/01:0??;=
801C)*>
-/019166<*801/01:D??##
&;=81*<:#=(?&;
/C801%2*)
*801):E#(???;
Results
)*801(%3
(F-%0(9
$/01#3*3*/01)'G
*)801:=?;/01#3
%1%0et al
/01(/013%801
H>*%>801*
(%1:=6;
().)+3, //#3, /%
>3/01801()
*)3%801:=6;%*,-.
B?)%*,-.?>(
*I!.):8&&&;
:&J;
Figure 11%*/01#3%(>:;%
:;%1/%*
:%(;
Figure 2,*.%*/01#3%1(9$<= >
:;*:%(;0<:;=
:%(;I!:%((;
Figure 3/0(3(2:/0#3/01#
3;,-.*:K;?:;B?FI!
:);3, /%>>
L/03M)9!
.),%%)3F*
%1:>%3)*;B?
Figure 4*,-.,-.?B?
)
Figure 5A )*2)I(:2I;
*B?),-.?K2I(
.#((>(K2I((
B?)
B?(F(%E/2I(
N,O(*%*B?
?0#22I)3I
)2II?(2I(.(*
2I
(,-.-.%%
((+2(%.*
%*
(%(*B?3%.
3:';2(%#
%B?
H*%*B?(*
(%B?
='1%(B?3
%()**%
:='$;
Figure5B.2I(*,?P>!P?6(B?
:;9),?P>!P?6*?*B?
333(
(,-.?
,-.)?%B?I!9
(+.)))
()**
).*801-(1
)5801:801;/01:)%/01;

1)5B'#6<Q*801?6B#
''&<Q*/016Q4567%)#?J<Q
*801?J#'?J<Q*/01*)801
/01)6:<Q;
:?<Q;)*%801/01)*
/01%(+
Figure 61)5?6B<QR''&<Q*/01
:K;B'#6<Q801)5H801
(>)**#>(
*0#%/01801%(0#
%/01%)%>>
.*>/01K+%
*0%%
)*801
4)), /%(28,4K()
>()(,-.>3
3O*)(3, //#3, /*
3, /%-801(/#3, /-801/01
3, /().+)((*
%)*/#3, /, /:, /(;
Figure 73, /*,-.O((802:K;/01:;
E/013 *>I%+I
802)>)/01%)%*
802%801/01O3, /
Figure 8/#3, /-801/01( F
(,-.>I%:K$;)B
%.)/01>#? K
(802+*8013)*
/#, //0:/;( FO801
 
Figure 9. Q*,-.Q*%F*J
 *?!*I>3, /:K;1%
'Q*:/;
Figure 10Q*,-.Q*%F*J
 *??*I>/#3, /:K;1%
'Q*:/;
Figure 11Q*+F *>
:K;(3, /+*%
3 ,-.O3*)(
(?>(,-.
*%'Q*
/01:/;
Figure 12.Q*+F *>
:K;(/#3, / K(>I
>)3, /(' F#
Q*:/;
Table 1456=%)!#?J<Q*801?J#
'?J<Q*/01
%)(%.(28/#3, />
*801)*3)
801.1+(801
))4567%1)52
(O))
(3))*/01()3%801#
3, /%
Figure 13.($>(%.28*>L)
/#3, /%#*)( 
(*801(*?*(*> *
S?T'*</#3, /)%<J*801
)'*<Q*!<Q*8011%801
802.*%3)(
(3)802456
1)5
(>(*%)J,-.(
3, //#3, /
Figure 14,-.$:;J,-./01
(+):$M;
Figure 15Q*Q**%F(C3, /:K;/#
3, /:;*I:;>:;J(
Table 2. *>I3, /:801%;)*
.%>*)*
)%/8 :I > ;)*>I
801T
Table 3. *>I/#3, /:/01U801;/)*/01V801
*!6V"V91(66VJ#B*91
Discussion
)*801)
H4561)5F
3)-)*91
*66801</013, //#3, /-)/01801)1#?
F(>I/#3, /V66
):?W"T6;2( F:'#B ;
(>3, //#3, /(2456
1801)(3, /%-
(802(*%3, /
*(4561(*%
%"!%802((
(3, /)801*
4561)5
3, /X%)801(
*%Y%801)=
+("#!%) ?
