3.29 Vaccines
how do mRNA vaccines work?
- Both Pfizer & Moderna vaccines used nucleoside modified mRNA LNP platform to develop COVID vaccines - Both generate robust immune responses with antibodies specific to the spike protein on the extracellular surface of the virus - The nucleoside modified mRNA encoding spike protein is synthesized through the method described in previous slide - Then mRNA encapsulated into LNPs - Vaccine injected intramuscularly into humans (into arm muscle) - LNPs deliver mRNA pay load to numerous cells, which translate it into spike protein - Immune system acknowledges foreign protein & produces copious amounts of antibodies against the antigen - If a vaccinated person is exposed to COVID, they now have developed memory B cells that can differentiate into plasma cells and secrete antibodies to neutralize the virus directly! FIGURE [slide 21 in 3.29 vaccines & mRNA] 1. spike protein DNA sequence is copied 2. DNA is copied into mRNA form 3. mRNA is encapsulated in a liquid nanoparticle 4. vaccine is injected into arm muscle 5. in the cell: - mRNA is released from the lipid & enters the target cell - mRNA is translated into antigen - new spike protein presents to the immune system 6. in circulation - antibodies against spike protein are made
live-attenuated vaccines
- most traditional method of vaccine development HOW MAKE - take pathologic virus / bacteria and weaken it by passing it in culture multiple times - weakened virus / bacteria then gets in cells, replicates, and makes proteins - immune response learns about it so when pathogen comes & attacks at later time, immune system has already seen it & recognizes it examples: measles, mumps, rubella, chicken pox, rotavirus, and yellow fever
problems with mRNA as a vaccine platform (2 major challenges)
1. Conventional mRNA has a very high inflammatory capacity - Major safety issue!! - Cells not accustomed to foreign mRNA so when see it, their RNA sensors are set off and immune cells respond, yielding high amounts of inflammation which can be dangerous 2. Conventional mRNA is highly unstable and generally degraded rapidly by RNases - Also, RNA is a large molecule and cellular uptake is difficult! - SO carrier molecule needed to help it enter cells - THUS a way to protect mRNA from degradation & carry it into cells was needed
7 benefits of mRNA as a vaccine platform
1. high efficacy in preclinical & clinical studies 2. easy to design & can be applied to numerous pathogens - simple design: once have components of circular plasma template in place, can insert an antigen sequence from any virus - the mRNA can be transcribed & purified from the plasma in a matter of weeks 3. quick to manufacture & scale up - straightforward production process - can be scaled up such that copious amounts of mRNA can be manufactured in short amount of time 4. no cells needed to produce - can decrease risk of contamination! 5. does not contain virus so cannot yield disease - since not live attenuated virus, recipient can't obtain disease after being immunized 6. does not alter DNA 7. degraded by natural cellular processes - only exist transiently in cells, where translated into protein before naturally degraded by cellular processes
list the 5 FDA-approved vaccine platforms
1. live-attenuated 2. inactivated 3. purified protein with or without adjuvant 4. viral-vectored (replication competent or incompetent) 5. mRNA
history of vaccine (probs won't need to know for exam)
1721: lady montagu - promote virulation practice (took scabs from patients with small pox, grind them up, and have others inhale them or scratch their skin with them) - Criticized when 2-3% of those inoculated died, but on way for vaccine! 1796: jenner - smallpox - previous exposure to cow pox confer protection against small pox - extract puss from lesions on hands of milk maid infected with cow pox - made cut on 8yo arm & introduce puss to site of incision - exposed boy to small pox 6 weeks later - didn't get sick - first vaccination developed! - used LIVE virus 1885: pasteur - rabies - 9yo boy bitten by rabid dog - 13 days: injected boy with weakened form of rabies virus - boy never developed it - so rabies vaccine created! - used weakened / attenuated virus 1955: salid - polio - Use polio virus killed with formalin (diluted form of formaldehyde) - SO used KILLED virus 2020: pfizer/moderna - covid19 Now have other platforms: purified proteins, viral vectored vaccines, first mRNA vaccine (COVID)
how make (synthesize) mRNA vaccines (4 steps)
1: cloning - recombinant DNA plasmid is made that contains a sequence encoding the antigen of interest (cDNA) 2: transcription - this plasmid then nicked with restriction enzymes such that it becomes a linear DNA molecule (mRNA) 3: capping - nucleosides including modified pseudo-uridine in place of uridine, RNA synthesizing enzymes, and 5' cap added to linear DNA molecule to transcribe linear mRNA (capped mRNA) 4: purification - this mRNA then purified & encapsulated in LNPs (purified capped mRNA) THUS nucleoside modified mRNA LNP vaccine is made!
who & how did they solve the mRNA vaccine inflammation problem?
