Precisely how does Pfizer's Covid-19 mRNA vaccine work?

In summary, Pfizer's Covid-19 mRNA vaccine works by using a small piece of genetic material called mRNA to instruct the body's cells to produce a harmless spike protein found on the surface of the virus. This triggers the body's immune system to create antibodies and T-cells to fight the virus. The vaccine does not contain any live or weakened virus, making it safe and effective. The mRNA is quickly broken down by the body, leaving no permanent changes to the genetic code. This innovative approach allows for a faster and more efficient production of the vaccine, providing a crucial tool in the fight against the Covid-19 pandemic.
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aheight
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TL;DR Summary
Precisely how biochemically, does Pfizer's mRNA vaccine work?
I have some questions about how the Pfizer mRNA vaccine BNT162b2 (BTN for short) works.

BTN codes for the SARS Covid-2 spike protein RNA sequence and is somehow delivered into cells, transcribed and then the proteins are delivered to the outer cell membrane or maybe just protrudes through the membrane. Since this is a "foreign" protein, it elicits a immune response, thereby "priming" the body for SARS Covid-2:

BNT162b2 uses messenger RNA that describes one of the spike proteins that stud the outer surface of SARS-CoV-2. Though human cells don’t make spike proteins, they can still read viral messenger RNA and follow its instructions. When someone receives a dose of BNT162b2, their body responds by producing the spike protein, but only the spike protein, and no other part of the virus.
Since spike proteins aren’t normally found in human cells, their presence triggers the immune system, leading to a defensive response where the proteins are removed. Now that the immune system’s had some practice, it’s ready for the real thing. If someone who was vaccinated against SARS-CoV-2 was exposed to the virus later on, their immune system is ready to react, and hopefully, fend off the virus.
Link to Pfizer vaccine description

I was wondering about the following:

(1) Naked RNA is rapidly destroyed if injected directly into the blood stream. BTN therefore has some additional machinery attached like proteins or sugars to mitigate this, not sure. How is BTN prepared to assure stability?

(2) How is BTN delivered into the cells? I don't think RNA by itself can do this. Must have protein machinery to bind to receptors and gain access. Was wondering if this is in fact, the S1 and S2 proteins the actual virus uses to gain entry or since the ACE2 receptors are naturally-occurring, proteins used by the body to accomplish this?

(3) I imagine the BTN-complex can't by itself get translated; the additional machinery attached to BTN would interfere? Is it first removed by some enzyme? Is there additional RNA attached to BTN to code for some type of CRISPR snipping?

(4) Once the spike protein (or at least part of it) is synthesized, how does it get to the outside of the cell? Is this by membrane-bound protein gates? I don't understand how a gate is going to recognize a "foreign" protein and let it through however. Does BTN include a sequence to code the necessary amino acids to open the gate. Is this reasonable?

(5) Since the spike proteins become an integral part of some cells, is it possible they in time will lead to a reduction in immunity if the immune system begins to recognize them as "normal" protein?

I have done a bit of searching but haven't found any specific enough to answer these question. I'll dig more.
 
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I think the answer to 1) question is micelles - very small lipid bi-layer balls with mRNA inside. The micelle merges with cell membranes and then release mRNA into the cytosol. Here is a video discussion aimed at medical professionals:

 
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  • #4
aheight said:
(1) Naked RNA is rapidly destroyed if injected directly into the blood stream. BTN therefore has some additional machinery attached like proteins or sugars to mitigate this, not sure. How is BTN prepared to assure stability?

As others have noted, mRNA vaccines are packaged in lipid nanoparticles (LNPs) that not only protect the mRNA from degradation, but also aid in the delivery of mRNA into cells: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439223/

(2) How is BTN delivered into the cells? I don't think RNA by itself can do this. Must have protein machinery to bind to receptors and gain access. Was wondering if this is in fact, the S1 and S2 proteins the actual virus uses to gain entry or since the ACE2 receptors are naturally-occurring, proteins used by the body to accomplish this?