:(;))*%
-.,)*
-/01()3())>
3, /
1)%4561)57)801
3)-))%>(0#%/01=
()((%
(%-*%3, //#3, /
91)
**91>)*801
/)801%801
())%*
C>E.coli801*):#=(
?&;$>E.coli801((3, / 6'
%%*1/# I#?
:Z??;(>3)%*
/01(3209#
)*/01801%1et al:1???;801
)*3>(O3209#*/
)*I!.),-.O
(**)(>*
)209#)%3801
.)*-
,3**Det al.%
3:8#9D??;(*C
).**3
+91:66<801</01;(456
1+=81:<;()
3, /(%:,;,
)*E.coliK#(,9+)
%+C):Z??;
1)*%>(*%
(%%(>/01>(
*801*)*/01
801/01%*>
3/0%3>%1*%
+))(>3801
/01)*)*3, /%%
)(%)%
>%-*80133
%/01
H3**>%
*802*
)*801H801
801%)>
)%4561%>((
7>(801/01>((0#
%-%*)%1(
 F(/#3, /3, /(>3
)*91)#)3, //#3, /
%*>>))
*
Methods
Purifying the mRNA from the LNPs
LiDs/SPRI puricaon
Q*(:<6<'*;
'Q*?X8(Q*I0,
Q*X2
?66Q*1:$>;
?'Q*?' ?:0(9$;
()+G*'*))
$(&"#(*(((?Q
*JX9L(K%*6Q*??Q*
(1)5
CTAB/Chloroform/SPRI puricaon of Vaccines
%3, /*((8<,/2-)*
K3%0,-)1
1$< *<,/2)().**3, /
1$7%6Q*I('Q* 1$: )1
12I1+2,-)>[!?!;(+*'
6B\ JQ**(+&*6
?'Q*3(?'Q*)$
*)F(+*'
)%(((BX9
()(-%6Q* )F
Simple boil preparaon for evaluang vaccine qPCR.
%>#Q*, /)
*%F:!??J;
"'\ *"
&'\ *?
Library Construcon for Sequencing
'Q*Q(/01#3*23
09$09$0+E228)/01%*2:09$[9BB";
**))(*'
=%)(+!?\ '
)
0/),%1(*
**%%(-*"%
*1)%)(
*/013%911
/01:]'X; /01
K)9218)8013F
*))(%&&&&XD2
(%%((**(

RNase A treatment of the Vaccines
/01%0#%>(/01#
L/01(%()+/01
%801*3I/01(%3
(6B\ *6(Q*?E<Q/0
1*09$/0)(-'G*12I1+:
[!?;(?Q?K801-)'Q(*801
3
DNase treatment of the vaccines
'Q* 1$-(6B\ *6(?Q802"Q*
802F:* [!?!6;?'Q*8%F(
80)/)(-"QX2!Q1'Q
 ?)()+)K*')%
(((JX9L
Whole genome shotgun of RNase’d Vaccines.
'Q*801(3%H>
H%)>>**801.
>*Y
Qubit™ 3 Fluorometry
4567%(*$1$/01$/>:[6B6;
$1)%8014)):[6"";
*)
E.coli qPCR
,99598)%:[!??;().
*)
qPCR and RT-qPCR Spike Assay
#P,-.PDPI+#>P=(
]11 1  1
#P,-.PDPI+#>P/
] 1  1 
#P,-.PDPI+#>P,
]<'"#=1< 111 1<Z90<11 1  11<621$>=4<
qPCR and RT-qPCR Vector Origin Assay
PI+#PLP=(
] 1 11     11
PI+#PLP/
  1 11 1
PI+#PLP,
<'9G<111 1 1<Z90< 1  1  <621$>=4<
928)
Make 50X primer-probe mix.
 ?'Q=(,
? ?'Q/,
6 ?'Q,
! 6B'Q*?
E'Q*+qPCR master mix(:'Q<
);
EQ*+RT-qPCR master mix(
Medicinal Genomics Master Mix kits used
 MV<<<3, /###6#?#+
? MV<<<>##3##>
Reacon setup for 30 reacons of qPCR
!Q9.%+:;
?!Q/)$F:;
?!"Q*?
'Q*,#,>
'Q*,#,L
E6JQ*+'Q*-:QI+801</01U!Q?* 
^';
Reacon setup for 34 reacons of RT-qPCR
?Q9.%+
&"Q*?
