Let's Discuss the "Chimeric Omicron Gain-of-Function" Study

And provide some much-needed context outside of the fear porn being pushed.

A few people have reported on this recent study¹ in which researchers created a chimeric Omicron by taking the spike gene from Omicron and inserting into the Wildtype (i.e. a D614G variant) variant, raising alarms as to this gain-of-function research being conducted and the possible havoc from such research.

With most people citing the abstract and focusing on the portion in bold below, which alone would appear rather concerning:

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

On the surface (not a spike protein joke!) this would seem concerning. A vaccine-escaping variant that killed 80% of the mice it infected? That sounds alarming!

Or it would sound alarming if not for the lack of any context. Suffice it to say, but there’s a lot more to this study.

So rather than conduct an abstract-only read of this study I’ll point out a few issues with this study, and one that appears to have been overlooked by many people who took a quick cursory glance.

Wildtype did better than either Omicron or Omi-S

So it is true that the researchers essentially stitched together a chimeric Omicron. They took the S gene from Omicron, swapped it into the Wildtype (labeled WT in the study) form of the virus, and then saw how it would do in both in vitro assays as well as in transgenic mice.

This new chimeric Omicron was denoted as Omi-S in this study. The diagram below from Fig. 2 illustrates this newfangled chimeric variant:

Everything remained WT aside from the S gene which was taken from Omicron, thus creating the new chimeric Omi-S.

Important to all studies, and something Brian Mowrey of Unglossed mentions very frequently, is that all studies are relative, in that they must be compared to either some control or some other standards (the all-important “compared to what?”)

The abstract alone doesn’t tell you much about this study, aside from the fact that this new Omi-S appears far more dangerous than Omicron.

That’s very concerning, but did you know that the researchers also tested Wildtype SARS-COV2?

The abstract never mentions this fact, and it completely obfuscates the important “compared to what?” issue, in that without mentioning the Wildtype results one may believe that this Omi-S variant is far more pathogenic and virulent than anything else we’ve ever seen.

The researchers own results suggest this to not be the case.

I've bolded the important numbers below based on the results of in vitro assays, but note which form of the virus it is referring to²:

The ancestral virus [hereinafter referred to as wild-type (WT)] and Omi-S spread fast in ACE2/TMPRSS2/Caco-2 cells, yielding 89% and 80% infected cells, respectively, at 24 hours post-infection (hpi) (Fig. 2b). In contrast, Omicron replicated slower, leading to 48% infected cells at 24 hpi. A similar pattern was seen in Vero E6 cells, where 60% and 41% of cells were positive for WT and Omi-S, respectively, at 48 hpi, in contrast to 10% positive cells for Omicron (Fig. 2c). The plaque assay showed that although both Omi-S and Omicron produced lower levels of infectious virus particles compared with WT, the viral titer of Omi-S was significantly higher than that of Omicron. In ACE2/TMPRSS2/Caco-2 cells, Omi-S produced 5.1-fold (p = 0.0006) and 5.5-fold (p = 0.0312) more infectious particles than Omicron at 12 hpi and 24 hpi, respectively (Fig. 2d). […] The higher infection efficiency of Omi-S relative to Omicron was also reflected in the plaque size; while WT produced the largest plaques (~ 4.1 mm), the size of Omi-S plaques (~2.2 mm) was 2- fold (p < 0.0001) larger than that of Omicron plaques (~1.1 mm) (Fig. 2g).

Given these in vitro results which included the WT form of SARS-COV2, it’s pretty clear that this new Omi-S fared better than Omi at infection but didn’t do better than WT.

All of these results show, at least as it relates to these assays, that the order of infectivity is as follows:

WT > Omi-S > Omicron

And this is reflected in the graphs shown below which look at the two difference cell lines (Caco-2³ & Vero E6⁴). Luckily for us, the researchers actually included the Omi-S results in the WT and Omi graphs in purple, which allows for easier comparison.

As you can see, WT had greater infectivity and viral replication as compared to Omi-S in these two cell lines:

This is also seen in the cell viability assay⁵ conducted with Caco-2 cell lines shown below, which shows that a marked decline in WT compared to either Omi-S and Omi.

Of course, the researchers don’t appear to show any statistical data for the comparison of WT here, but this is what their results show which we could infer shows statistically significant differences in the WT group when compared to either Omi-S or Omicron:

Lastly, the researchers challenged transgenic mice with each of the variants and examined their lung tissue for virus titer at 2 and 4 days post infection (dpi).

