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How are lipid-coated mRNA-based vaccines transported into cells for expression?

How are lipid-coated mRNA-based vaccines transported into cells for expression?



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In CNN's video Scientist says Coronavirus vaccine could be ready by 2021 after about00:25'Robin Shattock, the Head of Mucosal Infection and Immunity at Imperial College London' says:

We were able to access the sequence that was published by Chinese scientists and made globally available, which was a tremendous thing to do. And we went from that sequence to identifying part of the sequence that encodes for the surface proteins of the virus. And we're using that sequence to manufacture our vaccine.

We're using a particular approach where we make a synthetic vaccine based on RNA, so essentially it's essentially genetic code, we package that in essentially a lipid droplet, and use that to inject in a muscle; it expresses that protein, and the body recognizes that as foreign and it makes protective antibodies.

I'm assuming that the RNA mentioned is mRNA, and so once it reaches the cytoplasm of the vaccine recipients muscle cells it will be expressed and somehow returned to the cells' membrane where it will be recognized as foreign by passing lymphocytes.

Questions:

  1. Is this basically correct as far as it goes?
  2. If so, what causes the lipid droplet to fuse with muscle (or other) cells in the first place?

I can't give an authoritative answer on this because my PhD work was based on mRNA delivery using peptides instead of lipids, but many of the concepts are the same. I also don't have time to provide proper citations, but this is probably too much for a comment.

Anyway, mRNA is a very fragile molecule, it can be broken down extremely quickly in the blood by serum nucleases. Therefore we must protect the mRNA by mixing it with some other chemical, in this case a mixture of positively charged lipids. The positively charged lipids are attracted to the negatively charged phosphate backbone of the mRNA, and form a nanoparticle trapping the mRNA inside and hiding it from the nucleases. The lipids are often, but not always, PEGylated, which means that a molecule of polyethylene glycol (PEG) is attached to the lipid. The PEG groups form a layer on the outer surface of the nanoparticle that helps prevent protein binding.

These mRNA lipid nanoparticles are then injected into a patient. This will probably be an intramuscular injection so that the particles are most likely to be taken up by muscle cells. The exact mechanism explaining why lipid nanoparticles are attracted to cells isn't entirely clear, but it's likely a combination of electrostatic attraction between the positively charged nanoparticle and the negatively charged cell membrane and proteins that bind to the nanoparticle and are then recognized by receptors on the cell surface.

After the particle is taken up by a cell it is usually trapped inside a membrane-bound structure called an endosome. As the endosome matures it is acidified, and as the pH drops from about 7 to about 5.5 the nanoparticle is disrupted. The lipids that made up the nanoparticle can merge with the lipids that make up the endosomal membrane, disrupting that membrane and breaking open the endosome, allowing the mRNA to escape into the cytoplasm.

Once in the cytoplasm the mRNA will find a ribosome and produce protein. Just like you said, the protein in this case will be membrane bound and expressed on the outer surface of the cells for eventual immune recognition.


Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences

Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA.

Keywords: Cap analogs Cationic lipids Electroporation Immunotherapy Innate immunity Nucleoporation Poly(A) Protein expression Translational efficiency mRNA stability.


Pfizer, Biontech Covid-19 vaccine uses technology that could revolutionize future immunizations

Last week, Pfizer released preliminary findings that showed its vaccine candidate is more than 90 percent effective at preventing symptomatic Covid-19. On Monday, Moderna added to the encouraging news, with early results from its Phase 3 trial showing that its experimental vaccine is 94.5 percent effective at preventing the illness. Seeing such consistent results at this stage of the trials is a good sign, del Rio said.

“That makes me feel like, ‘gee, Pfizer wasn’t a fluke,’” he said. “This is for real. This is actually working.”

Though reassuring, the results are still preliminary — the full study results have not yet been published in a peer-reviewed journal for other scientists to scrutinize — and it’s not yet known how long the vaccines could offer protection, or whether they will perform well across all age groups and ethnicities.

One of the main differences between the two vaccine candidates is how they are stored. Both require two doses, but Pfizer’s vaccine has to be stored at temperatures of minus 94 degrees Fahrenheit or colder, which has raised practicality concerns about how they could be shipped and disseminated. Moderna’s vaccine does not require ultracold storage and can remain stable at regular refrigeration levels — between roughly 36 to 46 degrees Fahrenheit — for 30 days.

This distinction is probably because of how the vaccines’ synthetic mRNA, or messenger RNA, is packaged, according to Paula Cannon, an associate professor of microbiology at the University of Southern California's Keck School of Medicine. On its own, mRNA is a fragile molecule, which means it has to be coated in a protective, fatty covering to keep it stable.

The refrigeration conditions may have to do with how the mRNA was manufactured and stabilized, Cannon said, though those precise details are proprietary to the companies.

Dr. Drew Weissman, a professor of medicine at the University of Pennsylvania Perelman School of Medicine, has been an early pioneer in mRNA vaccine research and is now collaborating with BioNTech, a German biotechnology company that has partnered with Pfizer. He said work is ongoing to enhance the experimental vaccine — including improvements to its storage requirements.

“There are definitely improvements that are already being developed,” he said.

Both the Pfizer vaccine and the Moderna vaccine are made using synthetic messenger RNA. Unlike DNA, which carries genetic information for every cell in the human body, messenger RNA directs the body’s protein production in a much more focused way.

“When one particular gene needs to do its work, it makes a copy of itself, which is called messenger RNA,” Cannon said. “If DNA is the big instruction manual for the cell, then messenger RNA is like when you photocopy just one page that you need and take that into your workshop.”

The Pfizer vaccine and the Moderna vaccine use synthetic mRNA that contains information about the coronavirus’s signature spike protein. The vaccines essentially work by sneaking in instructions that direct the body to produce a small amount of the spike protein. Once the immune system detects this protein, the body subsequently begins producing protective antibodies.

“Those antibodies will work not just against the little bit of spike protein that was made following vaccination, but will also recognize and stop the coronavirus from getting into our cells if we’re exposed in the future,” Cannon said. “It’s really a clever trick.”

But as elegant a mechanism as this is in theory, mRNA vaccines have faced real biological challenges since they were first developed in the 1990s. In early animal studies, for instance, the vaccines caused worrisome inflammation.

“That became one of the big questions: How do you get this inside the body without creating an inflammatory response?” said Norman Baylor, president and CEO of Biologics Consulting and the former director of the FDA’s Office of Vaccines Research and Review.

Though neither company has reported any serious safety concerns so far, scientists will continue to monitor participants in both trials over time.

“There’s always a concern when you are trying to trick the immune system — which is what a vaccine does — that you could have unintended side effects,” Cannon said. “The immune system is incredibly complicated and it’s different from person to person.”


If participants in Phase 3 clinical trials are not intentionally exposed to the virus, how can vaccine efficacy be calculated?

People in the clinical trials were divided into two groups — those who got the vaccine and those who got a dummy shot. They would have gone to work and school and had social interactions as they normally do. Because Phase 3 clinical trials were mostly done in places with very active pandemics, such as the U.S. and Brazil, a certain number of participants would have been exposed to the virus that causes COVID-19 in their daily lives.

Efficacy was calculated by comparing the number of infections among those who got the vaccine and those who got the dummy shot.

Efficacy is how well the vaccine works in clinical trials. Effectiveness is how well a vaccine or a product will work in real world settings.


Results

Generation and characterization of SAM encoding influenza NP and M1 antigens

The full-length NP and M1 genes were amplified from the reverse-transcribed RNA genome of influenza virus A/PR/8/34 (H1N1), and then cloned into the DNA plasmid backbone as two monocistronic (SAM(NP) or SAM(M1)), and one bicistronic (SAM(M1-NP)) vectors (Fig 1A). The corresponding ssRNAs were synthesized in vitro by an enzymatic transcription reaction from a linear plasmid DNA template using a T7 RNA polymerase [31]. The in vitro activity of the monocistronic and bicistronic SAM replicons was measured after electroporation in BHK cells and compared to a control self-amplifying RNA of known potency (STD). The presence of intracellular dsRNA molecules, as markers of RNA amplification, was evaluated by flow cytometry (Fig 1B). The frequencies of dsRNA + BHK cells after transfection with the two SAM(NP) and SAM(M1) monocistronic or the SAM(M1-NP) bicistronic replicons were comparable or higher than that obtained with the STD, indicating that the new replicons self-amplified. The frequencies of dsRNA + and protein + cells were comparable for each replicon, suggesting that antigen expression paralleled RNA amplification.

(a) SAM(NP), SAM(M1) and SAM(M1-NP) constructs showing a 5’ cap, four non-structural genes (nsp1-4), a 26S subgenomic promoter (grey arrow), the vaccine antigen(s), and a 3’ polyadenylated tail. (b-d) Self-amplification of SAM replicons and antigen expression assessed after transfection of BHK cells with the different replicons. (b) Percentage of BHK cells positive for replicating SAM vectors (dsRNA + cells) and expressing the corresponding protein (protein + cells) was analyzed by flow cytometry and indicated as mean ± SD. (c) Cell lysates from BHK cells infected with the PR8 virus (0.1 multiplicity of infection) (lane 1), mock-transfected (lane 2), or transfected with SAM(NP) (top panel) and SAM(M1) (lower panel) (lane 3), or SAM(M1-NP) (lane 4) were analyzed by Western blot under reducing conditions. (d) Frequency of NP- and M1-expressing BHK cells transfected with mock, SAM(NP), SAM(M1) or with the bicistronic SAM(M1-NP) replicons were analyzed by flow cytometry. (e) Frequency (mean ± SD) and mean fluorescence intensity (MFI) of BHK cells expressing NP or M1 antigens after transfection with the different SAM/LNP formulations. Statistical analyses were performed using the Mann-Whitney U test. *p<0.05 compared to the SAM(NP)- or SAM(M1)-treated groups. Data shown are representative of three independent experiments.

Antigen expression by BHK cells after transfection with the different SAM replicons was further characterized by Western Blot (Fig 1C) and flow cytometry (Fig 1D). Both M1 and NP proteins expressed by the monocistronic (lane 3) or bicistronic (lane 4) vectors showed bands in the western blots with molecular weights equivalent to the respective proteins expressed by PR8 virus-infected BHK cells (lane 1) (Fig 1C). Finally, the percentage of NP or M1 expressing BHK cells was greater than 70% for monocistronic and bicistronic replicons, and the majority of BHK cells transfected with the bicistronic replicon co-expressed M1 and NP (Fig 1D). For in vivo studies, SAM vectors were encapsulated with LNPs. Mean particle size and polydispersity were measured by dynamic light scattering for SAM(NP)/LNP, SAM(M1)/LNP, [SAM(M1)+ SAM(NP)]/LNP, and SAM(M1-NP)/LNP. Z-average diameters ranged from 130 to 142 nm with a low polydispersity index (data not shown), indicating small uniform lipid particles able to encapsulate more than 95% of the mRNA [31]. Finally, flow cytometry analysis showed that the percentage and MFI of BHK cells expressing the NP or M1 antigens after transfection with the different SAM/LNP formulations were comparable between single antigen and combination groups (Fig 1E).

Immunogenicity of SAM(NP) and SAM(M1) vaccines

To assess the immunogenicity of SAM replicons expressing NP and/or M1 antigens, BALB/c mice were immunized i.m. twice, eight weeks apart, with 0.1 μg of SAM(NP), SAM(M1), a mixture of both SAM(NP)+SAM(M1), or SAM(M1-NP) and delivered with LNP. Infection with a low dose of the PR8 virus and treatment with PBS were used as positive and negative controls, respectively.

NP- and M1-specific IgG were already detectable in the sera of SAM-immunized mice after the first dose and were boosted by the second immunization (S1A Fig). Mice that had received the single antigens reached antibody titers 1.5–2 -fold higher (p<0.01) than those vaccinated with both antigens, suggesting that NP and M1 induce mild antigenic interference [38, 39]. Sera from SAM-immunized mice failed to neutralize PR8 virus infection of MDCK cells in vitro (S1B Fig), consistent with the internal location of these antigens in the virus [40].

Based on the key protective role played by NP- and M1-specific T cells against influenza disease [12, 14], we focused the next set of analyses on the characterization of antigen-specific T-cells by ICS and flow cytometry (Fig 2). Antigen-specific, cytokine-secreting cells were identified among the CD44 high CD8 + and CD4 + T cell subsets as previously described [30] in splenocytes of immunized animals stimulated in vitro with NP147-155 peptide (Fig 2A), recombinant NP protein (Fig 2B) or with M1-derived peptide pool (Fig 2C). NP-specific CD8 + T cells were already detectable 10 days after the first immunization and maintained at frequencies around 0.1–0.2% until week 6 (Fig 2A). A 10-fold increase was seen 10 days after the second immunization, with frequencies of NP-specific CD8 + T cells ranging from 1 to 2% of total CD8 + T cells and contracting to 0.6–0.9% at 6 weeks. The majority of NP-specific CD8 + T cells were IFN-γ + and IFN-γ + /TNF-α + , characteristic of an effector phenotype. No M1-specific CD8 + T-cells were detected in PR8-exposed mice and in any SAM(M1) vaccine group at any time point tested (data not shown).

(a-b) BALB/c mice (n = 24/group) were immunized i.m. twice, 8 weeks apart, with 0.1 μg of SAM(NP), SAM(M1), SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Ten days and 6 weeks after each immunization, the frequency of antigen (Ag)-specific, cytokine-secreting CD8 + (a) or CD4 + (b, c) T cells was determined by flow cytometry on splenocytes stimulated in vitro with the NP147-155 peptide (a), recombinant NP protein (b) or with the M1-derived peptide pool (c). Color code indicates the different combinations of cytokine produced by the respective cells. As control, a group of mice were infected with a low dose of influenza virus PR8. Data derived from two separate and merged experiments. Statistical analysis was performed using the Mann-Whitney U test. *p<0.05 compared to SAM(NP) (a-b) and SAM(M1) (c). Frequencies of Ag-specific CD4 + and CD8 + T cells were significantly higher (p<0.05) in all SAM-immunized and PR8-exposed groups than in PBS at all time points. Induction of antigen-specific memory T cells by SAM(NP) and SAM(M1) vaccines

Similarly, NP-specific CD4 + T cells were already detectable 10 days after the first immunization, and were predominantly Th0 (IL-2 + /TNF-α + , TNF-α + , and IL-2 + ) and multifunctional Th1 (IFN-γ + /IL-2 + /TNF-α + ) (Fig 2B). The second immunization expanded NP-specific CD4 + T cells in all the immunization groups, especially the multifunctional Th1 sub-population (IFN-γ + /IL-2 + /TNF-α + ). Frequencies of NP-specific CD4 + T cells ranged from 0.1–0.2% up to 6 weeks after the first immunization, increased up to 0.3–0.5% 10 days after the second immunization, and decreased to 0.1–0.3% at 6 weeks thereafter. No significant differences in terms of intensity or quality of the responses were observed between the different SAM vaccinated groups except for the 6 weeks post 2 time point when the intensity of NP-specific T cell responses were significantly reduced in mice immunized with SAM(M1-NP) compared to the other two SAM-vaccinated groups. M1-specific CD4 + T-cells showed kinetics and phenotype similar to NP-specific CD4 + T cells, albeit with lower frequencies (Fig 2C). No differences were observed in the frequencies of antigen-specific T-cells induced by SAM(M1) alone or in combination with SAM(NP).