?Q/02:;
!Q8:;
Q,#,>
Q,#,L
Q*+Q*I+801</01
Medicinal Genomics MIP DNA Puricaon Kit used
 MV<<<12I1+#801#9+)##?#)P?
 1$< *<,/2801</01)
Cycling condions
)(>*3, //#3, /0V'\ />(
3, / 3, /+>.%(F%
'\ =>*/01801(3, //#3, /
%((/*
% )*3, //#3, /
3**$>,) LT">T!
L
>
Sequencing Data
Raw Illumina Reads RNA-seq
,-.$I=(
,-.$I/
,-.$I?=(
,-.$I?/
I=(
I/
I?=(
I?/
/-?'":;81>
?'"*-L,P/9E/0*2**
-O)-(
,-.I=(
,-.I/
,-.I?=(
,-.I?/
I=(
I/
I?=(
I?/
Megahit Assemblies
,-.I
,-.I?
I
I?
Illumina Reads mapped back to Megahit Assemblies
,-.I$1= 2+=
,-.I?$1= 2+=
I$1= 2+=
I?$1= 2+=
Q30 Filtered Illumina Reads (use these for transcriponal error rate esmates)
=4#=(V]*3#-##*3*3
,-.I=(/
,-.I//
,-.I?=(/
,-.I?//
I=(/
I//
I?=(/
I?//
Q30 BAM les. Q30 Reads mapped against Megahit assemblies
,-.I36#$1- 2+=
,-.I?36#$1- 2+=
I36#$1- 2+=
I?36#$1- 2+=
IGVtools error by base on q30 reads
=T,)),):U;1U UUU0U8)U2)0)
#1# ###0#8)#2)
I
I?
,-.I
,-.I?
Analysis pipeline
/()+(
 #/23
 #)
*1/# I#?
 #$1-*(2I
)
$H1# #>*
K(#:(((;#E.+

2I #2I(.23
RNase Treated Libraries-BAM les
)-$1-(
(#)P#L$1])@+)@
)%
*P-+_`a#b<<<b_@>#cdP-+e##
@*)_(#cdP-+eabWf4ba#*?a#
b<0gV<<b_@b0()*cd)e<cP-+b@c
dP-+ec)]cdP-+e##<cd)e##+@@

,P/0PHP>!PB*
,P/0PHP>!PB
,P/0PHP>!PB
,?P/0PHP>!P?6*
,?P/0PHP>!P?6
,?P/0PHP>!P?6
Author contribuons
D# 33, /%4561
)5*%K
#L).801))53, /
#)+%)
Con@icts of interest- 1*%*(
**3, /801)>%
Acknowledgements.
HO>>Z*.3HO>>
D/.D>>%>,)%I)$M%
=%%8H,**
/*
1L*==8$ /8???
2/)*,-.$0 LI28#&/01I
$0"??2I Curr Issues Mol Biol44V'#?"
$,=, F%$$/>??
h9V )9+( LI28>,12%$0"??
:,-.#$0;I)8*1)V10
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... (v) The presence of LNP-encapsulated DNA contamination originating from residual plasmid DNA from DNA plasmids used during the manufacturing process of the Pfizer/BioNTech and Moderna modRNA genetic vaccines [58,59]. The residual DNA detected in the modRNA genetic 8 vaccines is high in copy number and contains elements such as: functional promoters, open reading frames (ORFs), origins of replication and nuclear targeting sequences [59]. ...
... The residual DNA detected in the modRNA genetic 8 vaccines is high in copy number and contains elements such as: functional promoters, open reading frames (ORFs), origins of replication and nuclear targeting sequences [59]. In the case of the Pfizer/BioNTech genetic vaccine, such plasmids have been engineered with a mammalian SV40 promoter-enhancer-ori from the oncogenic virus Simian Virus 40 (SV40) along with a nuclear targeting sequence (NTS) [58,59]. This human compatible promoter is not required for the expression of these plasmids in the E. coli bacterial expression system and its presence is highly unusual as it poses a significant oncogenic risk that is not needed for the plasmid's stated purpose. ...
... [60]. Additionally, the presence of the contaminating plasmids is far above the regulated limits for naked DNA contamination on vaccines [58,59]. As recently reported by Cancer Geneticist Prof. Buckhaults [61] and by Toxicologist and Molecular Biologist Janci Lindsay, Ph.D. ...