2005: Drew Weissman & Kariko (UPenn) DNA & RNA made up of nucleosides: nucleotides without the phosphate group; give cell instructions for protein synthesis DNA bases: ATGC mRNA bases: AUGC Uracil attached to a ribo sugar ring = nucleoside Uridine Uridine = nucleoside that causes RNA sensors to be set off, leading to inflammatory signals when foreign mRNA is delivered to the cell Discovered that by replacing uridine by "natural modified" nucleosides drastically reduce the inflammatory response Most commonly used modified nucleoside = pseudo-uridine (occurs naturally & can be found abundantly in cells, so body accustomed to it) Both Pfizer & Moderna COVID vaccines use pseudo-uridine Because this modified nucleoside is degraded by normal cellular pathways after some time, the risk of side effects is addressed!
central dogma of biology
DNA-transcription-RNA-translation-protein each component of central dogma can be used as vaccine platforms! (DNA, RNA, proteins)
principles review: vaccines + immunology [overview]
When vaccine administered (whether virus, protein, bacteria, or nucleic acid): body recognizes it & mounts immune response 1: innate immunity - macrophages recognize antigen (substance that causes an immune response in body) & ingest it (phagocytosis) Macrophages + other cells like dendritic cells can connect the innate immune response to the adaptive immune response Cells become antigen-presenting cells, which help activate B and T cells - Cytotoxic T cells kill infected cells - Helper T cells help w variety of things, like activating B cells - B cells can become plasma cells, which secrete highly specific antibodies, OR can become memory B cells, which can differentiate into plasma cells upon subsequent infection *Different vaccines depend on different correlates of protection: some focus on yielding a T cell response to kill a pathogen when it's entered cells while others focus on developing antibodies that respond to the pathogen outside of the cells through a number of mechanisms
what's an adjuvant (basic)?
agent that boosts an immune response to a particular antigen
inactivated vaccines
apply stress to the virus / bacteria to kill it - stress: heating / freezing, changing pH, radiation, dehydration, etc. HOW WORKS - killed pathogen interacts with host cells such that some of it gets into the cells, but since it's killed, it can't cause disease - SO the particle, whether it's extracellular or enters the cell, is enough for immune system to recognize the foreign antigen and mount an immune response to it ex: polio
purified protein vaccines
can be with or without an adjuvant HOW MAKE (e.g. hepatitis B vaccine) - DNA encoding a particular protein from the virus is isolated - then insert it into a bacterial plasmid that's been cut with restriction enzymes to allow for entry of the hepatitis B antigen producing gene - this recombinant DNA is introduced to yeast cells - fermented to produce copious amounts of the protein, which are then harvested & purified (other purified protein vaccines use bacterial cells like e. coli to produce these / purified (?) proteins) - purified protein then potentially combined with an adjuvant or conjugated to a polysaccharide to boost the recipient's immune response to the antigen
what makes mRNA unique compared to DNA vaccines?
doesn't have to get into the nucleus of the cell to function - INSTEAD mRNA is directly translated to proteins in the cytoplasm of the cell!
which is the newest vaccine platform approved by the FDA?
mRNA
history of mRNA vaccines (again probs don't need to know for exam, just for context)
mRNA vaccines not new concept - been around for awhile 1990: 1st publication demonstrating injecting synthesized mRNA into mice -> production of proteins 1992: mRNA encoding therapeutic protein could improve course of disease 1995: mRNA promising platform for cancer vaccination 1997: first mRNA company founded 2004-2005: also developed method to resolve the inflammation COVID-19 Pfizer & Moderna vaccines are delivered by a carrier molecule: lipid nano particle (LNP) 2015: modified mRNA LNP platform described! 2017: publications released illustrating effectiveness of mRNA LNPs as vaccines! Since then, numerous companies + academic institutions displayed efficacy & safety of this platform
how solved the mRNA unstable & hard to deliver problem
solution: package the mRNA in a liquid nanoparticle (LNP)! Once RNAses recognize it (foreign mRNA), it's rapidly degraded - Also large size of RNA makes it hard to enter cells - SO needed way to effectively deliver mRNA in way that it could be properly translated into protein in both animal & human models 2015: paper published demonstrating carrier molecules, lipid nano particles, LNPs (fat droplets), could be used to deliver large mRNA pay load & protect it from degradation by cellular processes! Nucleoside modified mRNA encapsulated in LNPs can efficiently enter cells where its translated into protein for the immune system to develop memory such that it can respond to subsequent infection 2017: studies showed this platform could be used to develop vaccines 2020: Pfizer & Moderna created their COVID vaccines using nucleoside modified mRNA LNPs (gained FDA authorization of this platform for the very first time!)
viral-vectored vaccines: replication competent
the virus is able to make copies of itself HOW MAKE: - take unrelated harmless virus (i.e. vesicular stomatitis virus, VSV, for ebola vaccine) and genetic material from the pathogen you want to target - then insert the gene you want to express from the pathogen into the virus vector HOW WORKS: - after injection, recombinant virus replications & particular antigens from the pathogen are synthesized - thus when someone infected with the pathogen, their immune response has already seen a portion of it and is able to respond
viral-vectored vaccines: replication incompetent
viral vector just expresses the protein of interest (virus unable to make copies of itself) - lacks viral genome feature for viral replication HOW WORK: - insert portion of genetic material from pathogen into harmless virus - BUT this time, virus not able to reproduce & there's no viral shedding / assembly - however, cells still able to take up viral vector encoding the antigen of interest, produce proteins, and mount an immune response such that they're produced if expressed to that pathogen later on
