Phage Hedges Its Bets With 10,000-Fold Reversion, and the First Humanized EBV Antibody
Phages hedge their bets through 10,000-fold reversion in SSRs, humanized EBV antibodies block tumors in vivo, and vaccine efforts advance on multiple fronts.
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Phages deliberately let some offspring break
Jolene presents a Nature Microbiology paper: homopolymeric nucleotide repeats (SSRs) in phage genes are prone to slippage during replication, causing insertions or deletions that shift the reading frame. This means a fraction of offspring always fail to make protein properly. That randomness is the basis of bet-hedging: most sequences are adapted to the current environment, but the low-level variants that persist could become the survivors if the environment shifts abruptly. The population trades a tiny cost for long-term survival capacity.
— Jolene RamseyNo need to pre-adapt: reversion is 10,000 times faster
The paper measured the reversion frequency in SSR regions and found it roughly 10,000 times higher than ordinary point mutations. That order of magnitude means phages can quickly turn a gene's function on or off within very few generations, rather than waiting for a rare point mutation. Jolene stresses that this mechanism lets the population avoid having to pre-adapt to environmental change; instead it continuously generates diversity and waits to be selected by the environment.
— Jolene RamseyT7 has not a single SSR, so betting is not standard issue
In the Basel phage collection, the T7 genome has no SSRs. Rich comments that T7 is "businesslike", straightforward, without these fancy mechanisms. This highlights that SSRs are not standard equipment for all phages, but an evolutionary strategy of certain lineages in particular niches. Even virulent phages under the same host can have completely different genome organization.
— Rich ConditThe hard part of humanization is not the variable region, it is the constant region
When converting a mouse antibody into a humanized antibody, there is a conflict between the variable and constant regions, and someone has to spend years adjusting the constant region to be compatible with the variable region before a therapeutic antibody can be made. Brienne says this was her epiphany moment; she originally did not understand why mice had to be used. Vincent adds that going directly to human antibodies may fail to find the antibody you want, whereas the advantage of the mouse platform is that you can screen large numbers of clones and engineer antigens, obtaining the full B cell clonal diversification process, after which classic hybridoma technology is still needed to obtain monoclonal antibodies.
— Brienne, VincentAfter hybridoma screening, only 10 antibodies remain
The research group immunized mice with recombinant GP350 and GP42, made hybridomas, plated 384-well plates, and used antigen-conjugated magnetic beads for high-throughput flow screening, testing binding and infection inhibition. Positive clones were subcloned and had heavy/light chain genes sequenced, then recombinant human IgG1 antibodies were expressed. In the end they obtained 2 GP350-binding antibodies and 8 GP42-neutralizing antibodies. The whole pipeline demonstrates the complete path from animal immunization to recombinant antibodies.
— VincentThe antibodies also blocked EBV challenge in vivo
Human CD34+ hematopoietic stem cells were engrafted into two kinds of immunodeficient mice, 500 micrograms of antibody (ATX42-2, ATX350-2, MO1 and others) was injected intraperitoneally, then EBV was challenged intravenously and the mice were observed for 11 weeks. The results showed these antibodies prevented tumor formation, suppressed viral DNA replication and blocked splenomegaly. In mice treated with ATX42-2 and MO1, no EBER-positive cells were detectable in the spleen. This means human antibodies protect against EBV challenge in vivo, not just neutralize in vitro.
— VincentAlum-adjuvanted subunit is dead, nanoparticle vaccines are still alive
The GP350 subunit vaccine with alum adjuvant failed to produce protective immunity in trials; GP42 is conformationally variable, making it hard to induce the right antibodies. But a GP350 ferritin nanoparticle vaccine has shown safety and immunogenicity in phase 1, with seropositive participants remaining uninfected with EBV out to 540 days. There are also GH/GP42 ferritin nanoparticles and Moderna's mRNA vaccine (containing four antigens) in development. Vincent expresses disappointment that the paper did not try combining GP350 and GP42 antibodies.
— VincentAnti-vaccine may not lack information, it lacks the will to think
Vincent revisits Thinking, Fast and Slow and quotes Joshua Reynolds: "There is no expedient to which a man will not resort to avoid the real labor of thinking." He suggests cognitive economy may partly explain anti-science and anti-vaccine attitudes: deep thinking is energy-expensive, and people lean toward the effortless Type 1 thinking. He immediately follows with "that is laziness, cognitive laziness." This view attributes the predicament of science communication to humans' default cognitive mode, rather than to a simple lack of information.
— VincentIn their own words · checked verbatim
the frequencies were quite different. I think they said it was 10,000fold different reversion frequency.
Jolene Ramsey21:19
Oh, and what I was disappointed that they didn't try the combination of the two, 350 and 42.
Vincent1:08:08
it's the first time that we've got these kinds of antibodies, human antibodies, right?
Vincent1:12:13
There is no expedient to which a man will not resort to avoid the real labor of thinking.
Vincent1:43:45
Figures
| Difference in reversion frequency | 10,000-fold | 21:19 |
| New EBV cancer cases per year | 358,000 | 42:36 |
| EBV deaths per year | 209,000 | 42:36 |
| Number of GP350-binding antibodies | 2 | 55:47 |
| Number of GP42-neutralizing antibodies | 8 | 55:47 |
| Antibody injection dose | 500 micrograms | 1:05:01 |
| Observation period | 11 weeks | 1:05:01 |
| Cassini launch year | 1997 | 1:38:41 |
| Time for Cassini to reach Saturn | 7 years | 1:38:41 |
| Cassini crash year | 2017 | 1:38:41 |
| Number of Saturn's moons | 260 | 1:39:41 |
Glossary
- SSR / simple sequence repeat
- A repeated nucleotide sequence in a gene that is prone to slippage during replication, causing insertions/deletions.
- bet-hedging
- An evolutionary strategy in which a population maintains multiple phenotypes simultaneously to cope with environmental change.
- contingency locus
- A genomic region that generates variation within a population through a high mutation rate.
- hybridoma
- An immortalized cell line produced by fusing myeloma cells with B cells, used to produce monoclonal antibodies.
How to listen
Researchers working on phages or antibodies, investors watching EBV vaccine progress, and engineers who want to understand the predicament of science communication.
The phage details in the first half hour are fairly basic; for antibody development alone, start at 50:44.