Preprint
Full-text available
In this report we describe the case of a healthy, young, athletic woman who developed acute lymphoblastic leukaemia (ALL)/lymphoblastic lymphoma (LBL) after receiving the second dose of the Pfizer/BioNTech modified mRNA (modRNA) COVID-19 genetic vaccine (marketed as Comirnaty®). The first dose of the genetic vaccine did not appear to illicit any noticeable side effects, but within 24 hours of the second dose the patient suffered widespread and intensifying bone pain, fever, vomiting, and general malaise. Due to the persistence of the symptoms, the patient underwent a series of tests and examinations including a full laboratory workup, a consult with a clinical immunologist and rheumatologist, a Positron Emission Tomography (PET) imaging, as well as an osteomedullary biopsy. These together led to a definitive diagnosis of ALL. A time interval of 16 weeks from the second vaccination to the diagnosis of cancer was noted. Several similar cases with identical pathology which developed after the modRNA COVID-19 vaccination, are described in case reports in the scientific literature. The massive and indiscriminate use of genetic vaccines to fight COVID-19 is raising serious concerns about their safety and about the technology platform as a whole for this purpose. Growing evidence is accumulating regarding the biodistribution and persistence of the modRNA which can reach, thanks to the lipid nanoparticles, a multitude of tissues and organs of the body, including the bone marrow and other blood-forming organs and tissues. Moreover, there is evidence that the modRNA vaccines display a particular tropism for the bone marrow, influencing the immune system at multiple levels and being able to trigger not only autoimmune-based pathologies, but also neoplastic mechanisms. The aim of this article is to assess, on the basis of the available scientific literature, the risk of developing haematopoietic cancers after modRNA vaccination, and to investigate the potential genetic mechanisms involved in the pathogenesis of disease.
... At least three genomics scientists claim to have discovered extremely high levels of plasmid DNA contamination in the Pfizer/BioNTech vials, namely, Kevin McKernan (McKernan et al., 2023), Phillip Buckhaults (see Demasi, 2023), and Jürgen O. Kirchner (see Kogon, 2023). If what they claim is true, then debates about whether mRNA from the "vaccines" can be reverse-transcribed and integrated into the genome of human cells recede in significance, because DNA was already present in the vials. ...
... Not unlike the Vanden Bossche open letter about the potential for the "Covid-19 vaccines" to lead to immune escape (see Chap. 4), we are faced here with some alarmist claims unsupported by reliable scientific evidence. McKernan et al. (2023) admit: "These vials were sent to us anonymously in the mail without cold packs" and "All of the monovalent vaccines [but not the bivalent vaccines] in this study are past the expiration date listed on the vial," implying no adequate chain of custody and tests conducted on vials which would not have been administered to patients. McKernan's second preprint (Speicher et al., 2023, p. 3), released in October 2023, notes that its findings need to be "replicated under forensic conditions." ...
Chapter
Full-text available
Totalitarianism is facilitated by what Hannah Arendt calls “gigantic lies and monstrous falsehoods,” which the masses are organised to believe. This tradition has a long history, even in the West. The “Covid-19 pandemic” was a Big Lie: there is no credible epidemiological evidence to support its existence. Rather, the “pandemic” was a media-driven social phenomenon that served to deflect attention from far-reaching technocratic agendas being advanced across every area of life. “Pandemic preparedness” provides cover for building the institutional architecture of global dictatorship under the pretext of public health. It is unclear whether “SARS-CoV-2” is real: problems exist regarding its alleged “isolation” (involving cytopathic effects, genome sequencing, and electron microscopy images). There is, however, evidence to suggest that “SARS-CoV-2” may, in part, have been influenza rebranded. The “vaccines,” which instead of protecting people have caused catastrophic harm, are likely bioweapons aimed at controlling the population in multiple ways. Yet, despite the “Covid-19” narrative being saturated with deceit, most people cannot and will not see it, owing to cognitive dissonance.