Mice pre-exposed to a low dose of the PR8 virus showed approximately 0.6% and 0.1% of NP-specific CD8 + and CD4 + T cells, respectively, that did not increase after a second exposure to the virus. It is likely that HA-specific antibodies induced by the first exposure to PR8 neutralized the second virus infection, thus preventing the recall and expansion of NP-specific T-cells. Low frequencies of M1-specific CD4 + T-cell responses, but no M1-specific CD8 + T cells were detected in infected mice, as was observed for SAM immunized mice. No NP- or M1-specific T cells were detected in PBS-treated mice. Similar immune profiles were observed when total numbers, rather than frequencies, of antigen-specific CD4 and CD8 T cells were reported (data not shown).

In addition to cytokine secretion, we characterized the memory phenotype of antigen-specific T cells in spleens of immunized mice by measuring the frequency of effector (TEFF, CD44 high /CD62L low /CD127 low ), effector memory (TEM, CD44 high /CD62L low / CD127 high ), and central memory (TCM, CD44 high /CD62L high /CD127 high ) T cells at different time points upon vaccination (Fig 3).

Ten days and 6 weeks after each immunization, the frequency of NP-specific (a-b) or M1-specifc (c) cytokine-secreting cells were determined within the central memory: TCM (CD44 high /CD62L high /CD127 high ), the effector memory: TEM (CD44 high /CD62L low / CD127 high ), and the effector: TEFF (CD44 high /CD62L low /CD127 low ) subsets. Arrows indicate the immunization times. Data derived from two separate and merged experiments. Statistical analyses were performed using the Mann-Whitney U test. *p<0.05 compared to SAM(NP) (a-b) or SAM(M1) (c).

Low frequencies of antigen-specific TCM, TEM and TEFF cells were measured after the first immunization with all SAM vaccines, while after the second immunization, the frequency of NP-specific CD8 + TCM cells was boosted up to 0.2% (Fig 3A), and some NP- and M1-specific CD4 + TCM cells were also detected (Fig 3B and 3C). The frequencies of CD8 + and CD4 + TEM and TEFF increased after the second vaccination, peaking at day 10 and contracting after 6 weeks. This kinetic was observed for both CD8 + and CD4 + , with no major differences among the immunized groups, except for NP-specific CD4 + TEM cells which showed a significantly reduced frequency in SAM(NP)+SAM(M1) and SAM(M1-NP) vaccine groups compared to SAM(NP) immunized mice. Altogether, these results suggest that SAM vaccines induce a strong activation of CD8 + than CD4 + T cells, and expansion of the effector memory compartment.

Induction of cytotoxic T cells by SAM(NP) vaccine

Finally, we characterized antigen-specific T cells induced by SAM vaccines for cytotoxic activity in vitro and in vivo (Fig 4). T cell cytotoxicity was evaluated by quantifying the surface expression of CD107a, as a measure of the degranulation process [41], upon in vitro antigen-stimulation of splenocytes from immunized animals. After two immunizations with SAM(NP) alone or in combination with SAM(M1), the majority of NP-specific CD8 + T cells were CD107a + (Fig 4A). The immunization with SAM(NP) alone induced higher frequency of CD107a + NP-specific CD8 T cells compared to the combination vaccines (p<0.05). We did not detect CD107a on NP- or M1-specific CD4 + T cells, suggesting that SAM formulations did not induce cytotoxic CD4 + T cells.

The induction of NP-specific CD8 + T cells by SAM(NP) alone or in combination with SAM(M1) was characterized 10 days after the second immunization. (a) Surface expression of CD107a on splenocytes stimulated in vitro with NP147-155 was assessed by flow cytometry. Data show the frequency of cytokine-secreting CD8 + T cells that express (black bars) or not (grey bars) CD107a. (b) Percentage of in vivo NP-specific target cell lysis calculated for each immunization group. (c) Representative histograms showing the frequency of influenza NP147-155-pulsed (CFSE + ) and HIV Gag197-2015-pulsed (CMTMR + ) target cells recovered in each immunization group 18 h after adoptive transfer. Statistical analyses were performed using the Mann-Whitney U test. *p<0.05 **p<0.01 compared to SAM(NP).

To evaluate the in vivo cytotoxic activity of NP-specific CD8 + T cells, an equivalent number of CFSE-labeled or CMTMR-labeled splenocytes were pulsed with the H2-K d -restricted NP147-155 peptide (0.5 μM CFSE) or with an unrelated HIV-Gag197-205 peptide (10 μM CMTMR), respectively, and were adoptively transferred in mice immunized with 0.1 μg of SAM(NP), 0.2 μg of SAM(NP)+SAM(M1) or 0.1 μg of SAM(M1-NP). The percentage of CFSE + and CMTMR + cells present in the spleens were measured by flow cytometry 18 h later (Fig 4B and 4C). A specific lysis of > 93% was measured in SAM(NP)-immunized mice, while 74% and 60% of specific lysis were detected in SAM(NP)+SAM(M1) and SAM(M1-NP) immunized groups, respectively. No specific lysis was detected in PBS treated mice confirming the antigen-specificity of the cytotoxic activity. These results demonstrated that the SAM formulations induced NP-specific CD8 + T cells with cytotoxic activity in vivo against target cells pulsed with the H2-K d -restricted immunodominant NP peptide. Furthermore, we observed an enhanced in vivo cytotoxic activity in SAM(NP) vaccinated mice compared to mice immunized with the combination vaccines, in agreement with the frequencies of CD107a + NP-specific CD8 + T cells observed in vitro in the respective immunization groups (Fig 4A).

Protective efficacy in mice against challenge with homologous and heterosubtypic influenza viruses

To explore the protective efficacy of SAM vaccines, BALB/c mice were immunized twice, eight weeks apart, with 0.1 μg of SAM(NP), SAM(M1), SAM(M1-NP), or 0.2 μg of SAM(NP)+SAM(M1) vectors formulated in LNPs, and challenged with a lethal dose of the mouse-adapted homologous PR8 influenza virus. As controls, we included two groups of mice previously exposed to a low dose of PR8 or HK68 influenza viruses. Survival, weight loss and clinical scores were measured for 14 days after challenge (Fig 5).

BALB/c mice (n = 18) were immunized i.m. twice, 8 weeks apart, with 0.1 μg of SAM(NP), SAM(M1), SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Four weeks after the last injection, mice were challenged with the homologous PR8 (a-d) or the heterosubtypic HK68 (e-g) influenza viruses. Mice were monitored for survival (a and e) body weight loss (b and f) and clinical score (c and g) for 14 days after infection and euthanized when the clinical score reached 4. Data shown are mean ± SD. (d) Viral titers measured in lungs collected at day 3, 6 and 17 after influenza challenge and expressed as fold-increase compared to pre-infected samples. Individual mice, mean and SD are reported. Data are derived from two independent and merged experiments. Statistical analyses were performed using the Log rank analysis (Mantel Cox test) (a, e), and the Mann-Whitney U test (b, c, d, f, g): *p<0.05, **p<0.01, ***p< 0.001 compared to the PBS-treated group.

Mice immunized with SAM replicons expressing NP, either alone or in combination with M1, and formulated with LNP were protected against lethal infection with the homologous PR8 virus, showing a substantially and significantly higher (p<0.0001) survival rate compared to PBS-treated control mice (Fig 5A). Mice immunized with SAM(NP), SAM(NP)+SAM(M1) and SAM(M1-NP) vaccines had survival rates of 71, 70, and 78%, respectively. In contrast, administration of the SAM(M1) vaccine alone was poorly protective (25% survival rate) and not statistically different from PBS-treated control mice (5% survival rate). As expected, PR8 pre-exposed mice were completely protected from the homologous challenge, while mice pre-exposed to heterosubtypic HK68 virus were partially protected (78%), with survival rates similar to those conferred by the SAM(M1-NP) vaccine. Transient body weight loss, as a sign of influenza disease, was observed in all the immunization groups. However, mice vaccinated with the NP-expressing replicons showed a more rapid recovery from disease compared to PBS-treated animals, as demonstrated by significantly reduced body weight loss (Fig 5B) and clinical scores (Fig 5C). Indeed, 3 days after influenza infection, animals immunized with SAM(NP) and combination (NP+M1 or M1-NP) vaccines had significantly reduced lung viral titers compared to PBS-treated mice (p<0.01), and completely cleared influenza viral particles from the lungs after 6 days (Fig 5D). Body weight loss and clinical scores indicated an intermediate grade of illness for SAM(M1)-immunized mice, in agreement with the survival results and the slower control of lung viral titers.

To determine the cross-protection conferred by SAM vaccines, we challenged mice with the heterosubtypic HK68 strain and assessed survival, loss in body weight, and overall clinical scores. All SAM vaccines were associated with 100% survival, reduced overall weight loss, and low clinical scores (Fig 5E–5G). In contrast, PBS-treated mice showed a poorer outcome with a 30% survival rate, over 15% body weight loss at the peak of the infection (days 6–8), and clinical scores above 3. The different protective efficacy of SAM(M1) vaccine in the homologous and heterosubtypic infection models could be due to the difference in virulence of PR8 and HK68 viruses [42], as suggested by the different survival rates observed in PBS-treated mice.

Effect of SAM vaccines on lung T-cell responses after influenza virus challenge

Vaccination might influence T-cell responses to influenza infection at the site of virus entry. Therefore, we characterized the lung T-cell composition on day 0, 3, 6 and 17 after PR8 challenge in SAM(NP), SAM(M1), SAM(NP)+SAM(M1) or SAM(M1-NP)-vaccinated mice (Fig 6).

BALB/c mice were immunized i.m. twice, 8 weeks apart, with PBS, 0.1 μg of SAM(NP), SAM(M1), SAM(M1-NP), or with 0.2 μg of SAM(NP)+SAM(M1). Four weeks after the second immunization, mice were infected with PR8 virus. NP-specific CD8 T cells recruited in the lungs after the infection were characterized by flow cytometry. (a) Numbers of NP-specific CD8 + T cells. Data are from individual mice (depicted as dots), while solid lanes indicate the mean±SD. (b) Cumulative frequency of Ag-specific, cytokine-secreting CD8 + T cells, indicated as absolute number per lung. The color code represents the different combinations of cytokine produced by the respective cells after in vitro stimulation with medium (m), NP147-155 peptide (NP), or M1 peptide pool (M1), as indicated. (c) Absolute number of NP-specific CD8 + T cells positive (black bar) or not (grey bar) for CD107a. Data derived from two independent and merged experiments. Statistical analyses were performed using the Mann-Whitney U test. *p<0.05 **p<0.01 compared to the PBS-treated group.

At the time of influenza challenge (day 0), H2-K d /NP147-155 pentamer + CD8 + T cells were already detectable in the lungs of mice immunized with SAM(NP) and combinations, but not with SAM(M1) or PBS. Their number increased in all immunization groups at day 6 after infection, and remained high at day 17 in SAM(NP) and combination groups (Fig 6A). IFN-γ + /TNF-α + , TNF-α + , and IFN-γ + NP-specific CD8 + T cells were detected in SAM(NP) and combination groups already at day 0. Their frequency increased at day 6 after infection, and showed a more complex IFN-γ + /IL-2 + /TNF-α + phenotype at day 17. Finally, M1-specific CD8 + T cells were detected at day 6 and 17 in the SAM(M1) vaccine group, but not in the combination formulation groups (Fig 6B). In agreement with the effector phenotype observed by ICS, most NP-specific CD8 + T cells found in the lungs of NP-immunized animals were CD107a + (Fig 6C).

Since antigen-specific CD4 + T cells can also have a role in mediating protection by contributing to the development of the effector functions of CD8 + T cells [43, 44], we characterized lung NP- and M1-specific CD4 + T cells after vaccination and subsequent influenza infection. The characteristic CD4 Th1 profile observed after systemic immunization (Fig 2) was maintained after infection by lung-infiltrating antigen-specific CD4 + T cells that expressed IFN-γ and TNF-α, alone or in combination (S2 Fig). Finally, histological analysis of the lungs of vaccinated mice showed that, the high number of effector CD8 + and CD4 + T cells present from day 6 onwards after challenge was not inducing overt pathology but was rather associated with a low histopathological score (S3 Fig).

Altogether, these data suggest that vaccination influenced the T-cell response in the lungs after influenza challenge. Mice immunized with SAM(NP) vaccines showed a rapid and enhanced recruitment of cytotoxic CD8 + T cells and polyfunctional CD4 + Th1 cells to the lungs that was associated with an efficient control of the virus, reduced lung lesions, and a significantly enhanced survival rate.

Co-administration of the SAM(M1-NP) vaccine with monovalent inactivated influenza vaccine

An ideal cross-protective influenza vaccine should induce both humoral responses versus the surface HA antigen and T-cell responses against the internal conserved influenza antigens (NP and M1) [45, 46]. Therefore, we evaluated the possibility to use RNA-based SAM(M1-NP) vaccine in combination with a monovalent inactivated influenza vaccine (MIIV) derived from A/California/7/2009 (H1N1) virus (Cal/H1N1). BALB/c mice were immunized i.m. twice, eight weeks apart, with 0.1 μg of SAM(M1-NP) or SAM(GFP) control vector in LNP, combined with a suboptimal dose of MIIV (0.1 μg) chosen to assess the possible synergy with SAM vaccines. PBS-treated and mice pre-exposed to a low dose of PR8 influenza virus were used as negative and positive control, respectively. One month after the last immunization, mice were infected with 10-fold the lethal dose of PR8 virus to increase the stringency of the model and were monitored for 14 days after infection.