Again, this comes from the LNPs that encapsulate the mRNA. LNPs seem to mimic low density lipoproteins (used to transport lipids and fats throughout the body) and are taken up through endocytotic pathways similar to those used for the uptake of the LDLs. The LNPs are also formulated to aid escape of the mRNA from endocytotic vessicles so that the mRNA can be delivered to the cytoplasm where translation of the mRNA into protein can occur (see the review article linked above).

While the spike protein encoded by the mRNA is used by the SARS-CoV-2 virus to gain entry into the cells, the spike protein is not involved in entry of the LNPs into cells (the LNPs do not contain spike protein, only mRNA that encodes the spike protein. The protein would not actually be made until the mRNA gains entry into the cytoplasm of cells).

(3) I imagine the BTN-complex can't by itself get translated; the additional machinery attached to BTN would interfere? Is it first removed by some enzyme? Is there additional RNA attached to BTN to code for some type of CRISPR snipping?

Putting a naked mRNA molecule into the cytoplasm of a cell is sufficient to get the mRNA molecule translated. The LNP that originally enveloped the mRNA gets absorbed in the membrane system of the cell after the mRNA gets released from the endocytotic vesicles, so the LNP does not interfere with translation of the mRNA.

(4) Once the spike protein (or at least part of it) is synthesized, how does it get to the outside of the cell? Is this by membrane-bound protein gates? I don't understand how a gate is going to recognize a "foreign" protein and let it through however. Does BTN include a sequence to code the necessary amino acids to open the gate. Is this reasonable?

Yes. All eukaryotic use a set of signal peptides on the N-terminus of proteins that tells the cell to traffic proteins to different locations in the cell (e.g. mitochondria, lysosomes, ER, plasma membrane, etc.). For more information see:
https://www.ncbi.nlm.nih.gov/books/NBK21471/
https://en.wikipedia.org/wiki/Signal_peptide

(5) Since the spike proteins become an integral part of some cells, is it possible they in time will lead to a reduction in immunity if the immune system begins to recognize them as "normal" protein?

No. The mRNA that is delivered does not permanently stay inside the body. Eventually, it gets decayed through normal mRNA turnover machinery in the cell, and cells will stop making the spike protein. Similarly, the spike protein gets removed through normal protein recycling mechanisms, so they too will go away some time after vaccine administration.
 
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Very interesting guys. Thanks. Seems the BTN vaccines code for modified versions of the S1 and S2 spike proteins. Also, looks like the modified versions include molecular bridges to hold the proteins in the necessary confirmation to elicit the desired immune effect. Really beautiful biochemical orchestration to be admired. :)
 
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aheight said:
Very interesting guys. Thanks. Seems the BTN vaccines code for modified versions of the S1 and S2 spike proteins. Also, looks like the modified versions include molecular bridges to hold the proteins in the necessary confirmation to effect binding to the ACE2 receptors. Really beautiful biochemical orchestration to be admired. :)

Where did you find that information? I know of the HexaPro version (paper below) but don't know whether the BTN vaccine uses that or another method.

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes by Hsieh et al
 
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I think the video has information, plus it also lists a site that has good details for each vaccine trial. The same site links to papers thaat are the design basis.
 
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jim mcnamara said:
I think the video has information, plus it also lists a site that has good details for each vaccine trial. The same site links to papers thaat are the design basis.

Thanks. From https://www.nejm.org/doi/full/10.1056/NEJMoa2027906, it seems that conformation stabilization for BNT162b1 relies on vaccines for the earlier SARS pandemic, while for BNT162b2 they cite https://science.sciencemag.org/content/367/6483/1260 which is earlier work from the group that authored the paper in post #6.
 
  • #9
atyy said:
Where did you find that information? I know of the HexaPro version (paper below) but don't know whether the BTN vaccine uses that or another method.
Regarding the molecular bridge? The video posted by Jim above alluded to this. That makes sense as I suspect naked S1 and S2 are not conformally suitable for eliciting an immune response to SARS Covid-2 but rather need framework to hold the proteins in the conformation as they are found on the virus coat. I would be curious if the 90% effectiveness of the Pfizer vaccine is a direct consequence of just how close the bridging, if in fact this is used, replicated the exact conformation of the spike proteins on the virus.
 