Similar to the in vitro assays, the results of the mice study suggest that WT-infected mice had higher viral titers than both Omicron and Omi-S⁶:

It’s important to remember that these results only pertain to the given assays— other studies may show different results (refer to Footnotes 2-5).

However, taken altogether the researchers’ own results suggest that WT is more infectious than either Omicron or Omi-S.

This is something you wouldn’t glean from reading just the abstract, which itself hardly provides any significant information.

And yet here it’s pretty clear that more is happening, such that the WT virus appeared to fare better than either Omicron or the new chimeric variant.

So why is it that coverage of this paper hasn’t mentioned this fact; that a virus we were exposed to at the early portion of the pandemic, based on the own researchers’ results, fared better than the construct they designed for this study?

Why the sudden histrionics that they are trying to make a more deadly virus when the virus that was created wasn’t better than the one that caused the start of the global pandemic?

This is an issue of “abstract-only” reading, such that all of the concerns about this paper were derived solely from the abstract.

Digging a little deeper we can see that a lot more is going on in, and in fact the evidence here, which should not be viewed as conclusive, would at least contradict some of the reports I have seen going around.

In Regards to the “80% Lethality”…

I’ve also seen that 80% lethality repeated several times, as if to say that the release of this chimeric Omicron will lead to the death of nearly everyone on Earth (aside from the lucky few, I suppose).

Again, this number lacks any context— what does 80% lethality mean here?

In one experiment the researchers challenged 26 mice with each of the 3 viruses:

• 6 were given WT

• 10 were given Omicron, and

• 10 were given Omi-S.

The researchers evaluated the mice using clinical scores (higher scores mean more symptoms and more severe infection) and body weight.

What’s interesting with this study is that it researchers put hard cutoffs for when to ethically euthanize the mice, such that mice that either showed 2 consecutive days of 80% body weight relative to the starting weight, or clinical scores above 4 for two consecutive days would be ethically killed:

Animals were considered moribund and humanly euthanized in case of weight loss greater than or equal to 20%, or if they received a clinical score of 4 or greater for two consecutive days.

I haven’t seen this in other studies before, but that may be me overlooking some of the methods for animal studies.

The researchers do mention that mice who died of infection were marked as a “day of” death, while mice who were humanely killed were marked as “day after” death i.e. if a mouse died on day 5 the death would be measured on day 5 while a mouse who was ethically killed on day 7 would be labeled as a death on day 8:

For the purpose of survival curves, animals euthanized on a given day were counted dead the day after. Animals found dead in cage were counted dead on the same day.

That is to say that the actual “death” here is rather muddied, in that it’s hard to tell which mice died from the virus or which ones were humanely euthanized given their clinical scores and weight (emphasis mine):

Since SARS-CoV-2 causes fatal infection in K18- hACE2 mice3,40,41, we leveraged this situation to compare the animal survival after viral infection. In agreement with the results of body-weight loss and clinical score, WT and Omi-S caused mortality rates of 100% (6/6) and 80% (8/10), respectively. In contrast, all animals infected with Omicron survived (Fig. 3c).

By all accounts the wording of the results infer that both the WT and Omi-S mice were humanely euthanized rather than died by their respective infections:

[…] inoculation with WT virus triggered a rapid decrease in body weight with all animals losing over 20% of their initial body weight by 8 days post-infection (dpi) (Fig. 3a). Importantly, 80% of animals infected with Omi-S also lost over 20% of their body weight by 9 dpi (Fig. 3a and Extended Data Fig. 2a).

But again, it’s hard to tell to what extent mice succumbed to the virus or met an artificial endpoint.

The figure for these results can be seen below for those who prefer a visual representation:

Survival rates of mice provided the various viruses listed above. Note that the actual cause of death in these cases are muddied by the researchers euthanizing the mice.

It’s also worth noting that none of the Omicron mice showed a reduction in body weight or an increase in clinical scores (Fig. 3A and 3B, respectively).

Taken altogether, I’d argue that this “80% lethality” number being touted around suffers from several problems. There’s definitive way of knowing which mice died from the virus or from being euthanized, and in the case of being euthanized we may not know how many mice would have recovered.

But let’s take this results for what they are and extrapolate from there.

Once again, given the results here, WT SARS-COV2 appears to be deadlier than Omi-S given that 100% of the mice challenged with WT died.

Of course, this assumption suffers from the huge fact that the researchers only included 6 mice in the WT group compared to the 10 in the Omi-S group— nearly half, meaning that it’s easy to game the percentages between these two groups (a 17% decline in survival among WT vs 10% decline in Omi-S).

But if we take this “100% lethality” value from WT could we extrapolate that to a real world setting?