SAM(M1-NP)+MIIV-immunized mice showed a survival rate significantly increased compared to PBS treated mice with 91% and 20%, respectively, while MIIV alone provided only partial protection in these experimental conditions (37% of survival) (Fig 7A). All immunized animals showed signs of disease during the course of the observation, with a transient weight loss peaking four days after infection (Fig 7B). To our surprise, 80% of the animals in the SAM(GFP)+MIIV vector control group survived the infection, although we previously showed that SAM(GFP) alone did not induce H1-specific immune responses nor did it protect mice against a challenge with the PR8 virus [30].

BALB/c mice (n = 20) were immunized i.m. twice, 8 weeks apart, with 0.1 μg of SAM(M1-NP) or SAM(GFP) in combination with 0.1 μg of MIIV (Cal/H1N1). Four weeks after the last injection, mice were challenged with 10-fold the lethal dose of heterologous influenza PR8 virus. Mice were monitored for survival (a) and body weight loss (b) for 14 days after infection. Data show mean of single mice ± SD. Data are derived from two separate and merged experiments. Statistical analyses were performed using the Log rank analysis (Mantel Cox test). ***p<0.001 compared to PBS. (c) Neutralizing titers against Cal and PR8 viruses in sera collected two weeks after the second immunization. (d, e) Ten days after the second immunization, the frequency of antigen-specific cytokine-secreting CD8+ (d) or CD4+ (e) T cells was determined by flow cytometry on splenocytes stimulated in vitro with a Cal/H1 peptide pool (d, e) or a PR8/H1 peptide pool (d, e). Data are derived from two independent and merged experiments. Statistical analyses with the Mann-Whitney U test were performed on total cytokines. *p<0.05 compared to MIIV.

To investigate a possible adjuvant effect [47, 48] of the ssRNA vector in vivo, we compared the adaptive immune responses induced by the different vaccine combinations by measuring antigen-specific functional antibody titers (Fig 7C) and T-cell frequencies (Fig 7D and 7E) two weeks after the second immunization. Virus neutralization titers against A/California/7/2009 (H1N1) vaccine strain ranged from 1.5x10 4 to 2x10 4 , and were not significantly different in sera from mice immunized with SAM(M1-NP)+MIIV or SAM(GFP)+MIIV and MIIV alone, while no neutralizing activity was found against the PR8 virus (Fig 7C), confirming previous observations [30]. In contrast, combining SAM(M1-NP) or SAM(GFP) with MIIV resulted in increased frequencies of Cal/H1 vaccine-specific and PR8/H1 cross-reactive CD8 + and CD4 + T cells compared to MIIV (Fig 7D and 7E). H1-specific CD8 + T cells showed a polyfunctional effector phenotype consisting of combinations of IFN-γ and TNF-α. Moreover, the co-administration of SAM replicons with MIIV shifted the usual Th0/Th2 phenotype elicited by MIIV and characterized by the production of IL-13/IL-4, to a Th0/Th1 profile dominated by the production of IFN-γ/TNF-α/IL-2 and IL-2/TNF-α. The similar T helper pattern observed when combining SAM vectors encoding M1-NP or GFP antigens with MIIV suggests that the polarization of the T-cell response was due to the replicon per se, and was likely not antigen-dependent. Finally, NP- and M1-specific T-cell responses were normally observed in mice immunized with SAM(M1-NP)+MIIV (S4 Fig).

These results demonstrated that co-administration of SAM(M1-NP) and MIIV induced broader immunity resulting in an enhanced protection against heterologous influenza viruses compared to MIIV alone. This is also the first evidence that the combination of ssRNA vectors and protein-based vaccines might be feasible to improve the efficacy of current seasonal and pandemic influenza vaccines.


Introduction

The focus of this review is on vaccine lot or batch release assays that are essential to monitoring critical quality attributes (CQAs) and ensuring that high quality and well-characterized vaccine products are manufactured consistently. It is especially important in this fast-moving landscape of development that control on quality and consistency is maintained as manufacturing scale up and global supply chains are progressed. Analytical bridging through demonstration of CQA-based comparability between lots will be needed all the more in COVID-19 vaccine programs in order to minimize the need for time-consuming and expensive clinical bridging.

The portfolio of COVID-19 vaccines in development is large and expanding by the day, utilizing all available novel and traditional vaccine platform technologies. Among these, the mRNA platform has recently delivered the first two COVID-19 vaccines, which also happen to be the first ever vaccine products out of this technology platform 1,2,3,4 . In addition, the viral vector platform, which earlier yielded two vaccines against Ebola, using both replicating and non-replicating vectors 5,6,7 , has now delivered vaccines against SARS-CoV-2 8,9,10,11,12 . Additional platforms being used include live attenuated viruses (LAV), inactivated viruses, and recombinant proteins and protein-based virus-like particles (VLPs), all of which have a long history of delivering approved vaccines against other viruses. Nevertheless, there are opportunities for implementing faster, more sensitive and robust batch release assays.

Interim analyses of data from phase 3 trials of mRNA vaccines have indicated excellent efficacy 13,14 . Within the mRNA platform, self-replicating or self-amplifying mRNA (sa-mRNA) constructs appear to offer the advantage of potential efficacy at a lower dose 15 . However, this has yet to be demonstrated especially in the context of SARS-CoV-2.

This review will outline opportunities to improve the speed of batch release testing without compromising quality. This is particularly important for vaccines against COVID-19, which will require a speedy release of vaccine batches to ensure urgent delivery. Robust and faster turn-around assays for potency and other selected CQA will also be important for monitoring long-term and accelerated stability. The requirements of toolboxes and assays are vaccine platform and product dependent, although there are commonalities. In particular, we will focus on potency assays, which are key to delivering safe and immunogenic doses of vaccines. Although assays are established for the proven platforms, such as LAV and recombinant proteins, faster and more robust in vitro assays can be developed for some CQA.

In addition to antigen, the final formulation of the vaccine drug product (DP) often contains adjuvants and excipients such as stabilizers or cryo-protectants. Batch release testing for DP must include key tests for these components. Furthermore, any potential interference of these components in antigen assays, e.g., potency, must be ruled out or addressed.

As of January 25th, 2021, according to CEPI’s ongoing landscape analysis 16,17 , approximately 58 candidate vaccines globally are in different phases of clinical trials and, additionally, several are very close to entering phase 1 human trial. Sixteen candidate vaccines are already in phase 2b/3 trials, while rest of them are in 1, 1/2, and 2 clinical phases. In the coming weeks, some of these data points may possibly change because of recent emergency use approval (after phase 3 completion) of a few vaccines, which may enter post licensure phase 4.

Over the years, regulatory agencies such as the Food and Drug Administration (FDA) of USA and the European Medicines Agency (EMA) as well as the World Health Organization (WHO) have provided guidelines and recommendations for quality control of vaccines produced by different platform technologies. General guidelines for vaccines against COVID-19 have been published 18,19,20,21,22,23,24,25 , and more specific recommendations are currently being drafted.

Principles of analytical evaluation based on CQA

CQA-based assays are, for the most part, product and platform specific. Additional critical assays, e.g., residual host cell and process-related impurities such as DNA, proteins, DNAase, trypsin, and serum albumin, may be generic but have impact on product safety and integrity. Quantitative and sensitive methods are well developed for such impurities and will not be discussed here. Microbiological testing including sterility is crucial to ensuring safety of any product. Traditional, culture based, sterility testing requiring a couple of weeks is often the slowest and rate-limiting step in vaccine lot release. Rapid and reliable sterility testing methods have been reported but, as of now, not received more than a limited degree of regulatory acceptance for the release of short shelf-life cell therapy products 26 . These may be further evaluated in the context of the faster batch release of vaccines against COVID-19.

Potency assays for LAV vaccines have traditionally utilized cell culture based infectivity testing, such as Median Tissue Culture Infectious Dose (TCID50) and plaque assays, which, depending on the virus, often take several days to produce definitive indication of cytopathic effects (CPE). These CPE-based methods are also being used for viral vectored vaccines. Alternative methods with increased sensitivity of detection, while keeping the essential cell incubation at the front end, have been extensively studied. These methods can significantly reduce turn-around time and offer higher throughput. In terms of rapid development and delivery of vaccines against SARS-CoV-2, there are now urgent needs and opportunities for implementing such assays with rigor and with a view to regulatory acceptance. It is possible and, indeed, highly desirable to combine speed with quality as well as to implement innovative analytical methods that will also improve precision and accuracy.

It appears that the majority of the developers have selected either the full-length spike glycoprotein (S-protein) or a part of it, such as the angiotensin converting enzyme-2 (ACE-2) receptor binding domain (RBD) as antigen, while over 50 vaccine candidates also included more than one target, e.g., S, M, E, and N proteins (see Fig. 1). For all nucleic acid- and viral vector-based projects, expression of the protein antigens corresponding to the respective transgenes should ideally be tested in appropriate cells as a part of potency assays. Antibodies raised in the sera of vaccine recipients are expected to contain SARS-CoV-2 binding and neutralizing antibodies. Published data suggest that the S protein could induce neutralizing antibodies 27 .

The number of projects is shown for each target antigen. Different platform technologies being used are coded with different colors as defined in the inset.

The quality of a vaccine must be evaluated by analytical methods that reflect its identity, purity, structural integrity, and biological activity as a measure of potency. These measurements and quantitative determination of doses to be delivered must be as accurate and precise as possible, though the particular assays for quantifying these CQA depend on the platform and the product being administered to the vaccine recipients. The in vitro potency assay, as one example, will vary for different technologies (see Table 1). Even for one developer using a particular platform, multiple sites around the globe will be used to enable the manufacturing and rapid distribution of hundreds of millions of doses in different parts of the world. Thus, setting and meeting specifications for quality indicating assays are of paramount importance in ensuring delivery of safe and effective vaccines of consistent quality worldwide.

There is significant expertise among many developers in analytical assays for batch release and characterization in the context of developing vaccines against pre-COVID-19 pathogens, using the same technology platforms. However, it is of utmost importance to focus on implementing those assays and tools that will be the most direct and precise indicators or surrogate measures of safety, potency, and immunogenicity. The development and execution of these methods may be dependent on the clinical phase. The critical assays such as those mentioned above should be qualified by the time of clinical entry (phase 1/2a) and, ideally, validated before phase 3. Development of reproducible, scientifically sound assays and standards during the pre-clinical phase allows efficient process optimization, facilitates regulatory interactions and entry into clinic.

In vitro methods are typically preferred for Chemistry, Manufacturing, and Control (CMC) batch release as they are more precise and robust than in vivo assays and have much shorter turn-around time. However, correlation between in vitro relative potency (IVRP) and in vivo immunogenicity (in a relevant animal model) may be desirable as a background rationale for a potency assay. Development of this correlation in a dose-dependent manner may be complementary to developing immunological assays that can detect and quantify virus-binding as well as virus-neutralizing antibodies in animals and, later, in clinical (human) sera samples.

Analytical comparability

CQA-based evaluation of lot-to-lot comparability is an important component of CMC activities. This ensures that vaccine lots being used for successive phases of clinical trials are equivalent based on key CQA of the product such as potency, purity, and physical chemical integrity. Maintaining comparability between smaller scale material such as that often used in pre-clinical toxicity study and phase 1 trial with later phase clinical trial material (CTM) produced by larger scale processes is a regulatory requirement. Any process modification and formulation change between these phases, such as addition of an approved stabilizer, would also need to be substantiated by CQA-based comparability. This is a part of Good Manufacturing Practice (GMP) and provides a safeguard against potentially costly and time-consuming clinical bridging. Demonstration of comparability between phase 3 and commercial lots is required and is especially critical if scale up or scale out is involved, even though drug substance (DS) and drug product (DP) processes were locked prior to phase 3.

Comparability analysis may have an additional dimension for COVID-19 vaccines because, even for a single product, technology transfer between a developer and a manufacturing partner with higher capacity will have to happen in many cases in order to meet large global demands. The establishment of a clear plan between partners involved, aided by appropriate regulatory advice, will allow ensuring comparability between processes and product batches.

Examples of CQA-based assays

A “potency assay” for a vaccine is, in reality, a biological activity assay that is a surrogate for immune response to be elicited by the antigen. This is typically a product and/or platform-dependent assay. While potency is intricately related to dose, quantitative methods to measure these attributes may be depending on the platform. For example, for a recombinant protein antigen, dose (or content) measurement is typically made by a quantitative protein assay or near-ultraviolet (UV) absorbance, while potency is measured by an immunoassay, at a series of pre-determined doses, as a surrogate of biological immune response. For LAV vaccines, on the other hand, both dose and potency can be expressed as infectious titer, although the content that a vaccinee is dosed with also contains non-infectious or defective viral genomes. In this case, the total number of viral particles or genome copies should be measured to track the ratio of infectious to total viral titer. For inactivated virus vaccines, the extent of inactivation is measured by following loss of infectious titer, while an immunoassay against a key epitope of the virus, which is still able to bind a specific mAb or polyclonal sera, can be used as a dose as well as a potency assay. For antigens inserted genetically in viral vectors, infectious titer of the vector has often been used as dose as well as potency, although the total number of viral particles has been reported as dose in other cases. In addition, expression of the target antigen encoded by the inserted gene in an appropriate cell line is expected as a potency assay. For DNA and RNA antigens, dose is readily measured by absorbance and fluorescence methods, or by quantitative PCR (qPCR). However, potency should be separately measured by transfection of appropriate cell lines and expression of the protein antigen.

Potency is also a primary stability indicator and will need to be monitored as a function of time for all candidate antigens in bulk (DS) as well as formulated DP. Other stability indicating CQA includes physical, chemical, and structural integrity. Aggregation, degradation or structural unfolding can cause loss of biological potency and may trigger toxicity issues, e.g., by inducing undesirable immune response. Depending on the nature of the antigen, i.e. the technology platform, these assays will be different, but the same basic principles apply.