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aheight said:
Regarding the molecular bridge? The video posted by Jim above alluded to this. That makes sense as I suspect naked S1 and S2 are not conformally suitable for eliciting an immune response to SARS Covid-2 but rather need framework to hold the proteins in the conformation as they are found on the virus coat. I would be curious if the 90% effectiveness of the Pfizer vaccine is a direct consequence of just how close the bridging, if in fact this is used, replicated the exact conformation of the spike proteins on the virus.

Thanks. Following the references, it seems BNT162b2 used earlier work from the group that introduced the HexaPro variant I mentioned in post #6. That earlier worked used proline substitutions: https://science.sciencemag.org/content/367/6483/1260.

https://cns.utexas.edu/news/covid-19-vaccine-with-ut-ties-arrived-quickly-after-years-in-the-making
 
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A Naive Question: Why is this mean technique more effective than traditional vaccines methods? Or why don't traditional vaccine methods suffice?
 
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The main advantage of mRNA vaccines is not efficacy (since they are relatively new, we don't really have much data to say whether they are any more or less effective than traditional vaccines). The main advantage is their versatility and the speed at which they can be prototyped and produced (evidenced by the fact that the mRNA vaccines were the first to reach clinical trials and the first to report results form those trials).

Traditional live attenuated vaccines or inactivated virus vaccines requires growing large quantities of the virus, a very time consuming process. Protein subunit vaccines can be produced more quickly, but different proteins from different viruses require different conditions for production that would have to be optimized for different viruses. In contrast, mRNA vaccines for different viruses would use almost the exact same processes for production as producing an mRNA encoding a Coronavirus protein would be very similar to producing an mRNA encoding a, say, Ebola protein.

The success of mRNA vaccines against SARS-CoV-2 suggests that if new pandemic viruses were to emerge, mRNA vaccines could be produced with similar speed provided we have a good understanding about the proper antigen to use in such a vaccine.

Here's a decent article discussing the topic in a bit more detail: https://jamanetwork.com/journals/jama/fullarticle/2770485
 
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Ygggdrasil said:
The main advantage of mRNA vaccines is not efficacy (since they are relatively new, we don't really have much data to say whether they are any more or less effective than traditional vaccines). The main advantage is their versatility and the speed at which they can be prototyped and produced (evidenced by the fact that the mRNA vaccines were the first to reach clinical trials and the first to report results form those trials).

Traditional live attenuated vaccines or inactivated virus vaccines requires growing large quantities of the virus, a very time consuming process. Protein subunit vaccines can be produced more quickly, but different proteins from different viruses require different conditions for production that would have to be optimized for different viruses. In contrast, mRNA vaccines for different viruses would use almost the exact same processes for production as producing an mRNA encoding a Coronavirus protein would be very similar to producing an mRNA encoding a, say, Ebola protein.

The success of mRNA vaccines against SARS-CoV-2 suggests that if new pandemic viruses were to emerge, mRNA vaccines could be produced with similar speed provided we have a good understanding about the proper antigen to use in such a vaccine.

Here's a decent article discussing the topic in a bit more detail: https://jamanetwork.com/journals/jama/fullarticle/2770485

So there is no advantage in mRNA vaccines in producing antigens. One might think that since they are constantly being produced in infected cells there would be a stronger or longer lasting effectiveness.
 
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ChinleShale said:
So there is no advantage in mRNA vaccines in producing antigens. One might think that since they are constantly being produced in infected cells there would be a stronger or longer lasting effectiveness.

We have so little data on mRNA vaccines that I don't think we have a clear answer to this question. Based on the limited data with Coronavirus vaccines we have, it seems like protein vaccines provoke stronger immune responses that mRNA vaccines, though again we only really have the SARS-CoV-2 mRNA vaccine as an example. Here's a relevant excerpt from a review article discussing early clinical data on the various SARS-CoV-2 vaccines under development:

For the vaccines in clinical trials for which Phase I/II data is available,
we observe both an immunogenicity and reactogenicity gradient.
In terms of immunogenicity, AdV5-based vaccines seem to rank
lowest, followed by inactivated and ChAdOx1 based vaccines, mRNA
vaccines, and finally adjuvanted, protein-based vaccines performing
best. Reactogenicity seems lowest in inactivated and protein based
vaccines, followed by mRNA vaccines, with vectored vaccines having
the highest rate of side effects. It is highly likely that the AstraZeneca,
Moderna and Pfizer vaccine candidates, which are along the furthest
in the US and Europe, all show sufficient efficacy and will be licensed if
sufficiently safe. However, it may also be that these vaccines will later
on be replaced by vaccines that show similar efficacy but have reactogenicity
profiles that are more tolerable. In addition, it is hard to
predict how availability and production capacity will shape the global
landscape of SARS-CoV-2 vaccines.