... The fact that this limit value was successfully met in the production of Comirnaty ® was generally accepted as a given after its authorisation. However, this dogma had to be reconsidered after the US scientist Kevin McKernan and his team made it public that they had found large quantities of DNA impurities in Comirnaty ® [8], most of which were present in quantities that were several hundred times higher than the applicable limit of 10 ng DNA per dose. Other scientists followed with their own results, including the Canadian group led by David Speicher [9] and the US cancer researcher Phillip Buckhaults, who presented his findings to the South Carolina Senate [10]. ...
Article
Full-text available
DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods. Experimental testing of this approach confirms that reliable values can be obtained in this way.
... An article from 2023 published in Medical Hypotheses (19) suggested that the accumulation of ModRNA and reverse-transcribed DNA molecules in the cytoplasm could potentially induce chronic inflammation, autoimmunity, DNA damage, and cancer in susceptible individuals (reviewed in 10,19). Geneticist researcher Kevin McKernan also found that ModRNA vaccines against COVID-19 can potentially be reverse-transcribed into DNA (20). McKernan and colleagues detected the Spike protein sequence from the COVID-19 ModRNA vaccine, which was reversetranscribed into DNA and integrated into chromosomes 9 and 12 in human cancer cell lines originating from the breast and ovary. ...
Preprint
Full-text available
There have been reports describing excess deaths in many countries during 2020 to 2023 in comparison with the mortality in 2019 or earlier. It has been suggested that the massive vaccination with ModRNA vaccines against covid may be associated with it. We describe 7 mechanisms by which ModRNA vaccines against Covid may increase the development and the excess deaths caused by different cancers.
... An article from 2023 published in Medical Hypotheses (19) suggested that the accumulation of ModRNA and reverse-transcribed DNA molecules in the cytoplasm could potentially induce chronic inflammation, autoimmunity, DNA damage, and cancer in susceptible individuals (reviewed in 10,19). Geneticist researcher Kevin McKernan also found that ModRNA vaccines against COVID-19 can potentially be reverse-transcribed into DNA (20). McKernan and colleagues detected the Spike protein sequence from the COVID-19 ModRNA vaccine, which was reversetranscribed into DNA and integrated into chromosomes 9 and 12 in human cancer cell lines originating from the breast and ovary. ...
Article
Full-text available
There have been documented excess mortality from 2020 to 2023 in comparison with 2019 or earlier years in Japan,Australia, several countries of Europe and USA. There seem to be evidence suggesting that this is related to the massive vaccination with ModRNA vaccines. Here, we present seven mechanism by which ModRNA vaccines anti-covid may increase the development and mortality due to cancer.
... The issue of batch variability is further complicated by recent findings of DNA contamination in the mRNA vaccines [151]. In an analysis of multiple vials of the bivalent Pfizer and Moderna mRNA products, McKernan et al. found "high levels of DNA contamination in both the monovalent and bivalent vaccines" that were "orders of magnitude higher than the EMA's limit" of 330 nanograms of DNA per milligram of RNA [152]. The DNA process-related impurities also exceeded the safety limits of the FDA (10ng/dose). ...
Article
Full-text available
The mRNA-LNP vaccine has received much attention during the COVID-19 pandemic since it served as the basis of the most widely used SARS-CoV-2 vaccines in Western countries. Based on early clinical trial data, these vaccines were deemed safe and effective for all demographics. However, the latest data raise serious concerns about the safety and effectiveness of these vaccines. Here, we review some of the safety and efficacy concerns identified to date. We also discuss the potential mechanism of observed adverse events related to the use of these vaccines and whether they can be mitigated by alterations of this vaccine mechanism approach.
Article
Full-text available
Our understanding of COVID-19 vaccinations and their impact on health and mortality has evolved substantially since the first vaccine rollouts. Published reports from the original randomized phase 3 trials concluded that the COVID-19 mRNA vaccines could greatly reduce COVID-19 symptoms. In the interim, problems with the methods, execution, and reporting of these pivotal trials have emerged. Re-analysis of the Pfizer trial data identified statistically significant increases in serious adverse events (SAEs) in the vaccine group. Numerous SAEs were identified following the Emergency Use Authorization (EUA), including death, cancer, cardiac events, and various autoimmune, hematological, reproductive, and neurological disorders. Furthermore, these products never underwent adequate safety and toxicological testing in accordance with previously established scientific standards. Among the other major topics addressed in this narrative review are the published analyses of serious harms to humans, quality control issues and process-related impurities, mechanisms underlying adverse events (AEs), the immunologic basis for vaccine inefficacy, and concerning mortality trends based on the registrational trial data. The risk-benefit imbalance substantiated by the evidence to date contraindicates further booster injections and suggests that, at a minimum, the mRNA injections should be removed from the childhood immunization program until proper safety and toxicological studies are conducted. Federal agency approval of the COVID-19 mRNA vaccines on a blanket-coverage population-wide basis had no support from an honest assessment of all relevant registrational data and commensurate consideration of risks versus benefits. Given the extensive, well-documented SAEs and unacceptably high harm-to-reward ratio, we urge governments to endorse a global moratorium on the modified mRNA products until all relevant questions pertaining to causality, residual DNA, and aberrant protein production are answered.