Well, we already lived through the emergence and spread of WT, and clearly we know that not everyone who was infected with WT died (I’m pretty sure CNN would have alerted us to that fact).

Therefore we absolutely know that these “% lethality” may hardly be comparable to the actual lethality or case-fatality rate if exposed to the general public.

Let’s also remember that people have also died from Omicron, although the numbers appear far better than prior variants.

In essence, this “80% lethality” can hardly tell us anything, and the fact that several people have reported on this high lethality rate is rather concerning.

Many people are already on edge, and such reports do nothing more than to stoke fear and increase anxiety.

It’s clear here the context tells us that far more is happening, and more importantly that it’s rather dangerous to associate these numbers to any real-life setting, especially given the fact that we have two forms of the virus (WT and Omicron) that we have dealt with already which contradict the results of these mice studies.

What does this study tell us?

I’ll avoid the neutralization part of this study, but again I’ll remind readers that people who are talking about this new Omi-S evading vaccine immunity is not reporting anything new.

Of course a virus given a distant spike (Omicron’s spike) would be able to evade immunity from prior variants. This is nothing new, and something we have known since Omicron emerged, and yet it appears to be reported as if this is some new revelation.

It’s strange how quickly some people’s prior knowledge can be completely overridden by so-called “new evidence”, even when the new evidence actually fits what we’ve known before and doesn’t change any of our prior assumptions.

This is a consequence of reporting that may obfuscate any of the necessary context and information and presents a parochial view of a study.

I will, however, criticize the researcher’s conclusion about their neutralization assay (emphasis mine):

Notably, Omi-S exhibited identical ND50 values to Omicron (11.5-fold 168 lower than that of WA1; p < 0.0001) (Fig. 4a,b), suggesting that the Omicron S protein, when incorporated into a WT virus, behaves the same way as in Omicron.

There’s no way of extrapolating the behavior of the spike from neutralization assays alone. All this assay is doing is measuring whether things that stuck to a different spike continue to stick to this distant spike, but in no ways can it tell how the spike is “behaving”.

Be careful with this sort of wording because it’s easy to take this and run away with this phrase.

And that brings us to the real question here: what the heck was the point of this study?

Among all the noise about gain-of-function and other fears, the actual intent of this study was to see if something more than the spike was responsible for the infectivity/pathogenicity of SARS-COV2.

By stitching together different parts of viruses, the researchers attempted to see if infectivity of cell lines or infectivity of mice would have changed.

If spike was the sole player in a virus’ ability to infect and kill off cells, the transplantation of the S gene into the WT virus would, hypothetically, provide the same results as Omicron given that they would share the same spike.

However, the results here would suggest something else is at play.

Unfortunately, the researchers never elucidated what exactly would be responsible for these differences: the study was only designed to suggest that more than spike can dictate how a virus infects cells, but it never explains what proteins or new pathways would be responsible.

To provide some context to this assumption, one recent review from Hossain, et al.⁷ shows that Omicron has many mutations in non-spike proteins, as outlined in the chart below (make note that a single * next to a mutation suggests that this mutation is found in Omicron only):

From Hossain, et al. Various mutations in non-spike proteins, with specific emphasis on Omicron mutations.

The authors provide this insight into their role of these non-spike proteins:

The non-spike proteins have multiple significant roles in immune regulation, transcriptional regulation, and viral pathogenesis. But, there reside gaps in knowledge regarding their specific mode of action and roles, especially in terms of unique mutations of Omicron. However, there have been some reports on the effect of non-spike protein mutations on viral fitness and disease severity in both experimental and clinical settings [87,100–102].

More important to this chimeric Omicron study is the role of epistasis, in which mutations in either the same or different viral proteins provide some additional features or synergistic behaviors to the virus. Essentially, mutations in non-spike proteins, when paired with spike protein mutations, may provide something new to the virus.

A few examples can be seen in the excerpt below:

The Delta R203 M mutation in N protein has a statistically significant synergistic effect with L452R Spike mutation, and the growth rate of the 203K(N)/484K(S)/501Y(S) is consistently higher than the 203R(N)/484K(S)/501Y(S) variants. Supported by clinical data, the Spike D614G + NSP12 L323P may have increased the replication rate [104]. More importantly, this association of non-spike mutations found in other variants thus opens windows for more similar studies for the Omicron variant. The presence of unique mutations such as in NSP6, in M protein, and in ORFs, which sets the two subvariants apart, may also contribute to these impacts through a chain of epistatic effects [ [105]s]. Notably, there are however robust literatures on the plausible epistasis of non-spike mutations based on computational studies [88,106–108].