Krammer, F. SARS-CoV-2 vaccines in development. Nature (2020). https://doi.org/10.1038/s41586-020-2798-3
 
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I wonder if taking statin drugs degrades the LNPs,
 
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Is there a possibility it works only temporary like 6 months and one is no longer immuned or require yearly Pfizer Covid vaccinations? Or is it one time like polio vaccine that last a lifetime?
 
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Excellent information. Lipid nano particles. LPN. How the mRNA vaccines get into the cell. For some reason the name Lieber comes to mind.
 
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swampwiz said:
I wonder if taking statin drugs degrades the LNPs,
Why do you think taking statin drugs would degrade the LNPs?

jake jot said:
Is there a possibility it works only temporary like 6 months and one is no longer immuned or require yearly Pfizer Covid vaccinations? Or is it one time like polio vaccine that last a lifetime?
From studies of coronaviruses that cause common colds, we know that immunity to those coronaviruses wanes over time (over the course of ~ 6 months to a few years). However, subsequent infections led to more mild or asymptomatic infections. Vaccines, however, likely induce stronger immunity than viral infections as viruses contain components that try to reduce the body's immune response to its antigens while vaccines are designed to elicit a strong immune response. I would doubt that the vaccine would provide lifetime immunity, but we still need more data to know how long (on average) immunity lasts (and whether the immunity is sterilizing -- preventing infection-- or functional -- preventing sympotmatic disease but not stopping infection and transmission).

Here's a good piece that discusses these issues: https://www.statnews.com/2020/08/25/four-scenarios-on-how-we-might-develop-immunity-to-covid-19/

Phil Core said:
Excellent information. Lipid nano particles. LPN. How the mRNA vaccines get into the cell. For some reason the name Lieber comes to mind.

LNPs seem to mimic low density lipoproteins (used to transport lipids and fats throughout the body) and are taken up through endocytotic pathways similar to those used for the uptake of the LDLs. The LNPs are also formulated to aid escape of the mRNA from endocytotic vessicles so that the mRNA can be delivered to the cytoplasm where translation of the mRNA into protein can occur (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439223/ for more information).
 
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Ygggdrasil said:
Why do you think taking statin drugs would degrade the LNPs?
Uh, because the statins' effect is to hinder lipoprotein levels?
:rolleyes:
 
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@Ygggdrasil

If the virus surpasses immune response does that mean that vaccination after being infected is less effective than before any infection?
 
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swampwiz said:
Uh, because the statins' effect is to hinder lipoprotein levels?
:rolleyes:

Statins inhibit a key enzyme in the cholesterol biosynthesis pathway, lowering the amount of cholesterol produced by the body. In addition, they also seem to increase uptake of LDL from the bloodstream. If anything, this would be expected to increase the effectiveness of the mRNA vaccine since the mRNA vaccine is thought to be taken up into cells via a similar pathway as the uptake of LDL.

ChinleShale said:
@Ygggdrasil

If the virus surpasses immune response does that mean that vaccination after being infected is less effective than before any infection?

No. Viruses have components that help them "hide" from the immune system, but these components should not persist after the virus is eliminated from the body.
 
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Ygggdrasil said:
Statins inhibit a key enzyme in the cholesterol biosynthesis pathway, lowering the amount of cholesterol produced by the body. In addition, they also seem to increase uptake of LDL from the bloodstream.
I was merely pointing out that it would be legitimate to speculate on statins having a (negative) effect on vaccine efficacy. That it actually helps is great news!
 