Recombinant protein-based vaccines

For subunit and VLP antigens and, indeed, for all classes of antigens, a dose-dependent correlation between in vitro potency and immune response in animal models often forms the basis for a potentially efficacious and safe dose selection in clinical trials. However, as a lot release assay, an in vitro potency assay is typically preferred for a few reasons including higher precision, lower inter-assay variability, faster turn-around and higher throughput. Indeed, this appears to be the preference among developers and regulatory agencies for vaccines being developed against SARS-CoV-2. For antigens that work predominantly by a humoral pathway, immune response or “immunogenicity” can be determined by measuring levels of antibodies in animal sera that bind to target epitopes on the antigen. Immunoassays such as binding and competitive enzyme-linked immunosorbent assays (ELISA), and Surface Plasmon Resonance (SPR) are typically employed. T-cell mediated responses or Cell Mediated Immunity (CMI) is determined by evaluating induction of cytokines such as interferons (IFN), interleukins (IL), and tumor necrosis factors (TNF). Further pre-clinical work, outside of CMC, may include testing for neutralization of the whole virus at a relevant titer by antibodies raised in animal sera.

There are several examples of recombinant protein-based subunit and VLP antigens for which in vitro potency assays have been successfully developed. For example, for approved VLP vaccines against hepatitis B virus (HBV) and human papilloma virus (HPV), the correlation between in vitro ELISA and in vivo production of neutralizing antibodies have been well-established 28,29,30 . IVRP assays have been accepted by regulatory agencies. For a trimeric post-fusion F-protein-based Respiratory Syncytial Virus (RSV) vaccine, which was in clinical development (but not an approved product), the correlation between a sandwich ELISA IVRP and in vivo immunogenicity was established 31 , and IVRP was used as a lot release potency assay.

While ELISA, in various forms such as direct binding, competitive, or sandwich, has been a widely used and reliable technology, newer immunoassay technologies provide fast turn-around, high throughput, and good precision. An example is VaxArray, introduced by InDevR 32 . In addition, SPR and Bio-Layer Interferometry (BLI) are alternative options for direct antigen-antibody binding assays 33,34 . The mAb CR3022, originally developed against the S-protein of SARS-CoV, also binds with high affinity to SARS-CoV-2 (although not neutralizing) and can be used for ELISA or any of these alternative immunoassays as a lot release potency assay. SARS-CoV-2 S-protein RBD specific mAbs have been reported 27 , and several are commercially available. A surrogate SARS-CoV-2 virus neutralization assay has been published, which is based on antibody-mediated inhibition of interaction between S-protein and angiotensin converting enzyme-2 (ACE-2) receptor 35 .

The reliability of a potency immunoassay depends on the accuracy and precision of an independent assay to measure the content (dose) of the antigen being used. For recombinant protein antigens, spectrophotometric methods (such as absorbance at 280 nm or A280nm) or a dye-based protein assay may be used. Measurements of A280nm should include corrections for light scattering that may result from protein aggregation.

Physicochemical and structural integrity of protein antigens are other CQA that can affect both potency and safety. Preservation of key epitopes can be determined by binding of specific mAbs, which can also give an indication of overall integrity and stability of the tertiary structure. Direct measurements of stability of secondary structures and thermal unfolding of protein antigens can be monitored by far-UV circular dichroism (CD) and differential scanning calorimetry (DSC). These are relatively quick and supportive tools, although not of high resolution. As a part of extensive higher order structural characterization of, cryo-electron microscopy was performed on a candidate antigen, NVAX-CoV2373, which is a stabilized form of pre-fusion spike glycoprotein 36 . During development, leading up to a final process, detailed structural characterization provides a strong foundation for structure-function correlation for protein antigens. Once this foundation is established, a simple functional assay (e.g., antibody binding) that consistently correlates with structural perturbation can serve as a surrogate CQA assay. In addition to initial quantitation of the level of purity, protein degradation and post-translational modification at the primary structure level should be watched for and integrated in the stability program. While VLPs constitute self-associated protein units of well-defined sizes, the possibility of non-specific protein aggregation should be monitored by well-described hydrodynamic methods, e.g., light scattering based techniques. Aggregation of HPV 11 and HPV16 L1 VLPs was directly correlated to loss of potency as measured by antibody production in mice 37 . Non-specific aggregation can potentially also cause undesirable immune response.

Historically, recombinant protein-based vaccines have always required adjuvants for optimal immune response. Aluminum salts such as aluminum hydroxide, phosphate, and hydroxy-phosphate-sulfate have been the only approved adjuvants for decades. In recent years several new adjuvants, based on lipid A such as monophosphoryl lipid A and glucopyranosyl lipid A in a stable emulsion (MPLA, GLA-SE), squalene (AS03, MF59), and saponins (Matrix-M), have been approved in vaccine products or for clinical trials. Another example is Cytosine phosphoguanine (CpG) synthetic DNA. The current pipeline of candidate vaccines against SARS-CoV-2 includes stabilized pre-fusion form of the surface protein in combination with adjuvants such as AS03, CpG 1018, MF-59, and Matrix-M 38,39,40,41 . In these cases, vaccine DP release will need to include purity profiles of such adjuvants.

Nucleic acid-based vaccines: pDNA and mRNA platforms

Commonalities and differences

Nucleic acid-based vaccines code for selected protein antigens and rely on human cells to produce these antigens after administration. Both pDNA and mRNA technologies are being used in the development of vaccines against SARS-CoV-2, based on nucleotide sequences that would express the S-protein in human cells. Neutralization by antibodies raised in animal sera as well as protection against virus challenge in vaccinated animals were used to support selection of potentially safe and immunogenic dose for initial clinical trials. In the early phases of clinical trials (phase 1/2a), it may be sufficient, from a regulatory perspective, to move forward with a CMC package containing CQA-based tests such as genetic identity, conformational purity, and content. This could be supported by the demonstration of antigen-specific immune response in animal models using a serological assay such as the plaque reduction neutralization test. However, in phase 3, a potency assay is expected to demonstrate the expression of the protein antigen in a relevant cell line and be correlated with in vivo expression. WHO guidelines and regulatory documents from EMA and FDA have recently provided these recommendations 42,43,44 . Also, a guideline listing appearance, identity, potency, and integrity analyses for Official Control Authority Batch Release (OCABR) of mRNA vaccines has been published 23 .

Antigen expression in transfected cells can be demonstrated qualitatively, e.g., by Western blot analysis using antibodies against SARS-CoV-2. During vaccine development efforts targeted at SARS-CoV, this was accomplished for an experimental DNA vaccine that utilized the nucleoprotein (N-protein) coding sequence 45 . Quantitative determination of the levels of antigen can be obtained by use of fluorescently labeled SARS-CoV-2 S-protein antibodies for pDNA (and mRNA) projects that are using the coding sequence of this antigen. Flow cytometry has been used for quantitative detection of antigen expression in nucleic acid-based vaccine candidates 46 . Transgene expression of pDNA vaccines may also be quantified at the RNA level in transfected cells by RT-PCR 47 .

Transfection efficiencies, as a measure of potency of mRNA constructs, in dendritic cells and several other cell lines have been determined using fluorescently labeled mRNA and flow cytometric detection 48 . Examples include mRNA transcripts encoding Rabies and Zika antigens 49,50 .

There is a fundamental difference in the mechanism of action between pDNA and mRNA antigens. This leads to several important differences in the characteristics of the resulting products. pDNA antigens require intracellular transport to accomplish transcription in the cell nucleus, followed by exit into the cytosol where translation happens. mRNA antigens, on the other hand, are delivered directly into the cell cytoplasm for translation to the corresponding protein. This difference is the most likely reason why required doses for pDNA vaccines are typically much higher than those of mRNA vaccines. pDNA vaccines are best delivered by electroporation using a special device after intramuscular or subcutaneous injections. The device requires regulatory approval, but once approved, can be used for electroporation of all pDNA antigens unless changes are made in its design. pDNA antigens usually have higher stability and formulation requirements are simpler, while mRNA antigens are typically less stable physically and require encapsulation by lipid nanoparticles (LNP) to protect them from degradation by RNAses. This also results in a relatively larger number of release and characterization testing for mRNA-based DPs.

Bioanalytical advances in mRNA platform

Self-replicating RNA or sa-mRNA is a further development of the mRNA platform with potential to achieve equivalent immune response at a lower dose than needed for standard mRNA 51 . An in vitro potency assay for a sa-mRNA construct was developed to measure replication efficiency by capturing intermediate double-stranded RNA (dsRNA) and comparable protein expression in individual cells by an antigen-specific antibody. This assay is based on the indirect antibody labeling approach where protein expression proficiency measured by transfection of antigen encoded mRNA into Baby Hamster Kidney (BHK) cells followed by staining with fluorochrome tagged primary antibody 52 . The frequencies of fluorescent positive cells indicate the level of protein expression, confirming antigen identity within 48 h. This procedure could easily allow detecting multiple proteins in a single cell and could be adaptable as a platform-based approach by using secondary antibodies to detect multiple antigen-specific antibodies. Intracellular dsRNA produced during the replication cycle is a marker of RNA amplification. BHK cells transfected with influenza and Zika virus sa-mRNA reported high frequencies of dsRNA positive cells stained (as measured by flow cytometry) with anti-dsRNA antibody, unveiling launch of self-amplification 52,53 . Interestingly, immunogenic profile of Zika virus sa-mRNA vaccine was found to be comparable with frequencies of dsRNA positive BHK cells and protein expression 53 . Similarly, direct and sandwich ELISA could also be developed using electroporated cells with RNA vaccines. The current portfolio of mRNA-based COVID-19 vaccine candidates includes sa-mRNA candidates.

Developers of mRNA vaccines have paid close attention to stability and efficient delivery of antigens. As capping at the 5′-end may have an impact on stability and translation, the percentage of capped RNA is a CQA that should be measured quantitatively. Methods typically employed for this purpose have been reviewed 48 . For drug product formulation and delivery, LNPs have been extensively used with the objective of stimulating innate immune response, and these studies include clinical trials 54 . More recently, mRNA transcripts encoding for pre- and post-fusion RSV F-protein and formulated in LNP have been found to elicit protective immune response in rodent models 55 . These formulations typically involve encapsulation of mRNA in LNP. Therefore, encapsulation efficiency should also be determined as a CQA. The mean hydrodynamic size and size distribution (polydispersity) of LNP should be kept within target specification range. Dynamic or multiangle light scattering techniques are suitable for monitoring these parameters. The amount and purity of each lipid used in the LNP need to be monitored as these parameters are likely contributors to effectiveness and safety. Formulation of two candidate sa-mRNA vaccines against Zika virus with a cationic nanoemulsion (CNE) has been reported to induce potent immunity in mice and non-human primates 53 . In this case, a simple mixture of the mRNA antigen with the CNE adjuvant was used.

Removal of dsRNA in the final product resulting from in vitro transcribed mRNA transcripts, as completely as possible, is important as dsRNA is known to cause undesirable local, injection site, immune response. A facile method for the removal of dsRNA has been reported 56 . This is another CQA that should be quantitatively monitored as it relates to safety.

In the context of the physical stability of mRNA-based vaccines, significantly improved thermal stability was achieved for a freeze-dried, candidate Rabies glycoprotein mRNA vaccine 57 . Genetic stability should also be monitored over time during storage.

Recent deliveries of COVID-19 vaccines from mRNA platform

mRNA-based technologies constitute a relatively new platform. Two of the modified mRNA-based vaccines against SARS-CoV-2 have advanced rapidly through clinical phases 58,59,60,61 , and have recently delivered products that have received Emergency Use Authorization (EUA) by several regulatory agencies. These products, BNT-162b2 and mRNA-1273, were developed by Pfizer-BioNTech and Moderna Therapeutics, respectively. Both are based on non-replicating mRNA sequences encoding the full-length S-protein, packaged in LNP to provide protection against RNAses. The EMA Assessment Report on BNT162b2 provides a comprehensive summary of tests that were performed on this product 3 . Product-specific tests include in vitro bioanalytical batch release and characterization assays for the antigen and LNP.

Viral vector-based vaccines

Viral vectored vaccines constitute a significant part of the global portfolio with 60 vaccine candidates being developed against SARS-CoV-2. Candidate vaccines are based on replicating and non-replicating vectors including, but not limited to, vesicular stomatitis virus (VSV), measles, modified vaccinia ankara (MVA), adenovirus 26 and 5 (Ad26, Ad5) and chimpanzee adenovirus Oxford construct 1 (ChAdOx1). These vaccines are being evaluated either in a homologous or a heterologous prime-boost regimen, or in a single dose regimen. Prior to COVID-19, two products, both against Ebola, using viral vector platforms have been licensed. In addition, clinical trials demonstrating acceptable safety profiles have been reported for all of the above-mentioned vectors 62,63,64,65,66 . Assessing safety of a viral vectored vaccine is based on the same principles as those applied to LAV. In addition, it is necessary, from a CMC standpoint, to monitor the concentration of replication competent viral particles that may appear during the manufacturing of a replication-deficient viral vector. The potency of a viral vectored vaccine should ideally reflect both infectivity and transgene expression 67,68,69,70 . The European pharmacopoeia expert group (Group 15) has provided guidance on appropriate analytical strategies for viral vectored vaccines 71 and, in addition, OCABR guidelines have been published for analyses of non-replicating human and chimpanzee adenovirus vectored vaccines 21,22 .

Viral vectored vaccines are typically dosed based on infectivity and/or total viral particles. Assays to determine infectivity of viral vectored vaccines are based on the same cell-based principles and experience the same challenges as described below for LAV. Of the currently approved products for ebola, the recombinant VSV-based vaccine against the Zaire strain of ebola virus (rVSV-ZEBOV) utilizes a combination of infectivity determined by TCID50 assay and total viral particles assessed by droplet digital polymerase chain reaction (ddPCR) assay 72 . Similarly, the Ad26.ZEBOV/MVA-BN®-Filo heterologous prime-boost Ebola vaccine prime is dosed on the basis of total viral particles determined by qPCR and qPCR-based potency assay (QPA). QPA combines qPCR with a tissue culture-based infectivity assay to quantify the adenovirus (prime) potency, while the boost MVA-BN®-Filo potency is determined solely on infectivity through a flow cytometry-based method 73 . The throughput and turn-around time of the latter infectivity assays are improved compared to more classical TCID50, because the higher sensitivity of detection allows reduction of cell-virus incubation time from 7 days or longer to 2 days. Furthermore, accuracy and precision of analysis are also improved, and qPCR allows automated analysis 74 .