Strangely, the researchers of this chimeric Omicron study may have evidence of epistasis, as the results of their mouse study in which they assessed the bronchioles of mice infected with these viruses show some form of tropism of Omi-S for bronchiole epithelium, although these results should be taken with a good deal of caveats (note the typo as it should state Days not hours):

From the figure caption: “The percentage of S-positive bronchioles in the lungs of infected mice. Each dot represents 665 an infected animal.” Note here that this may have been a typo, in that it should have stated Days rather than hours.

We may infer that Omi-S may have some tropism for bronchioles based on the combination of Omicron S and WT non-spike proteins, but once again there’s no way of assessing what mechanisms are occurring here, or if the results of this current study provide circumstantial evidence.

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In any case, the actual role of this Omi-S seems to have led to some unnecessary histrionics.

Is this Omi-S a stronger Omicron, or is it a weaker WT? If the purpose was to kill people off why not just take a crack at releasing WT and see how that goes again given that these results suggest WT would be the more dangerous variant?⁸

The main issue here is that we aren’t living in a non-SARS-COV2 world where such research would raise serious concerns.

The fact that scientists may have attempted to construct a virus that may (or may not) appear some time in the future in order to create therapeutics or vaccines which may (or may not) possibly deal with the virus raises serious concerns.

Such gain-of-function research which may construct new viruses is egregiously unethical.

But let’s also be realistic in stating that this study isn’t occurring in a vacuum- it’s occurring in a post-COVID world. The fact that the researchers tried to stitch together a virus from parts we have already seen, and may have made a mediocre middle-ground virus because of it doesn’t raise the same arguments of novelty here as it would for a virus we have never experienced.

This is a reminder that Team Skeptic should still remain level-headed rather than succumbing to fear porn, especially over things that they may not have properly assessed. Proper concerns should be raised, but proper readings of studies are also critical. The fact that several people have reported on this study solely on the abstract continues to show that there’s an incentive to rush to report rather than spending time to read these studies.

Let’s not lose our heads over things we didn’t take time to make sense of. We already know this is happening far-too often already.

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1. Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron

Da-Yuan Chen, Devin Kenney, Chue-Vin Chin, Alexander H Tavares, Nazimuddin Khan, Hasahn L Conway, GuanQun Liu, Manish C Choudhary, Hans P Gertje, Aoife K OConnell, Darrell N Kotton, Alexandra Herrmann, Armin Ensser, John H Connor, Markus Bosmann, Jonathan Z Li, Michaela U Gack, Susan C Baker, Robert N Kirchdoerfer, Yachana Kataria, Nicholas A Crossland, Florian Douam, Mohsan Saeed

bioRxiv 2022.10.13.512134; doi: https://doi.org/10.1101/2022.10.13.512134

2. Measurements for N-positive were based on anti-N antibodies and the use of flow cytometry.

3. Caco-2 cell lines are cancerous cells of the intestinal epithelium. Here they were made to express ACE2 and TMPRSS2. Keep this in mind when looking at such assays, as the cell lines used should at least closely resemble the cells that would be targeted by the virus. Assays with Caco-2 may not properly reflect the actual cells in humans, and the expression of ACE2 and TMPRSS2 can bias results. Remember that Omicron is believed to not use the TMPRSS2 as a method of entryway into cells.

4. Vero E6 cell lines are taken from the kidneys of an African Green Monkey. Similar to Caco-2 cell lines keep in mind that both ACE2 and TMPRSS2 expression in these cell lines may differ compared to other cell lines, meaning that these results may not be reflective of the actual path and rate of infection in human lung epithelial cells or an actual infection.

5. Note that cell viability assays measure how many cells are so-called “alive” after introducing the virus. Therefore, lower cell viability is assumed to be associated with cell death of some capacity.

6. The researchers here consider the difference in virus titers between WT and Omi-S to be “not statistically significant”, as denoted by the ns in the graph. However, this may be due to one mouse which appears to show very low virus titer at 2-dpi within the WT group, and I would bet that this outlier is influencing the average for the WT 2-dpi group.

7. Hossain, A., Akter, S., Rashid, A. A., Khair, S., & Alam, A. (2022). Unique mutations in SARS-CoV-2 Omicron subvariants' non-spike proteins: Potential impacts on viral pathogenesis and host immune evasion. Microbial pathogenesis, 170, 105699. https://doi.org/10.1016/j.micpath.2022.105699

8. This doesn’t take into account the fact that this may have already occurred with WT, so I’m being a little sarcastic here.