  • #23
swampwiz said:
I was merely pointing out that it would be legitimate to speculate on statins having a (negative) effect on vaccine efficacy. That it actually helps is great news!
I don't think we have any evidence to say whether it helps or hurts efficacy, merely that there's just as much legitimate reason to speculate that it could help efficacy.
 
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Are the nucleotides used in the mRNA vaccine the same as in humans or are they chemically modified? If modified, as the mRNA breaks down can these nucleotides be incorporated in our RNA and DNA?
 
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bz2731 said:
Are the nucleotides used in the mRNA vaccine the same as in humans or are they chemically modified? If modified, as the mRNA breaks down can these nucleotides be incorporated in our RNA and DNA?

Yes, I believe that the mRNA vaccines do contain modified nucleosides (e.g. here's a publication from Moderna about how modified nucleosides can help the delivered mRNAs avoid the innate immune system). It's worth noting that there are over 160 modified nucleosides known to exist in natural organisms, and the modification discussed in the Moderna publication above (1-methylpseudouridine) is found in the ribosomal RNA of eukaryotes.

Generally, the RNA and DNA polymerase enzymes that synthesize RNA and DNA are fairly selective about which nucleotides they use, so they generally avoid using any modified nucleotides that might be present inside of the cell. Furthermore, the cell maintains fairly large amounts of the standard nucleotides inside of the cell that any modified nucleotides that result from degradation of modified mRNAs would constitute a very small fraction of that pool, further lowering the chance of misincorporation.

I would not expect the modified nucleotides in the mRNA vaccines to be incorporated into our RNA or DNA in any appreciable amount to cause any adverse effects.
 
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That's well and good for one or two doses of mRNA vaccines. However, this will quickly become the go to method of making vaccines since they can be developed in a matter of months rather than the years taken for traditional vaccine development. Do you think over time as we start getting more of these types of vaccines with modified nucleosides we could start incorporating them, particularly when perhaps having to get them every year or maybe multiple times a year since we don't know how long the various antibodies last? Granted, the nucleosides used in making each type of mRNA vaccine will not necessarily be the same but I can imagine a single company would tend to use the same ones. That actually brings to mind another question. Do they get all the types of antibodies from the mRNA vaccine as generated by the actual virus?
 
  • #27
bz2731 said:
That's well and good for one or two doses of mRNA vaccines. However, this will quickly become the go to method of making vaccines since they can be developed in a matter of months rather than the years taken for traditional vaccine development. Do you think over time as we start getting more of these types of vaccines with modified nucleosides we could start incorporating them, particularly when perhaps having to get them every year or maybe multiple times a year since we don't know how long the various antibodies last? Granted, the nucleosides used in making each type of mRNA vaccine will not necessarily be the same but I can imagine a single company would tend to use the same ones.

I don't think the half-life of the modified nucleotides in the body would be so long that they would cause problems from yearly vaccines. Regardless, we already have safety data from tens of thousands of people receiving mRNA vaccines over a few months showing few adverse events, and soon we'll have safety data from millions of people getting mRNA to get better ideas of any unexpected long term safety issues.

That actually brings to mind another question. Do they get all the types of antibodies from the mRNA vaccine as generated by the actual virus?
Yes, data from the clinical trials of the Pfizer vaccine show that the vaccine is able to activate both humoral and cellular immunity: https://www.nature.com/articles/s41586-020-2814-7
 
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Is the sequence of the mRNA (including modified nucleotides) in the public domain? Does it code any enzymes in addition to the spike protein?
 
  • #29
markar said:
Is the sequence of the mRNA (including modified nucleotides) in the public domain? Does it code any enzymes in addition to the spike protein?