Quantifying virus particles may also be done using various particle sizing instruments. For instance, Nanoparticle Tracking Analysis (NTA), e.g., NanoSight ® (Malvern Instruments Ltd) and ZetaView ® (Particle Metrix GmbH), tracks the Brownian motion of particles through a flow cell in real-time. From the video parameters such as particle size and number are determined that can be correlated to VP quantity 75 . Another rapid analysis method for viral particle quantification is high-performance liquid chromatography (HPLC) where the column allows separation of intact virus particles from other cellular contaminants or fragmented virus particles 76 .

For adenovector-based vaccines, which are being widely used for COVID-19 response, absorbance measurements are commonly used for quantitation of virus particles, using methods based on the correlation between the protein content of adenovirus preparations and absorbance at 260 nm (for viral DNA) with appropriate controls 77,78 . The use of such methods enables in-process controls of adenovirus particle concentration and high throughput analysis for release. In addition, there are several commercial kits available utilizing the production of viral hexon proteins to analyze infectious titers of adenoviral stocks in 48 h.

Initially, semi-quantitative assays such as gel electrophoresis and Western blot are accepted to show the transgene expression of viral vectored vaccines, although for late-stage clinical trials and licensure more quantitative methods will be required to assess potency. A variety of methods may be applied, including biochemical (e.g., protein binding, enzymatic reactions), immunochemical (e.g., flow cytometry, ELISA), and molecular (e.g., PCR, microarray). For instance, the Ebola vaccine licenced by Janssen vaccines uses ELISA to determine transgene expression in the adenoviral prime vaccine and flow cytometry for the MVA boost 79,80 .

As viral vectored vaccines are recombinant products the genetic stability is an important CQA to prove that the gene of interest, as well as the vector, is uncompromised. In general, this is tested in characterization studies prior to licensure and not for batch release. This is done by passaging the virus, normally several passages beyond what the manufacturing passage will be, and sequencing several subsequent passages using either Sanger or Next Generation sequencing (NGS).

Recent advances in viral vector-based COVID-19 vaccine delivery

Interim analysis of phase 3 clinical trial for AstraZeneca-Oxford’s ChAdOx1 based vaccine (AZD1222) have been published 81 , and this vaccine has received EUA in the UK and many other countries 82 . Phase 3 clinical trial was slowed down, especially in the U.S., due to an adverse event, although observed at an extremely low frequency and with no demonstrated linkage to the vaccine. This demonstrates the critical role of bioanalytical characterization to the fullest extent, although response in each individual cannot be predicted with 100% certainty even for a placebo injection. Interim results of Ad26.SARSCoV-2.S vaccine trials by Johnson and Johnson have been reported 83,84 . This vaccine has the advantage of refrigerator (2–8 °C) storage stability and offers the possibility of protective efficacy from a single dose. This vaccine has now received EUA by FDA 85 .

Live attenuated virus vaccines

This platform has produced some of the most effective vaccines for decades and has a sound track record for safety as exemplified by global usage of the measles and rubella vaccine in infants. The older technology of attenuation by multiple passaging through cells and the more recent introduction of attenuation by genetic engineering are effective. However, safety concerns include reaching the balance between eliciting protective immune response and causing vaccine-induced infection. For engineered viruses, the absence of reversion mutants must be monitored and NGS is an effective tool in this regard. The issue of potency versus safety is best addressed by potency assays that are as accurate and precise as possible. Traditional infectious titer assays such as TCID50, which are widely accepted by regulatory agencies, often suffer from large standard deviations. Depending on the virus, these assays may have a long turn-around time due to the low sensitivity of CPE detection, which requires a large number of replication cycles for a sufficient percentage of the cell population to be killed by infection. As described above in the section on viral vector-based vaccines, PCR or fluorescence-based detection techniques provide more sensitive detection and, thereby, allows reduction of the incubation time with cells. Published examples of these applications to several LAVs include but are not limited to measles, rotavirus, VSV, and HIV-1 86,87,88 . Fluorescent focus assay (FFA), a fluorescent antibody-based infectious titer assay, offers the advantage of sensitivity, automated foci counting and relatively high throughput. Compared to plaque and TCID50 assays, infection of cells can be quantitatively detected earlier in the process instead of having to await cell membrane lysis. Therefore, infectious titer, as focus forming unit, can be determined significantly faster compared to a CPE-based assay. FFA is used as a batch release potency assay for an approved LAV influenza vaccine 89 . However, it is important to establish that focus formation is a result of infection and one is not observing simply a binding event that may not lead to CPE. Demonstration of concordance with plaque or TCID50 assay can be a part of the FFA validation process 90 . With appropriate controls in place, FFA could provide a sensitive and reasonably fast cell-based potency assay for SARS-CoV-2 LAV projects. An emerging label-free technology for infectious measurements is based on laser force cytology to detect and quantify virus-infected cells. It appears to be more sensitive and much faster than the TCID50 assay, and may offer a higher precision 91 . This method can also be used for rapid in-process testing during manufacturing.

Non-clinical and clinical assays

Outside of CMC, specific assays are developed in the pre-clinical and clinical trial phases to test immune response induced in animals and humans, respectively. These assays are further tested for robustness, qualified and validated in later stages to support the vaccine development process. Often, challenges including variability in in vivo biology and technical differences between laboratories can make comparison of data produced by individual laboratories in late phases and multicenter trials difficult. Additionally, multiple assays developed for vaccines of various modalities also augment complexities. To mitigate some of these challenges to reliable immunological profiling of each vaccine candidate based on different platforms, and to provide robust assays to facilitate the regulatory process, CEPI has established a global network of seven Central Laboratories (CLs). Immune responses elicited by different vaccines during the pre-clinical and clinical phases will be analyzed by implementing common protocols, antibody standards and equivalent key reagents across these laboratories 92,93 .

The antibody-mediated immune response relies on B-cell recognition of antigens and the release of antibodies. ELISA is often used to evaluate humoral responses. CEPI selected ELISA methods that exploit and capture signals of full-length SARS-CoV-2 S, RBD, and N-protein IgG antibody/antigen interactions. ELISA only measures binding antibodies while detection of functional antibodies is critical for vaccine efficacy and regarded as the golden standard. Robust pseudovirus and wild-type virus-based neutralization assays have been developed to detect neutralizing antibodies to SARS-CoV-2, as they are designed to detect antibodies capable of inhibiting viral replication. These assays are either validated or qualified.

CMI response relies primarily on T-cells and is often assessed with enzyme-linked immunospot (ELISPOT) or flow cytometry. The CEPI supported ELISPOT is an immunostaining assay that focuses on a quantitative measurement of the frequency of the cytokine secreting T-cells. This ELISPOT assay allows the detection of specific TH1 (IFN-γ) and TH2 (IL-5) cytokines-producing T-cells in peripheral blood mononuclear cells (PBMCs) stimulated with SARS-CoV-2 peptides spanning full-length S protein. These cytokines have been detected frequently with individuals infected with SARS-CoV-2 and predict immune protection 94 .

Clinical assays are used to quantify immune responses generated in human subjects after immunization, while potency assays are designed to measure optimal doses and surrogate biological activities that would potentially result in desired levels of immune responses. In setting up a potency assay, it is desirable to have knowledge-based hypothesis regarding the mechanism of action of a vaccine that may correlate with a clinically relevant immune response. However, for SARS-CoV-2 vaccines correlation of protection in humans is not yet known, although protection in animal models has been demonstrated for some candidate vaccines 47,58 . While such indication of protective efficacy, typically obtained in a handful of animals, is certainly promising, it is not a guaranteed predictor of protective efficacy in humans. Still, it is very encouraging that early clinical trials have demonstrated that the doses selected on the basis of potency assays used for CMC batch release have yielded high levels of immune responses 58,95 . The development of non-clinical immunoassays could synergistically help in establishing functionally meaningful and qualitative potency assays.

Most of the SARS-CoV-2 vaccine developers are using ‘’in-house” assays, reagents and panel of reference regents. This makes it difficult to compare immune responses across different vaccines. Therefore, it is critical to standardize assays and reagents to support advanced phase clinical trials. To address this issue, CEPI has facilitated the development of a WHO endorsed international antibody reference standard 96 , and vaccine developers are encouraged to include this standard in immunological assays. Furthermore, CLs in CEPI’s global network of laboratoriess are using common key reagents including coating protein, SARS-CoV-2 virus strain, virus pseudoparticles, peptide pools for PBMC stimulation, panel of standards and controls. This dual approach enables impartial comparison of data generated across vaccine trials.

Closing comments

The COVID-19 pandemic has galvanized vaccine development efforts globally with an unprecedented sense of urgency. Developers from biopharmaceutical industry, small and large, and academic laboratories have stepped up with a mission to end the current pandemic. All vaccine platform technologies developed to date, old and new, are being employed. Regulatory agencies have prioritized reviews of investigators’ applications seeking entry into and progression through clinical trials. At this time, the first two mRNA vaccines have been given to more than 25 million people under EUA. Four viral vectored vaccines have closely followed in getting approval for emergency use in different countries, as well as four inactivated coronavirus vaccines. As clinical trials for many more candidate vaccines are progressing to advanced phases involving tens of thousands of volunteers, quality attributes of vaccine candidates that may be critical to safety and immunogenicity are being evaluated by developers and stringently reviewed by regulators in a closely interactive manner. Based on the large body of data already gathered from human clinical trials and from ongoing vaccination with EUA approved products in several countries, it is clear that safety profiles of vaccines produced using newer platforms have been excellent. In order to meet the urgent demands of virtually the entire global population, analytical assays of high reliability and short turn-around times should be implemented for testing CQA of each product. Such assays have been described in the literature in the context of development or study of other vaccines and can be adapted for vaccines against SARS-CoV-2, for example, in measurements of biological activity or potency. Batch release assays should preferably be performed in vitro. This is not only appropriate for adhering to 3R principles, but also preferable for practical considerations including savings in time and reducing irreproducibility associated with animal-based tests, which often leads to costly and unnecessary rejection of good quality batches. Reagents for in vitro immunochemical and biochemical assays, including the mAb CR3022, RBD of the S-protein, and human ACE2 are now being made available to eligible developers in collaborative arrangements with PATH and NIBSC. Robust and faster assays for CQA will expedite technology transfers between manufacturing sites, which are required for many COVID-19 projects in order to meet large supply needs among all populations of the world. A well-designed CMC plan will also facilitate regulatory reviews and approvals. Furthermore, selected CQA-based bioanalytical evaluation methods, outlined in part in this review, will allow comparing consistency in quality between vaccine batches used in different clinical phases and forming a valuable bridge that will extend to commercial products.


6. Pre-Clinical and Clinical Applications of LNPs as Delivery Systems for RNA-Based Vaccines

The application of LNPs as delivery systems for RNA-based vaccines is beneficial for reaching the full potential of the vaccine as these systems serve to protect the large encapsulated nucleic acid molecule against nuclease degradation [1,121]. As delivery systems, LNPs pass through the cell membrane for cellular uptake (by endocytosis) and deliver their enclosed mRNA into the cytosol only after endosomal escape. LNPs may also affect the innate immune response and provide the mRNA vaccines with synergistic adjuvant effects [2].

The various application roles of RNA for both preventive and therapeutic uses have led to different employments of RNA-based vaccines against both infectious pathogens as well as cancer. Many research studies that have focused on LNPs as delivery systems for self-amplifying mRNA and conventional mRNA against various infectious diseases have shown robust and rapid immune stimulation in different animal species [9,27,29,30,122,123,124]. Examples of mRNA vaccines in various lipid-based formulations developed for preclinical studies against infectious diseases and cancer are summarized in Table 1 .

In addition, different mRNA vaccine formulations developed for protection against infectious conditions have entered clinical studies to evaluate their effectiveness. Table 2 summarizes the status of these clinical trials. The clinical study conducted by Bahl et al. [27] and sponsored by Moderna Therapeutics is considered as the first human trial ( <"type":"clinical-trial","attrs":<"text":"NCT03076385","term_id":"NCT03076385">> NCT03076385) that used an LNP-formulated mRNA vaccine encoding the HA antigen of influenza H10N8. This study revealed that all 31 participants developed specific antibodies titers of � against the HA antigen of influenza H10N8 after two intramuscular immunizations (using 100 µg of the vaccine) separated by 3 week intervals, indicating good immunogenicity of the vaccine. In addition, the study showed that virus-neutralizing antibodies titers of � were present in the serum of 87% of the vaccinated participants after 43 days of vaccination. The results obtained from human trials were considered satisfactory despite low values of titers compared to those obtained from animal models [27]. Similar findings were reported by the group of Feldman et al. [125]. The work of these authors demonstrated that LNP-formulated mRNA vaccines encoding full-length HA from the H10N8 and H7N9 influenza strains were safe and able to produce robust humoral immune responses in healthy adults after intramuscular vaccination with two doses 3 weeks apart at 100 and 25 µg dose levels [125]. The authors also reported that the safety and reactogenicity profiles of both vaccines, used at doses up to 100 µg, were comparable to those obtained for licensed vaccines formulated with or without adjuvants [125].

Table 2

mRNA vaccines that entered clinical studies against infectious diseases and cancer.