Preprint
Full-text available
Background: In vitro transcription (IVT) reactions used to generate nucleoside modified RNA (modRNA) for SARS-CoV-2 vaccines currently rely on an RNA polymerase transcribing from a DNA template. Production of modRNA used in the original Pfizer randomized clinical trial (RCT) utilized a PCR-generated DNA template (Process 1). To generate billions of vaccine doses, this DNA was cloned into a bacterial plasmid vector for amplification in Escherichia coli before linearization (Process 2), expanding the size and complexity of potential residual DNA and introducing sequences not present in the Process 1 template. It appears that Moderna used a similar plasmid-based process for both clinical trial and post-trial use vaccines. Recently, DNA sequencing studies have revealed this plasmid DNA at significant levels in both Pfizer-BioNTech and Moderna modRNA vaccines. These studies surveyed a limited number of lots and questions remain regarding the variance in residual DNA observed internationally.Methods: Using previously published primer and probe sequences, quantitative polymerase chain reaction (qPCR) and Qubit® fluorometry was performed on an additional 27 mRNA vials obtained in Canada and drawn from 12 unique lots (5 lots of Moderna child/adult monovalent, 1 lot of Moderna adult bivalent BA.4/5, 1 lot of Moderna child/adult bivalent BA.1, 1 lot of Moderna XBB.1.5 monovalent, 3 lots of Pfizer adult monovalent, and 1 lot of Pfizer adult bivalent BA.4/5). The Vaccine Adverse Events Reporting System (VAERS) database was queried for the number and categorization of adverse events (AEs) reported for each of the lots tested. The content of one previously studied vial of Pfizer COVID-19 vaccine was examined by Oxford Nanopore sequencing to determine the size distribution of DNA fragments. This sample was also used to determine if the residual DNA is packaged in the lipid nanoparticles (LNPs) and thus resistant to DNaseI or if the DNA resides outside of the LNP and is DNaseI labile. Results: Quantification cycle (Cq) values (1:10 dilution) for the plasmid origin of replication (ori) and spike sequences ranged from 18.44 - 24.87 and 18.03 - 23.83 and for Pfizer, and 22.52 – 24.53 and 25.24 – 30.10 for Moderna, respectively. These values correspond to 0.28 – 4.27 ng/dose and 0.22 - 2.43 ng/dose (Pfizer), and 0.01 -0.34 ng/dose and 0.25 – 0.78 ng/dose (Moderna), for ori and spike respectively measured by qPCR, and 1,896 – 3,720 ng/dose and 3,270 – 5,100 ng/dose measured by Qubit® fluorometry for Pfizer and Moderna, respectfully. The SV40 promoter-enhancer-ori was only detected in Pfizer vials with Cq scores ranging from 16.64 – 22.59. In an exploratory analysis, we found preliminary evidence of a dose response relationship of the amount of DNA per dose and the frequency of serious adverse events (SAEs). This relationship was different for the Pfizer and Moderna products. Size distribution analysis found mean and maximum DNA fragment lengths of 214 base pairs (bp) and 3.5 kb, respectively. The plasmid DNA is likely inside the LNPs and is protected from nucleases.Conclusion: These data demonstrate the presence of billions to hundreds of billions of DNA molecules per dose in these vaccines. Using fluorometry, all vaccines exceed the guidelines for residual DNA set by FDA and WHO of 10 ng/dose by 188 – 509-fold. However, qPCR residual DNA content in all vaccines were below these guidelines emphasizing the importance of methodological clarity and consistency when interpreting quantitative guidelines. The preliminary evidence of a dose-response effect of residual DNA measured with qPCR and SAEs warrant confirmation and further investigation. Our findings extend existing concerns about vaccine safety and call into question the relevance of guidelines conceived before the introduction of efficient transfection using LNPs. With several obvious limitations, we urge that our work is replicated under forensic conditions and that guidelines be revised to account for highly efficient DNA transfection and cumulative dosing.