AFAIK, the sequence information is not public. Here's how they describe the vaccine in one of their papers:

Manufacturing of RNA.
BNT162b2 incorporates a Good Manufacturing Practice (GMP)-grade mRNA drug substance that encodes the trimerised SARS-CoV-2 S glycoprotein RBD antigen. The RNA is generated from a DNA template by in vitro transcription in the presence of 1-methylpseudouridine-5’- triphosphate (m1ΨTP; Thermo Fisher Scientific) instead of uridine-5’-triphosphate (UTP). Capping is performed co-transcriptionally using a trinucleotide cap 1 analogue ((m2 7,3’- O)Gppp(m2’-O )ApG; TriLink). The antigen-encoding RNA contains sequence elements that increase RNA stability and translation efficiency in human dendritic cells11,12 . The mRNA is formulated with lipids to obtain the RNA-LNP drug product. The vaccine was transported and supplied as a buffered-liquid solution for IM injection and was stored at -80 °C.
https://www.medrxiv.org/content/10.1101/2020.12.09.20245175v1
 
  • #30
Ygggdrasil said:
AFAIK, the sequence information is not public. Here's how they describe the vaccine in one of their papers:

Pretty amazing too how fast the sequence was created:

35-year old contribution to Covid vaccine

Just two days after the first complete genome of the virus was mapped and publicly posted online in early January, Moderna's COVID-19 vaccine candidate, mRNA-1273, was finalized.

I assume the " trimerised SARS-CoV-2 S glycoprotein RBD antigen " is the spike protein consisting of three protein chains?

Really beautiful how biology has advanced in 30 years!
 
  • #31
markar said:
Is the sequence of the mRNA (including modified nucleotides) in the public domain? Does it code any enzymes in addition to the spike protein?

Here's a good piece describing the components of the vaccine (though, again, the exact sequence of the mRNA appears to be proprietary): https://www.technologyreview.com/20...-the-ingredients-of-pfizers-covid-19-vaccine/

aheight said:
I assume the " trimerised SARS-CoV-2 S glycoprotein RBD antigen " is the spike protein consisting of three protein chains?
Yes. The S glycoprotein is another name for the spike protein (S stands for spike). A glycoprotein is a protein that has sugars attached to it. Most proteins on the surfaces of cells have sugars attached to them, and the spike protein in no exception. The RBD stands for the receptor binding domain, and refers to a specific part of the spike protein that binds to the ACE2 protein on the surface of cells. You want antibodies to target the RBD of the spike protein because these antibodies could interfere with attachment of the virus to cells. On the surface of the viruses, three spike proteins come together to form an active trimer, so timerization of the antigen in the vaccine helps to mimic how the spike protein looks on the outside of viruses.
 
  • #32
markar said:
Is the sequence of the mRNA (including modified nucleotides) in the public domain? Does it code any enzymes in addition to the spike protein?

Here's a source that apparently has the sequence of the Pfizer mRNA vaccine:
 

Related to Precisely how does Pfizer's Covid-19 mRNA vaccine work?

1. How does Pfizer's Covid-19 mRNA vaccine work exactly?

The Pfizer Covid-19 mRNA vaccine works by delivering a small piece of genetic material, called messenger RNA (mRNA), into the body. This mRNA contains instructions for the body to produce a harmless piece of the virus's spike protein. This protein triggers an immune response, teaching the body's immune system to recognize and fight off the virus if it encounters it in the future.

2. Is the mRNA in Pfizer's Covid-19 vaccine the same as the virus's genetic material?

No, the mRNA in the Pfizer vaccine is not the same as the virus's genetic material. The mRNA in the vaccine only contains instructions for the body to produce a small piece of the virus's spike protein, not the entire virus. This means that the vaccine cannot cause a person to become infected with Covid-19.

3. How does the body's immune system respond to the mRNA in Pfizer's Covid-19 vaccine?

When the body receives the mRNA from the vaccine, it recognizes it as a foreign substance and begins to produce antibodies and activate immune cells to fight it off. This immune response is what teaches the body to recognize and fight off the virus if it encounters it in the future.

4. How long does it take for the body to develop immunity after receiving the Pfizer Covid-19 vaccine?

It typically takes a few weeks for the body to develop immunity after receiving the Pfizer Covid-19 vaccine. This is because it takes time for the immune system to produce enough antibodies and for them to become effective at fighting off the virus.

5. How effective is the Pfizer Covid-19 vaccine in preventing infection?

According to clinical trials, the Pfizer Covid-19 vaccine has been shown to be approximately 95% effective in preventing infection. This means that out of 100 people who receive the vaccine, 95 of them will be protected from getting Covid-19. It is important to note that no vaccine is 100% effective, but the Pfizer vaccine has shown to be highly effective in preventing infection.

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