Sponsoring ManufacturermRNA VaccineDelivery SystemTargetTrial NumberStageStatusReference
Infectious diseases
Moderna Therapeutics/National Institute of Allergy and Infectious Diseases (NIAID)mRNA-1273 (perfusion stabilized S protein mRNA vaccine)LNPCOVID-19 <"type":"clinical-trial","attrs":<"text":"NCT04470427","term_id":"NCT04470427">> NCT04470427Phase IIIActive, not recruiting[126]
BioNTech / Pfizer BNT162
(3 LNP–mRNA vaccines)
LNPCOVID-19 <"type":"clinical-trial","attrs":<"text":"NCT04537949","term_id":"NCT04537949">> NCT04537949Phase IIIRecruiting[126]
CureVacCV7202 (sequence-optimized)LNPRabies <"type":"clinical-trial","attrs":<"text":"NCT03713086","term_id":"NCT03713086">> NCT03713086Phase IActive, not recruiting, PCD: January 2022[127]
Moderna TherapeuticsmRNA-1440 (nucleoside-modified)LNPInfluenza H10N8 <"type":"clinical-trial","attrs":<"text":"NCT03076385","term_id":"NCT03076385">> NCT03076385Phase ICompleted PCD: October 2018[27,125]
Moderna TherapeuticsmRNA-1851 (nucleoside-modified)LNPInfluenza H7N9 <"type":"clinical-trial","attrs":<"text":"NCT03345043","term_id":"NCT03345043">> NCT03345043Phase IActive, not recruiting, PCD: February 2020[27,125]
Moderna TherapeuticsmRNA-1653 (nucleoside-modified)LNPHMPV/HPIV3 <"type":"clinical-trial","attrs":<"text":"NCT03392389","term_id":"NCT03392389">> NCT03392389Phase ICompleted, PCD: July 2019[2]
Moderna TherapeuticsmRNA-1325 (nucleoside-modified)LNPZika <"type":"clinical-trial","attrs":<"text":"NCT03014089","term_id":"NCT03014089">> NCT03014089Phase ICompleted, PCD: July 2019[123]
Moderna TherapeuticsmRNA-1893 Zika <"type":"clinical-trial","attrs":<"text":"NCT04064905","term_id":"NCT04064905">> NCT04064905Phase IActive, not recruiting, PCD: February 2021[123]
Moderna TherapeuticsmRNA-1647 and mRNA-1443 (nucleoside-modified)LNPHCMV
<"type":"clinical-trial","attrs":<"text":"NCT03382405","term_id":"NCT03382405">> NCT03382405Phase IActive, not recruiting, PCD: July 2020[2]
Moderna TherapeuticsmRNA-1388 (nucleoside-modified)LNPChikungunya <"type":"clinical-trial","attrs":<"text":"NCT03325075","term_id":"NCT03325075">> NCT03325075Phase ICompleted, PCD: November 2019[2]
Cancer immunotherapy
BioNTech RNA Pharmaceuticals GmbHmRNA lipoplex (Lipo–MERIT)LiposomesTAAs (advanced melanoma) <"type":"clinical-trial","attrs":<"text":"NCT02410733","term_id":"NCT02410733">> NCT02410733Phase IActive, not recruiting[128]
BioNTech AGmRNA lipoplex (TNBC–MERIT)LiposomesTAAs (triple-negative breast cancer) <"type":"clinical-trial","attrs":<"text":"NCT02316457","term_id":"NCT02316457">> NCT02316457Phase IActive, not recruiting[129]

HCMV, human cytomegalovirus hMPV, human metapneumovirus HPIV3, human parainfluenza virus type 3 LNP, lipid nanoparticle PCD, estimated primary completion date TAAs, tumor-associated antigens.

6.1. RNA/LNP Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

Nowadays, RNA-based vaccines have become one of the most effective vaccine technologies developed to protect against the pandemic due to coronavirus (COVID-19), which emerged in December 2019 due to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [122].

Various pre-clinical studies [130,131,132,133,134] were conducted to evaluate the efficacy and immunogenicity of vaccines based on LNP–mRNA encoding for the SARS-CoV-2 spike protein or the spike receptor binding domain. For example, immunization of mice with self-amplifying RNA encoding the virus spike protein encapsulated in LNP formulation produced markedly high SARS-CoV-2-specific antibodies and induced a robust cellular immunity response compared to electroporated pDNA vaccine. These observations were attributed to the nature of the LNP formulation that was used [132].

Another preclinical study evaluated the immunogenicity of nucleoside-modified mRNA encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. It was observed that the two vaccines induced strong T and B cell responses in addition to potent antibody responses after a single dose [130]. Further, an mRNA vaccine (mRNA-1273) encoding the spike protein of SARS-CoV-2 in the LNP formulation was developed by Moderna Therapeutics, Cambridge, MA, USA. Immunization of non-human primates with this vaccine produced high neutralizing activity and remarkably elevated S-protein-specific antibodies [135].

The results obtained from preclinical studies on mRNA vaccines formulated in LNPs were promising in providing a vaccine solution for COVID-19, and enabled the transfer of RNA-based vaccines to the level of clinical studies. Moderna mRNA-1273 is considered to be the first vaccine that entered phase I clinical studies (ClinicalTrials.gov identifier <"type":"clinical-trial","attrs":<"text":"NCT04283461","term_id":"NCT04283461">> NCT04283461) only 42 days after identification of the genetic sequence of SARS-CoV-2 [122]. The manufacturing company recently announced this vaccine to be 94% effective based on the first interim analysis of phase III clinical studies (ClinicalTrials.gov identifier <"type":"clinical-trial","attrs":<"text":"NCT04470427","term_id":"NCT04470427">> NCT04470427) [136].

Different institutions and pharmaceutical manufacturers are exploiting different LNPs as platform technologies to develop RNA-based vaccines against COVID-19. For example, the mRNA vaccine (BNT162) developed by BioNTech (Mainz, Germany) in collaboration with Pfizer (New York, NY, United States) was designed to comprise four different mRNA formats that target antigens of the S-protein and receptor binding domain and formulated as an LNP formulation [122,137,138]. As of 9 November 2020, BioNTech (Mainz, Germany) and Pfizer (New York, NY, United States) reported that the BNT162 vaccine was more than 90% effective against COVID-19 based on the first interim efficacy analysis from the phase III clinical studies (ClinicalTrials.gov Identifier: <"type":"clinical-trial","attrs":<"text":"NCT04368728","term_id":"NCT04368728">> NCT04368728) [139].

Overall, the use of LNPs to formulate mRNA-based vaccines can be considered a promising approach for vaccination against SARS-CoV-2. These lipid-based formulations may be rapidly manufactured and, hence, accelerate vaccine development. Different mRNA-based vaccines against COVID-19 that are formulated in LNPs were shown to be safe and immunogenic in various clinical trials. The data obtained from animal studies revealed that LNP–mRNA vaccines induced a strong neutralizing antibody response and provided high protection against SARS-CoV-2.

6.2. RNA/LNP Vaccines against Influenza Virus Infection

Influenza vaccines based on mRNA are one of the most extensively studied mRNA vaccines because of the ease of evaluating their efficacy in small animals in addition to the possibility of measuring induced T and B cell responses. It was reported that a self-amplifying mRNA vaccine can be produced within a short period of time once the genetic sequence coding of influenza hemagglutinin (HA) antigen is identified [4]. Based on this, a self-amplifying mRNA vaccine against a strain of H7N9 influenza in China was produced (encapsulated in LNPs) within 8 days after identification of the gene sequence of HA antigen and demonstrated to be immunogenic in mice [4].

Lindgren et al. [28] also developed modified non-replicating mRNA vaccine encoding HA of a pandemic H10N8 influenza strain in an LNP formulation. Intramuscular and intradermal immunization of rhesus macaques with this vaccine produced an expansion of B cell responses along with the formation of germinal centers (GCs) in draining lymph nodes after each vaccination. In addition, an increase in the level of H10-specific T follicular helper cells was observed and correlated with high-avidity antibody responses, which take place after seasonal influenza vaccination in humans, indicating seroconversion [28].

A universal influenza vaccine was developed using an mRNA–LNP formulation to induce a potent immune response against conserved epitopes (chimeric and headless hemagglutinin structures) of different virus strains. This universal influenza vaccine raised antibodies against the stalk domain of hemagglutinin and showed good protection of animals against a wide range of influenza viruses [140]. Another broadly protecting influenza vaccine was produced and evaluated by Pardi et al. [29] in which nucleoside-modified mRNA–LNPs that express the full-length influenza virus HA were used. A single immunization with the formulated vaccine resulted in HA stalk-specific antibody responses in different animal models (mice, rabbits, and ferrets) and, hence, provided protection against homologous, heterologous, and heterosubtypic influenza virus infections in mice.

Further, LNPs were utilized for delivery of a modified mRNA vaccine encoding the HA of either H10N8 or H7N9 influenza strains [27]. The formulated vaccine produced robust, rapid, and long-lasting immune responses in mice, ferrets, and cynomolgus monkeys.

6.3. RNA/LNP Vaccines against Rabies Virus Infection

LNPs were employed to formulate mRNA vaccines against rabies virus infection. A sequence-optimized, unmodified mRNA vaccine, encoding rabies virus glycoprotein (RABV-G) was developed by Lutz et al. [9]. A single immunization of cynomolgus monkeys with this vaccine resulted in induction of the virus neutralization titers which exceeded the reference values set by the World Health Organization (0.5 IU/mL) to correlate with protection in humans. These titers were dose-dependent and were further enhanced by a 20 fold increase following the second immunization of the animals performed at day 28. The authors noticed that the protection against rabies remained stable during the observation period of 1 year [9].

6.4. RNA/LNP Vaccines against Zika Virus Infection

Utilization of mRNA/LNPs vaccines for protection against Zika virus has been described in the literature by different research groups [30,31,123]. The group of Pardi et al. [30] have shown that single intradermal vaccinations of mice (30 µg dose) or rhesus macaques (50 µg dose) with nucleoside-modified mRNA/LNPs vaccine encoding the pre-membrane and envelope (prM𠄾) glycoprotein of Zika virus resulted in potent and persistent protective immunity in both animal models with induction of anti-Zika virus neutralizing antibodies. Similar results were reported by Richner et al. [123] upon intramuscular vaccination of mice with two 10 µg doses of modified mRNA/LNPs vaccine encoding the prM𠄾 glycoprotein of Zika virus. Further, the groups of Richner et al. [31,123] engineered an mRNA/LNP vaccine with mutations destroying the conserved fusion-loop epitope of the E protein. This mutant mRNA vaccine was found to be protective against Zika virus infection, and reduced production of antibodies enhancing dengue virus infection (which is closely related to Zika virus infection) in both cell culture and mice. The same vaccine was evaluated for its ability to protect the fetus against congenital malformation that may occur during pregnancy due to the transmission of Zika virus. The authors reported that two immunizations with the vaccine protected the pregnant mouse against maternal, placental, and fetal infection by Zika virus [31].

Furthermore, vaccines based on mRNAs can be designed to deliver multiple mRNAs encoding different antigens in order to produce immunity against multiple pathogens or against different antigens of the same infecting pathogen after a single immunization. These multivalent mRNAs encoding different antigens are particularly useful for stimulating different immunity responses or to target antigens expressed in multiple life cycles of the infecting pathogen [2]. For example, the work of John et al. [32] described the production of LNPs encapsulating nucleoside-modified mRNAs encoding the five different subunits of the human cytomegalovirus (CMV) pentameric protein complex and glycoprotein B (gB). The produced vaccine was efficiently delivered in vivo and resulted in potent immune responses and broadly neutralizing antibodies in both mice and non-human primates after intramuscular immunization. The authors also formulated an additional LNP/mRNA vaccine encoding the immunodominant CMV T cell antigen, pp65. Administration of this conventional vaccine with the pentameric protein and gB vaccine resulted in multi-antigenic or broad T cell responses and did not interfere with the levels of antibodies produced by vaccinating mice with the multivalent pentameric protein [32]. The mRNA vaccine expressing the pentameric proteins of human CMV has entered the clinical evaluation step and is currently in phase I clinical trials [2].

6.5. RNA/LNP Vaccines against Cancer

Cancer vaccines act either as a prophylactic (to prevent infections by cancer-causing viruses) or therapeutic (to treat existing cancer). The first therapeutic cancer vaccine, Sipuleucel-T (provenge) was approved in 2010 for treatment of prostate cancer [141,142]. The clinical benefits of cancer vaccines to decrease the recurrence of cancer and to improve the overall survival of patients have been established in different studies [141,143].

Various antigens, such as tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), can be encoded into cancer vaccines. TAAs comprise proteins that are overexpressed in cancer cells but also present in normal cells. e TSAs are expressed only in tumor cells and are derived from oncogenic proteins of viruses or from proteins produced upon gene mutations or rearrangements [23]. In addition, mutations in tumor cells during progression and carcinogenesis may lead to the production of altered proteins termed neoantigens. These types of proteins can be recognized by the analysis of genetic mutations in an individual cancer cell. Utilization of neoantigens may allow the production of personalized neoepitope cancer vaccines that could be advantageous in enhancing tolerance and limiting normal tissue toxicity, as well as to improve antitumor immune response compared to conventional cancer vaccines [23,142]. Personalized RNA mutanome vaccines [144] and personalized peptide vaccines [145] are examples of personalized vaccines that have shown promising results in phase I clinical trials.

The safety, immunogenicity, and tolerability of the first personalized IVAC MUTANOME (BioNTech RNA Pharmaceuticals GmbH), which is a poly-neoepitope-coding RNA vaccine, have been evaluated in phase I clinical trials (ClinicalTrials.gov identifier: <"type":"clinical-trial","attrs":<"text":"NCT02035956","term_id":"NCT02035956">> NCT02035956) targeting mutant neoantigens for the treatment of patients with melanoma. A strong immune response against the vaccine antigens was observed. In addition, T cell response was generated against 60% of the 125 selected neoepitopes with no adverse drug reactions, indicating good tolerability of the vaccine by enrolled patients [146].

The possibility of obtaining RNA from a tumor sample, to produce patient-specific antigens and to then formulate a personalized vaccine is considered among the advantages of mRNA cancer vaccines compared to other types of vaccines. In addition, signals provided through the Toll-like receptors TLR3, TLR7, and TLR8 may reflect the adjuvanticity of mRNA to enhance immune response [23]. Furthermore, mRNA vaccines do not possess the risk of infection, and their manufacture is rapid, scalable, and inexpensive [14,142,147].

mRNA vaccines stimulate a specific immune response when the encoded antigen is translated to proteins in the cytosol of antigen-presenting cells or APCs (either dendritic cells (DCs) or macrophages). The expressed proteins are presented on major histocompatibility complex (MHC) class I molecules to CD8+ T cells, stimulating the cellular response. Induction of supportive CD4+ T helper cell response, which is crucial in cancer immunotherapy, may take place by fusion of the mRNA-encoded antigen to MHC class II trafficking signals derived from lysosomal proteins [23,147,148,149,150]. Therefore, mRNA vaccines can be developed to encode cancer-specific antigens to produce a specific T cells immune response against tumor cells [25].

Clinical trials and in vivo administration of personalized mRNA cancer vaccines utilize safe and biocompatible nanoparticle systems, such as lipid nanoparticles and liposomes, to formulate and optimize mRNA delivery [25,142]. The application of nano-particulate systems as carriers for mRNA cancer vaccines provides advantages in protecting the mRNA from degradation in addition to the enhancement of antitumor responses by the possibilities of co-delivery of the vaccine with an adjuvant and the utilization of ligands to target dendritic cells. In addition, nanoparticulate systems may provide the possibility to control the release and distribution of the vaccine [44,142].