Preprint
Full-text available
Background: In vitro transcription (IVT) reactions used to generate nucleoside modified RNA (modRNA) for SARS-CoV-2 vaccines currently rely on an RNA polymerase transcribing from a DNA template. Production of modRNA used in the original Pfizer randomized clinical trial (RCT) utilized a PCR-generated DNA template (Process 1). To generate billions of vaccine doses, this DNA was cloned into a bacterial plasmid vector for amplification in Escherichia coli before linearization (Process 2), expanding the size and complexity of potential residual DNA and introducing sequences not present in the Process 1 template. It appears that Moderna used a similar plasmid-based process for both clinical trial and post-trial use vaccines. Recently, DNA sequencing studies have revealed this plasmid DNA at significant levels in both Pfizer-BioNTech and Moderna modRNA vaccines. These studies surveyed a limited number of lots and questions remain regarding the variance in residual DNA observed internationally. Methods: Using previously published primer and probe sequences, quantitative polymerase chain reaction (qPCR) and Qubit® fluorometry was performed on an additional 27 mRNA vials obtained in Canada and drawn from 12 unique lots (5 lots of Moderna child/adult monovalent, 1 lot of Moderna adult bivalent BA.4/5, 1 lot of Moderna child/adult bivalent BA.1, 1 lot of Moderna XBB.1.5 monovalent, 3 lots of Pfizer adult monovalent, and 1 lot of Pfizer adult bivalent BA.4/5). The Vaccine Adverse Events Reporting System (VAERS) database was queried for the number and categorization of adverse events (AEs) reported for each of the lots tested. The content of one previously studied vial of Pfizer COVID-19 vaccine was examined by Oxford Nanopore sequencing to determine the size distribution of DNA fragments. This sample was also used to determine if the residual DNA is packaged in the lipid nanoparticles (LNPs) and thus resistant to DNaseI or if the DNA resides outside of the LNP and is DNaseI labile. Results: Quantification cycle (Cq) values (1:10 dilution) for the plasmid origin of replication (ori) and spike sequences ranged from 18.44 - 24.87 and 18.03 - 23.83 and for Pfizer, and 22.52 – 24.53 and 25.24 – 30.10 for Moderna, respectively. These values correspond to 0.28 – 4.27 ng/dose and 0.22 - 2.43 ng/dose (Pfizer), and 0.01 -0.34 ng/dose and 0.25 – 0.78 ng/dose (Moderna), for ori and spike respectively measured by qPCR, and 1,896 – 3,720 ng/dose and 3,270 – 5,100 ng/dose measured by Qubit® fluorometry for Pfizer and Moderna, respectfully. The SV40 promoter-enhancer-ori was only detected in Pfizer vials with Cq scores ranging from 16.64 – 22.59. In an exploratory analysis, we found preliminary evidence of a dose response relationship of the amount of DNA per dose and the frequency of serious adverse events (SAEs). This relationship was different for the Pfizer and Moderna products. Size distribution analysis found mean and maximum DNA fragment lengths of 214 base pairs (bp) and 3.5 kb, respectively. The plasmid DNA is likely inside the LNPs and is protected from nucleases. Conclusion: These data demonstrate the presence of billions to hundreds of billions of DNA molecules per dose in these vaccines. Using fluorometry, all vaccines exceed the guidelines for residual DNA set by FDA and WHO of 10 ng/dose by 188 – 509-fold. However, qPCR residual DNA content in all vaccines were below these guidelines emphasizing the importance of methodological clarity and consistency when interpreting quantitative guidelines. The preliminary evidence of a dose-response effect of residual DNA measured with qPCR and SAEs warrant confirmation and further investigation. Our findings extend existing concerns about vaccine safety and call into question the relevance of guidelines conceived before the introduction of efficient transfection using LNPs. With several obvious limitations, we urge that our work is replicated under forensic conditions and that guidelines be revised to account for highly efficient DNA transfection and cumulative dosing.
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