Phages use replication slippage to stock variants, betting bacterial defenses will escalate
Slippage is not a replication fault but a phage hedge: in any population a minority of individuals shift reading frame at repeat sequences, generating variants that selection has not picked yet. Most of the population fits the current environment; the moment bacterial defenses escalate, the low-frequency backups take over.
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What replication slippage leaves behind is standing variation
The phage does not simply wait for random mutation. Its genome contains ‘simple sequence repeats’, and during DNA replication the polymerase occasionally slips, adding or deleting one or two bases, changing the reading frame and switching a particular protein between on and off. Unlike frameshifting at the translation stage, this is a permanent change at the DNA level, and it is inherited by the progeny. Jolene's own words: getting the polymerase to slip here or there is far easier and far faster than acquiring a new gene or repairing a broken one.
— Jolene RamseyLow-level variation is a bet placed on the future
‘Bet-hedging’ means this: most of the sequences fit the current environment, but a few low-frequency variants may be exactly right for the next one. In the arms race between bacteria and phages, rather than waiting for a random mutation to be selected, it is better to let slippage keep producing low-frequency switching. Jolene compresses the logic into a single line — you always have a batch of low-level variation on hand, and it may be better suited to the next environment.
— Jolene RamseyAdenine repeat length decides which defense the phage switches to
The AGT gene is itself a trade-off between two risks: the alpha-glucosyltransferase it encodes modifies phage DNA, which lets the phage evade the bacterium's type IV restriction-modification system, but the modified DNA is in turn easily recognized by certain other defense mechanisms. A stretch of adenine repeats inside the gene comes in three lengths, 6, 7 and 8; a different length means a different reading frame, so the protein's function is either on or off. One slip lets the phage change gears quickly between the two classes of defense, with no need to maintain two separate genes.
— Jolene RamseyReversible reversion runs about ten thousand times more often than point mutation
T4's R2A gene was chosen to test this: it carries a run of six adenines, and the phenotype can be tracked — on hosts expressing the Rx defense system, once R2 function is lost, no plaques grow. The experiments show that the reversion frequency in this SSR region is about ten thousand times higher than that of ordinary point mutation. Put differently, the phage can switch the gene off by slippage to escape the defense it currently faces, and use the same mechanism to switch it back on quickly.
— Jolene RamseyThe best-matched antibody has no in vitro shortcut
The study wanted human monoclonal antibodies able to neutralize EBV, and rather than relying on an in vitro library such as phage display, it went back to hybridomas: fuse B cells from immunized mice with myeloma cells, select in HAT medium, and screen for the target in 384-well plates. The reason is that the somatic hypermutation and junctional diversity that occur as B cells mature add or delete bases in CDR3, which is hard to imitate in vitro. To get a perfectly matched antibody, first let the B cells run the complete germinal center reaction inside the mouse, then take the heavy- and light-chain genes back into cultured cells and re-express them.
GP42 antibodies neutralize only B cell infection
All eight antibodies directed at GP42 came from two clonal lineages, and the neutralization mechanism is to stop EBV's GP42 protein from binding HLA class II; it therefore blocks only B cell infection and does nothing against epithelial cells, because epithelial entry runs through a different entry mechanism. The strongest of the eight is ATX42-2. In the humanized mouse experiments that followed — 500 micrograms injected intraperitoneally, 11 weeks of observation — ATX42-2 prevented tumor formation, viral DNA and splenomegaly.
Vaccine design is beginning to fix on the critical epitope
GP350 subunit with an alum adjuvant did not induce protective immunity, and GP42, because its conformation varies, is hard to use to raise the correct antibodies through conventional immunization. This paper's contribution is to define the critical epitope and the structural target on GP42 as it binds HLA class II, so that vaccine design can be reverse-engineered. Since then a GP350 ferritin nanoparticle vaccine has been shown to be safe and immunogenic in phase I, with seronegative participants followed to 540 days without infection; Moderna is at the same time working on an mRNA vaccine containing gHgL, GP42 and GP220.
Anti-science may be the brain economizing on energy
Vincent uses Kahneman's ‘Thinking, Fast and Slow’ to explain anti-science: the brain defaults to type one, which costs little energy, while checking something in depth is type two, which requires extra energy and a start-up cost. The line from Joshua Reynolds on his wall — there is no expedient to which a man will not resort to avoid the real labor of thinking — has become the footnote to this observation. Someone with no background in statistics hears ‘Tylenol causes autism’ and a lazy type one simply accepts it, instead of going to the literature. He stresses that this is only speculation, not a causal conclusion.
— VincentIn their own words · checked verbatim
So that you have a low-level variation always there that could be the better fit for the next environment.
Jolene Ramsey14:36
And just slipping the DNA polymerase here and there is way easier and faster than having to acquire a new gene or fix a gene that is broken.
Jolene Ramsey32:35
It's the fact that you're getting a full sort of diversification process in the mouse.
There is no expedient to which a man will not resort to avoid the real labor of thinking.
Vincent1:42:39
That's lazy type one thinking instead of going to the literature and reading it yourself.
Vincent1:46:40
Figures
| Adenine repeat lengths in the AGT gene | 6, 7, 8 | 18:48 |
| SSR reversion frequency versus point mutation | 10,000 times | 20:51 |
| SSRs in phage T7 | 0 | 26:01 |
| GP350-binding antibodies | 2 | 54:55 |
| GP42 neutralizing antibodies | 8 | 54:55 |
| Antibody injection dose | 500 micrograms | 1:04:26 |
| Weeks of observation | 11 weeks | 1:04:26 |
Glossary
- simple sequence repeats (SSR)
- Tandem repeats of a short motif in a genome; polymerase slippage during replication changes the repeat number and switches a gene's function on or off.
- bet-hedging
- Keeping in a population, over the long run, a minority of variants that do not fit the present environment but may fit a future one, as insurance against unpredictable change.
- hybridoma
- An immortalized cell line formed by fusing an immunized B cell with a myeloma cell; it secretes a single antibody continuously.
- HAT medium
- A selective medium containing hypoxanthine, aminopterin and thymidine, used to screen for successfully fused hybridoma cells.
- humanized mice
- Mice engrafted with human hematopoietic stem cells or immune cells, used to partially reconstruct human immune processes in vivo.
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