Personalized mRNA cancer vaccines encoding different antigens have been formulated in lipid nanosystems and have already entered clinical studies ( <"type":"clinical-trial","attrs":<"text":"NCT03897881","term_id":"NCT03897881">> NCT03897881, <"type":"clinical-trial","attrs":<"text":"NCT02316457","term_id":"NCT02316457">> NCT02316457, <"type":"clinical-trial","attrs":<"text":"NCT03313778","term_id":"NCT03313778">> NCT03313778, <"type":"clinical-trial","attrs":<"text":"NCT03480152","term_id":"NCT03480152">> NCT03480152, <"type":"clinical-trial","attrs":<"text":"NCT03323398","term_id":"NCT03323398">> NCT03323398) [23,25]. The successful application of LNPs to encapsulate personalized mRNA vaccines has been described for the treatment of patients with melanoma [144]. All patients demonstrated T cell responses against the neoepitopes of the vaccine. In addition, highly reduced rates of metastases were observed in two subjects after the start of vaccination, which led to the enhanced survival of patients [144].

In order to induce a strong cytotoxic CD8 T cell response, the group of Oberli et al. [44] developed LNPs for the delivery of an mRNA vaccine encoding the model immunology protein, ovalbumin (OVA). The authors identified an optimum formulation that contains an ionizable lipid (cKK-E12) and an additive (sodium lauryl sulfate). The optimal formulation showed increased T cell response upon reducing the molar ratio of cKK-E12 from 35% to 10%. Immunization of model mice with transgenic OVA-expressing tumor or with aggressive B16F10 melanoma using the formulated mRNA vaccine encoding the corresponding antigens resulted in strong CD8 T cell immunity activation in addition to slow tumor growth, shrinkage of tumor and, consequently, extended survival of treated mice [44].

Enhancement of the LNP potency through delivery of their encapsulated mRNA can be achieved by attachment of different moieties to the surface of LNPs for targeting specific receptors on the surface of immune cells. Alternatively, co-administration of LNPs with adjuvants may enhance the stimulation of immune response [1,47,112]. For instance, incorporation of TLR4 agonist (lipopolysaccharide LPS) in LNPs reduced tumor growth and provided longer survival in mice with B16F10 melanoma [44]. Similarly, Verbeke et al. [112] demonstrated that co-delivering the nucleoside-modified mRNA with TLR4 agonist (monophosphoryl lipid A MPLA) inside DOTAP𠄼holesterol mRNA lipoplexes induced innate immunity and allowed high antigen expression in vivo. In addition, the group of Lee et al. [47] incorporated the lipopeptide tripalmitoyl-S-glyceryl cysteine (Pam3), which is a TLR1 and TLR2 agonist, as an adjuvant in LNPs encapsulating OVA mRNA. Using Pam3–LNP formulation for intramuscular immunization of mice resulted in high expression of tumor antigens with enhanced cellular immune stimulation [47].

Further to the benefits of LNPs in the delivery of mRNA cancer vaccines, LNPs can be designed to deliver mRNAs encoding cytokines to activate immunity response and kill tumor cells without causing toxicity or side effects to healthy cells [25]. The efficiency of mRNA–LNPs encoding interleukin-12 (IL-12), an example of cytokines with anticancer activity, was examined by the group of Lai et al. [151] for suppression of tumor growth in transgenic mouse models of hepatocellular carcinoma (HCC). The systemic administration of the formulated mRNA–LNPs did not result in toxicity of healthy tissues but reduced the growth of the liver tumor and increased the survival of treated mice [151].

Another strategy to increase the specificity of therapeutic mRNAs was investigated by the group of Jain et al. [45]. The authors examined the possibility of using therapeutic mRNAs to program diseased or cancerous cells to synthesize a toxic protein that will cause self-destruction of these cells without harming healthy cells. For this purpose, microRNA (miRNA) target sites were incorporated in modified mRNAs encoding toxic or apoptotic proteins like caspase or PUMA (p53 upregulated modulator of apoptosis). The presence of miRNA binding sites will allow targeting of miRNAs that are present only in healthy cells and then enable these cells to recognize and degrade the toxic mRNA. It was found that intra-tumoral administration of LNPs loaded with these miRNA–mRNA combined sequences in mice prevented the expression of toxic proteins from the mRNA of healthy cells but selectively triggered apoptosis in tumor cells without causing systemic toxicity [45].


MRNA Technology Gave Us the First COVID-19 Vaccines. It Could Also Upend the Drug Industry

I had been staring her in the eyes, as she had ordered, but when a doctor on my other side began jabbing me with a needle, I started to turn my head. “Don’t look at it,” the first doctor said. I obeyed.

This was in early August in New Orleans, where I had signed up to be a participant in the clinical trial for the Pfizer-BioNTech COVID-19 vaccine. It was a blind study, which meant I was not supposed to know whether I had gotten the placebo or the real vaccine. I asked the doctor if I would really been able to tell by looking at the syringe. “Probably not,” she answered, “but we want to be careful. This is very important to get right.”

I became a vaccine guinea pig because, in addition to wanting to be useful, I had a deep interest in the wondrous new roles now being played by RNA, the genetic material that is at the heart of new types of vaccines, cancer treatments and gene-editing tools. I was writing a book on the Berkeley biochemist Jennifer Doudna. She was a pioneer in determining the structure of RNA, which helped her and her doctoral adviser figure out how it could be the origin of all life on this planet. Then she and a colleague invented an RNA-guided gene-editing tool, which won them the 2020 Nobel Prize in Chemistry.

The tool is based on a system that bacteria use to fight viruses. Bacteria develop clustered repeated sequences in their DNA, known as CRISPRs, that can remember dangerous viruses and then deploy RNA-guided scissors to destroy them. In other words, it’s an immune system that can adapt itself to fight each new wave of viruses&mdashjust what we humans need. Now, with the recently approved Pfizer-BioNTech vaccine and a similar one from Moderna being slowly rolled out across the U.S. and Europe, RNA has been deployed to make a whole new type of vaccine that will, when it reaches enough people, change the course of the pandemic.

Up until last year, vaccines had not changed very much, at least in concept, for more than two centuries. Most have been modeled on the discovery made in 1796 by the English doctor Edward Jenner, who noticed that many milkmaids were immune to smallpox. They had all been infected by a form of pox that afflicts cows but is relatively harmless to humans, and Jenner surmised that the cowpox had given them immunity to smallpox. So he took some pus from a cowpox blister, rubbed it into scratches he made in the arm of his gardener’s 8-year-old son and then (this was in the days before bioethics panels) exposed the kid to smallpox. He didn’t become ill.

Before then, inoculations were done by giving patients a small dose of the actual smallpox virus, hoping that they would get a mild case and then be immune. Jenner’s great advance was to use a related but relatively harmless virus. Ever since, vaccinations have been based on the idea of exposing a patient to a safe facsimile of a dangerous virus or other germ. This is intended to kick the person’s adaptive immune system into gear. When it works, the body produces antibodies that will, sometimes for many years, fend off any infection if the real germ attacks.

One approach is to inject a safely weakened version of the virus. These can be good teachers, because they look very much like the real thing. The body responds by making antibodies for fighting them, and the immunity can last a lifetime. Albert Sabin used this approach for the oral polio vaccine in the 1950s, and that’s the way we now fend off measles, mumps, rubella and chicken pox.

At the same time Sabin was trying to develop a vaccine based on a weakened polio virus, Jonas Salk succeeded with a safer approach: using a killed or inactivated virus. This type of vaccine can still teach a person’s immune system how to fight off the live virus but is less likely to cause serious side effects. Two Chinese companies, Sinopharm and Sinovac, have used this approach to develop vaccines for COVID-19 that are now in limited use in China, the UAE and Indonesia.

Another traditional approach is to inject a subunit of the virus, such as one of the proteins that are on the virus’s coat. The immune system will then remember these, allowing the body to mount a quick and robust response when it encounters the actual virus. The vaccine against the hepatitis B virus, for example, works this way. Using only a fragment of the virus means that they are safer to inject into a patient and easier to produce, but they are often not as good at producing long-term immunity. The Maryland-based biotech Novavax is in late-stage clinical trials for a COVID-19 vaccine using this approach, and it is the basis for one of the two vaccines already being rolled out in Russia.

The plague year of 2020 will be remembered as the time when these traditional vaccines were supplanted by something fundamentally new: genetic vaccines, which deliver a gene or piece of genetic code into human cells. The genetic instructions then cause the cells to produce, on their own, safe components of the target virus in order to stimulate the patient’s immune system.

For SARS-CoV-2&mdashthe virus that causes COVID-19&mdashthe target component is its spike protein, which studs the outer envelope of the virus and enables it to infiltrate human cells. One method for doing this is by inserting the desired gene, using a technique known as recombinant DNA, into a harmless virus that can deliver the gene into human cells. To make a COVID vaccine, a gene that contains instructions for building part of a coronavirus spike protein is edited into the DNA of a weakened virus like an adenovirus, which can cause the common cold. The idea is that the re-engineered adenovirus will worm its way into human cells, where the new gene will cause the cells to make lots of these spike proteins. As a result, the person’s immune system will be primed to respond rapidly if the real coronavirus strikes.

This approach led to one of the earliest COVID vaccine candidates, developed at the aptly named Jenner Institute of the University of Oxford. Scientists there engineered the spike-protein gene into an adenovirus that causes the common cold in chimpanzees, but is relatively harmless in humans.

The lead researcher at Oxford is Sarah Gilbert. She worked on developing a vaccine for Middle East respiratory syndrome (MERS) using the same chimp adenovirus. That epidemic waned before her vaccine could be deployed, but it gave her a head start when COVID-19 struck. She already knew that the chimp adenovirus had successfully delivered into humans the gene for the spike protein of MERS. As soon as the Chinese published the genetic sequence of the new coronavirus in January 2020, she began engineering its spike-protein gene into the chimp virus, waking each day at 4 a.m.

Her 21-year-old triplets, all of whom were studying biochemistry, volunteered to be early testers, getting the vaccine and seeing if they developed the desired antibodies. (They did.) Trials in monkeys conducted at a Montana primate center in March also produced promising results.

Bill Gates, whose foundation provided much of the funding, pushed Oxford to team up with a major company that could test, manufacture and distribute the vaccine. So Oxford forged a partnership with AstraZeneca, the British-Swedish pharmaceutical company. Unfortunately, the clinical trials turned out to be sloppy, with the wrong doses given to some participants, which led to delays. Britain authorized it for emergency use at the end of December, and the U.S. is likely to do so in the next two months.

Johnson & Johnson is testing a similar vaccine that uses a human adenovirus, rather than a chimpanzee one, as the delivery mechanism to carry a gene that codes for making part of the spike protein. It’s a method that has shown promise in the past, but it could have the disadvantage that humans who have already been exposed to that adenovirus may have some immunity to it. Results from its clinical trial are expected later this month.

In addition, two other vaccines based on genetically engineered adenoviruses are now in limited distribution: one made by CanSino Biologics and being used on the military in China and another named Sputnik V from the Russian ministry of health.

There is another way to get genetic material into a human cell and cause it to produce the components of a dangerous virus, such as the spike proteins, that can stimulate the immune system. Instead of engineering the gene for the component into an adenovirus, you can simply inject the genetic code for the component into humans as DNA or RNA.

Let’s start with DNA vaccines. Researchers at Inovio Pharmaceuticals and a handful of other companies in 2020 created a little circle of DNA that coded for parts of the coronavirus spike protein. The idea was that if it could get inside the nucleus of a cell, the DNA could very efficiently churn out instructions for the production of the spike-protein parts, which serve to train the immune system to react to the real thing.

The big challenge facing a DNA vaccine is delivery. How can you get the little ring of DNA not only into a human cell but into the nucleus of the cell? Injecting a lot of the DNA vaccine into a patient’s arm will cause some of the DNA to get into cells, but it’s not very efficient.

Some of the developers of DNA vaccines, including Inovio, tried to facilitate the delivery into human cells through a method called electroporation, which delivers electrical shock pulses to the patient at the site of the injection. That opens pores in the cell membranes and allows the DNA to get in. The electric pulse guns have lots of tiny needles and are unnerving to behold. It’s not hard to see why this technique is unpopular, especially with those on the receiving end. So far, no easy and reliable delivery mechanism has been developed for getting DNA vaccines into the nucleus of human cells.

That leads us to the molecule that has proven victorious in the COVID vaccine race and deserves the title of TIME magazine’s Molecule of the Year: RNA. Its sibling DNA is more famous. But like many famous siblings, DNA doesn’t do much work. It mainly stays bunkered down in the nucleus of our cells, protecting the information it encodes. RNA, on the other hand, actually goes out and gets things done. The genes encoded by our DNA are transcribed into snippets of RNA that venture out from the nucleus of our cells into the protein-manufacturing region. There, this messenger RNA (mRNA) oversees the assembly of the specified protein. In other words, instead of just sitting at home curating information, it makes real products.

Scientists including Sydney Brenner at Cambridge and James Watson at Harvard first identified and isolated mRNA molecules in 1961. But it was hard to harness them to do our bidding, because the body’s immune system often destroyed the mRNA that researchers engineered and attempted to introduce into the body. Then in 2005, a pair of researchers at the University of Pennsylvania, Katalin Kariko and Drew Weissman, showed how to tweak a synthetic mRNA molecule so it could get into human cells without being attacked by the body’s immune system.

When the COVID-19 pandemic hit a year ago, two innovative young pharmaceutical companies decided to try to harness this role played by messenger RNA: the German company BioNTech, which formed a partnership with the U.S. company Pfizer and Moderna, based in Cambridge, Mass. Their mission was to engineer messenger RNA carrying the code letters to make part of the coronavirus spike protein&mdasha string that begins CCUCGGCGGGCA … &mdashand to deploy it in human cells.

BioNTech was founded in 2008 by the husband-and-wife team of Ugur Sahin and Ozlem Tureci, who met when they were training to be doctors in Germany in the early 1990s. Both were from Turkish immigrant families, and they shared a passion for medical research, so much so that they spent part of their wedding day working in the lab. They founded BioNTech with the goal of creating therapies that stimulate the immune system to fight cancerous cells. It also soon became a leader in devising medicines that use mRNA in vaccines against viruses.

In January 2020, Sahin read an article in the medical journal Lancet about a new coronavirus in China. After discussing it with his wife over breakfast, he sent an email to the other members of the BioNTech board saying that it was wrong to believe that this virus would come and go as easily as MERS and SARS. “This time it is different,” he told them.

BioNTech launched a crash project to devise a vaccine based on RNA sequences, which Sahin was able to write within days, that would cause human cells to make versions of the coronavirus’s spike protein. Once it looked promising, Sahin called Kathrin Jansen, the head of vaccine research and development at Pfizer. The two companies had been working together since 2018 to develop flu vaccines using mRNA technology, and he asked her whether Pfizer would want to enter a similar partnership for a COVID vaccine. “I was just about to call you and propose the same thing,” Jansen replied. The deal was signed in March.

By then, a similar mRNA vaccine was being developed by Moderna, a much smaller company with only 800 employees. Its chair and co-founder, Noubar Afeyan, a Beirut-born Armenian who immigrated to the U.S., had become fascinated by mRNA in 2010, when he heard a pitch from a group of Harvard and MIT researchers. Together they formed Moderna, which initially focused on using mRNA to try to develop personalized cancer treatments, but soon began experimenting with using the technique to make vaccines against viruses.

In January 2020, Afeyan took one of his daughters to a restaurant near his office in Cambridge to celebrate her birthday. In the middle of the meal, he got an urgent text message from the CEO of his company, Stéphane Bancel, in Switzerland. So he rushed outside in the freezing temperature, forgetting to grab his coat, to call him back.

Bancel said that he wanted to launch a project to use mRNA to attempt a vaccine against the new coronavirus. At that point, Moderna had more than 20 drugs in development but none had even reached the final stage of clinical trials. Nevertheless, Afeyan instantly authorized him to start work. “Don’t worry about the board,” he said. “Just get moving.” Lacking Pfizer’s resources, Moderna had to depend on funding from the U.S. government. Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases, was supportive. “Go for it,” he declared. “Whatever it costs, don’t worry about it.”

It took Bancel and his Moderna team only two days to create the RNA sequences that would produce the spike protein, and 41 days later, it shipped the first box of vials to the National Institutes of Health to begin early trials. Afeyan keeps a picture of that box on his cell phone.

An mRNA vaccine has certain advantages over a DNA vaccine, which has to use a re-engineered virus or other delivery mechanism to make it through the membrane that protects the nucleus of a cell. The RNA does not need to get into the nucleus. It simply needs to be delivered into the more-accessible outer region of cells, the cytoplasm, which is where proteins are constructed.

The Pfizer-BioNTech and Moderna vaccines do so by encapsulating the mRNA in tiny oily capsules, known as lipid nanoparticles. Moderna had been working for 10 years to improve its nanoparticles. This gave it one advantage over Pfizer-BioNTech: its particles were more stable and did not have to be stored at extremely low temperatures.

By November, the results of the Pfizer-BioNTech and Moderna late-stage trials came back with resounding findings: both vaccines were more than 90% effective. A few weeks later, with COVID-19 once again surging throughout much of the world, they received emergency authorization from the U.S. Food and Drug Administration and became the vanguard of the biotech effort to beat back the pandemic.

The ability to code messenger RNA to do our bidding will transform medicine. As with the COVID vaccines, we can instruct mRNA to cause our cells to make antigens&mdashmolecules that stimulate our immune system&mdashthat could protect us against many viruses, bacteria, or other pathogens that cause infectious disease. In addition, mRNA could in the future be used, as BioNTech and Moderna are pioneering, to fight cancer. Harnessing a process called immunotherapy, the mRNA can be coded to produce molecules that will cause the body’s immune system to identify and kill cancer cells.

RNA can also be engineered, as Jennifer Doudna and others discovered, to target genes for editing. Using the CRISPR system adapted from bacteria, RNA can guide scissors-like enzymes to specific sequences of DNA in order to eliminate or edit a gene. This technique has already been used in trials to cure sickle cell anemia. Now it is also being used in the war against COVID. Doudna and others have created RNA-guided enzymes that can directly detect SARS-CoV-2 and eventually could be used to destroy it.

More controversially, CRISPR could be used to create “designer babies” with inheritable genetic changes. In 2018, a young Chinese doctor used CRISPR to engineer twin girls so they did not have the receptor for the virus that causes AIDS. There was an immediate outburst of awe and then shock. The doctor was denounced, and there were calls for an international moratorium on inheritable gene edits. But in the wake of the pandemic, RNA-guided genetic editing to make our species less receptive to viruses may someday begin to seem more acceptable.

Throughout human history, we have been subjected to wave after wave of viral and bacterial plagues. One of the earliest known was the Babylon flu epidemic around 1200 B.C. The plague of Athens in 429 B.C. killed close to 100,000 people, the Antonine plague in the 2nd century killed 5 million, the plague of Justinian in the 6th century killed 50 million, and the Black Death of the 14th century took almost 200 million lives, close to half of Europe’s population.

The COVID-19 pandemic that killed more than 1.8 million people in 2020 will not be the final plague. However, thanks to the new RNA technology, our defenses against most future plagues are likely to be immensely faster and more effective. As new viruses come along, or as the current coronavirus mutates, researchers can quickly recode a vaccine’s mRNA to target the new threats. “It was a bad day for viruses,” Moderna’s chair Afeyan says about the Sunday when he got the first word of his company’s clinical trial results. “There was a sudden shift in the evolutionary balance between what human technology can do and what viruses can do. We may never have a pandemic again.”

The invention of easily reprogrammable RNA vaccines was a lightning-fast triumph of human ingenuity, but it was based on decades of curiosity-driven research into one of the most fundamental aspects of life on planet earth: how genes are transcribed into RNA that tell cells what proteins to assemble. Likewise, CRISPR gene-editing technology came from understanding the way that bacteria use snippets of RNA to guide enzymes to destroy viruses. Great inventions come from understanding basic science. Nature is beautiful that way.

Isaacson, a former editor of TIME, is the author of The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race, to be published in March. After the Pfizer vaccine was approved, he opted to remain in the clinical trial and has not yet been “unblinded.”


And here it comes, the claim that COVID-19 vaccines cause cancer

I did forget one common antivaccine claim, though. It’s one that I hadn’t yet seen about the COVID-19 vaccine, even though it’s a claim that dates back decades about early versions of the polio vaccine used in the late 1950s and early 1960s, namely that vaccines cause cancer. (It’s a claim that’s morphed and metastasized to invoke “chronic inflammation”—which can contribute to cancer—from vaccines in general as a cause of cancer.) You remember that hoary old claim, don’t you, that because early versions of the polio vaccine were contaminated with a virus (SV40), those polio vaccines given to children over 60 years ago are responsible for a wave of cancer over the last two decades. I discussed in my inimitable detail how that claim was investigated and found to have no scientific basis way back in 2013. Now that claim has been repurposed for the COVID-19 vaccine. No, antivaxxers are not claiming that the COVID-19 vaccines by Moderna, Pfizer/BioNTech, or Johnson & Johnson are contaminated with SV40. Rather, they are focusing their false claim that COVID-19 vaccines will cause a wave of cancer in decades to come on the mRNA-based design of these vaccines and a cherry picked study from Memorial Sloan-Kettering Cancer Center from 2018 (to give their misinformation the patina of scientific respectability). I haven’t seen this claim widely disseminated (yet), but as a trained molecular biologist I thought I’d try to nip it in the bud before it grows too much.

Let’s start with this Tweet:

Basically, DNA replicates from a DNA template and results in a double-stranded molecule that is very stable, as it has complementary sequences that tightly bind to each other in a sequence-specific fashion. This DNA template is unwound by enzymes that use the template to make RNA strands, which are single-stranded, which is then used by a ribosome to make protein out of amino acids. Again, to put it simply, each nucleotide equals one letter of the code each three-nucleotide sequence (codon) equals one “word” that translates to an amino acid. Given that there are four nucleotides, there are 64 possible codons. Since there are only 20 amino acids, that means that most amino acids are encoded by more than one combination of nucleotides or more than one codon i.e., the genetic code is redundant. Of course, it’s more complicated than that, as this diagram shows:

After the genetic code was cracked 60 years ago, it soon became apparent that the RNAs encoding for proteins are often not fully formed right after they’re transcribed. Often RNA starts out as a longer precursor RNA (a pre-mRNA) that is spliced to the final mRNA sequence before being transported out of the nucleus into the cytoplasm to be used to drive the production of protein in the cytoplasm. In brief, the precursor RNA that is initially transcribed contains sequences known as “exons” and “introns”. In genes, exons contain the nucleotide sequences that encode actual protein, while introns contain nucleotide sequences that do not code for anything but can have important sequences that regulate gene production and activity. Here’s an illustration of the splicing process from Wikipedia:

This diagram is actually fairly simple, with two exons and one intron. Some genes have many exons and introns, requiring multiple splices, as in this diagram:

Did I forget to mention that mRNAs are also processed to have a “cap” at one end and a stretch of As (a poly-A tail) at the other end? The poly-A tail is very important in regulating mRNA stability and therefore its half-life in the cytoplasm. In any event, as with any biological process, things can go wrong with these splicing events. Splice site mutations, for instance, can result in mis-spliced mRNAs and proteins lacking exons:

I could go on and on and on. There are normal genes can produce more than one protein through alternative splicing:

Sometimes when splicing goes awry, it can result in a truncated protein lacking one end. For instance, if an intron is left attached to two exons, chances are that the ribosome (the enzyme complex that translates mRNA into protein) will hit a “stop” codon (three nucleotide code that tells transcription to stop) long before it reaches the other end of the intron, at which point transcription will just stop. All of this is not even counting the other molecular modifications that the RNA can undergo on its journey from transcription to pre-mRNA through splicing to the final “mature” mRNA.

Unsurprisingly, if these sorts of errors occur in genes important to processes regulating cell growth and invasion, cancer can result, either from a mis-splicing removing a regulatory region in the protein that keeps it in check or by producing a protein that doesn’t function as it should. Examples of cancers that are caused or accelerated by a splice site mutation are accumulating. It is this latter possibility, a truncated protein that doesn’t function, that the paper being misapplied by Wells examines. The proteins compromised by the truncation are tumor suppressor proteins, whose function is to shut down growth or other processes that can result in cancer when they are overly active.

So what did the paper show? What was interesting about the paper is that it showed the existence of splicing errors in tumor suppressor genes in a specific cancer, chronic lymphocytic leukemia, without mutations in splice sites to explain how these proteins became truncated due to splicing errors, or, as the authors put it in the manuscript:

We discovered widespread upregulation of truncated mRNAs and proteins in primary CLL cells that were not generated by genetic alterations but instead occurred by intronic polyadenylation

The truncated proteins generated by intronic polyadenylation often lack the tumour-suppressive functions of the corresponding full-length proteins (such as DICER and FOXN3), and several even acted in an oncogenic manner (such as CARD11, MGA and CHST11). In CLL, the inactivation of tumour-suppressor genes by aberrant mRNA processing is substantially more prevalent than the functional loss of such genes through genetic events.

So what does this all mean? First, to reiterate and simplify, the authors detected truncated mRNAs and proteins for a number of tumor suppressor genes in CLL that could not be explained by DNA mutations in the genes themselves, such as splice site mutations. They did a lot of other controls, such as making sure that the explanation for the truncated proteins was not cleavage by proteases, enzymes that cut proteins at specific amino acid sequences. After ruling out other possibilities, the authors demonstrated that these mRNAs and proteins were truncated because of a process called intronic polyadenylation. But what is that?

Polyadenylation is the process of adding a bunch of adenosines (As) to the 3′ end of an RNA molecule. It’s how the poly-A tail is added to the end of an mRNA, but it turns out that it’s a common process for polyadenylation to occur in introns. This process is very widespread and was appreciated well over a decade ago. It’s involved in diversifying the products of immune cell mRNAs, the process explained thusly:

In the splicing literature, isoforms generated through recognition of an IpA signal are often described as ‘alternative last exon’ events. Genes that generate IpA isoforms are thought to harbor competing splicing and polyadenylation signals, producing a full-length messenger RNA (mRNA) when splicing outcompetes polyadenylation and otherwise producing a truncated mRNA. As the defining event is the recognition of an IpA signal, we call these transcripts IpA isoforms. It is now possible to recognize the widespread expression of IpA isoforms through the analysis of 3ʹ-end sequencing data.

Or, to put it more simply, whether there is a truncated protein or a full-length protein depends on the balance of splicing to poly-adenylation at the site in the intron. If there’s more splicing activity, you get much more of the whole protein. If there’s more polyadenylation, you get much more of the truncated protein. What Mayr’s lab found was that too much polyadenylation can result in truncated tumor suppressor proteins in CLL, contributing to the development of the cancer, which is why, according to the MSKCC press release which explains the findings pretty well:

These findings help explain a long-standing conundrum, which is that CLL cells have relatively few known DNA mutations. Some CLL cells lack even known mutations. In effect, the mRNA changes that Dr. Mayr’s team discovered could account for the missing DNA mutations.

Because CLL is such a slow-growing cancer and people with CLL often live for many years, it’s too early to say whether these mRNA changes are associated with a poorer prognosis.

There are some important differences between the mRNA changes and a bona fide DNA mutation. Most important, the inactivation of tumor suppressors through mRNA is usually only partial only about half of the relevant protein molecules in the tumor cells are truncated. But in many cases this is enough to completely override the function of the normal versions that are present. And because this truncation could apply to 100 different genes at once, the changes can add up.

So why does none of this have anything to do with mRNA-based vaccines causing cancer? I’m glad you asked and hope you don’t mind that I took this opportunity to geek out a bit, in a molecular biology sense. The biology being abused by Wells is actually quite complex and fascinating, and I don’t get to discuss pure molecular biology very often any more. I hope I didn’t lose too many readers with the explanation, but I also bet more than a few of you have already figured out why what Wells is peddling is utter nonsense. If not, here we go.


An mRNA vaccine for influenza

An influenza vaccine made of stabilized mRNA may allow vaccine design and manufacture to keep pace with viral evolution.

In the world of infectious disease, influenza remains a continuing scourge. The genetic mutability of the virus ensures an endless game of immunological cat and mouse, requiring a complex monitoring system to chase viral changes. One way of countering the plasticity of influenza virus would be to increase the plasticity of vaccine design and production. In this issue, Petsch et al. 1 suggest that this could be achieved using mRNA vaccines, which can be rapidly modified and manufactured. The first application of mRNA vaccines to an infectious disease, the study demonstrates protection against influenza in several animal models, paving the way for future clinical evaluation.


Watch the video: How Vaccines Are Made and Manufactured. mRNA-Based Platform (August 2022).