29 August 2021 Ep 167 Damed Delta Part 2 Molecular virology and evolution

Dear colleagues,

Over the last month, there is an increasing number of articles in the lay press and in the preprint scientific literature, pointing to the emergence of more evolved and potentially “dangerous” delta variants, such as “delta plus”, “delta D” and “AY.3”, which in several parts of the world, including US and Australia are beginning to “take over” the delta epidemic. 

Combined with the observations on breakthrough infections and the apparent lowering efficacy of vaccines, it is tempting to speculate that the SARS-CoV-2 virus is such a rapidly moving target that soon may escape vaccination to such an extent that it gets useless.  

To me, this is a tricky part, because the definitions of the delta subvariants seems to be a moving target too…. I just try to summarize some recent papers, hoping that you may be able to make sense of it….  

According to EDCD website on 26 Aug, these are the essential mutations in Spike protein

Alpha:                             N501Y, D614G and P681H

Betha:   K417N, E484K, N501Y, D614G and A701V

Gamma:K417T, E484K, N501Y, D614G and H655Y

Delta:    L452R, T478K,               D614G and P681R

According to CDC  on 29 Aug : https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html there are a few more in Spike: T19R, (V70F*), T95I, G142D, E156 del, F157del,   R158G, (A222V*), (W258L*), (K417N*), L452R, T478K, D614G, P681R, D950N

According to Pango on 29 Aug  https://cov-lineages.org/global_report_B.1.617.2.html

• Spike: T19R; L452R; T478K; P681R; D950N
• ORF3a: S26L
• Membrane: I82T
• ORF7a = immune-modulatory protein): V82A ; T120I
• Nucleoprotein D63G; R203M; D377Y

Ep 167-1: Baral in BBRC showing that L452R/ T478K mutations have a role in increased receptor binding and escape from neutralizing antibodies.

Ep 167-2: According to several papers, summarized here, P681R is “the mutation that helps delta spread like wildfire”: it is facilitating the S1-S2 split by the host enzyme furin, thus enhancing fusion. Nevertheless, “context” is important, since the same mutation is present in the kappa VOC and a similar one (P681H) in the alpha VOC, while those are less “fusogenic” than delta.

Ep 167-3: Adi Stern on 7 Aug in medRxiv shows that among Delta, the D variant is dominant worldwide. Delta D is characterized by an excess of non-synonymous mutations outside S, mostly occurring in ORF1a/b (=polymerase), and also T140I in ORF7b (= immune modulatory), and G215C in Nucleocapsid.

Ep 167-4: Case report of Linsemeyer in medRxiv 10 Au on “cryptic transmission” of delta AY.3 variant with high viral load in hospital setting.  The index patient (asymptomatic and vaccinated with Moderna) identified by routine just before discharge infected the following contacts:

• 6/38 patients (5 vaccinated, but no masking)
• 1/6 visitors (all vaccinated)
• 1/168 staff (vaccinated and masked) 
• All asymptomatic at diagnosis hence “cryptic”

Conclusion:

- For HCW: Despite the high viral load of their patients, masking combined with vaccination may have protected HCW during this outbreak.

- Patients; Encouragement of patient masking might further decrease nosocomial Delta infection.

- Unclear whether the AY.3 variant is more transmissible.  

Ep 167-5: Internet paper by JP Santiago 23 July on the “subvariants” of delta.  See nice figure

Ep 167-6: Mc Callum in medRxiv 12 Aug with instructive Table S1 p. 17

From both overviews I understand

• that delta and delta AY.3 have very similar mutations in Spike, but AY.3 has some additional ones that are sometimes present, but it is not clear to me what the exact definition is   
• That Delta plus is characterized by K417N: an immune-escape mutation, shared with the beta (South-African) variant.
  • AY.1 has the additional W258L
  • AY.2 the additional A222V

Ep 167-7: Kannan on 11 August provides a further level of complexity by describing a number of additional mutations that are characteristic of Delta and/or Delta-plus (AY.1).  A signature in spike for both delta and delta-plus  is T95I, while in delta plus W258L is also present.  Table 1 provides a list of mutations in other ORFs.

Ep 167-8 : Zhang on 17 Aug medRxiv show that membrane fusion is particularly efficient with delta. They suggest, but are unable to prove that the N-terminal domain of the S1 is responsible. 

They admit that more efficient functioning of RNA polymerase could also have a role. 

Ep 167-9: Foster on 27 Aug describes in medRxiv the rapid spread in Australia of a delta variant with a 17-nucleotide deletion in ORF7a has spread rapidly.  The advantage is not immediately clear to the virus as this would inactivate a viral protein that counteracts a host anti-viral factor (Tetherin).  Therefore, the question is whether this is just a “founder effect” (in the Australian context with little viral circulation) or whether this variant has ab unexpected competitive advantage.

Ep 167-10 : A medRxiv paper by Liu et al with the worrying title: The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines.

Starting observation: Delta has mutations in both receptor-binding domain RBD and N-terminal domain (NTD) of Spike:

• RBD mutations are limited and the most dominant L452R and T478K are also present in other VOC, which are NOT as infectious.
• NTD mutations such as T19R, G142D, E156G, F157del and R158del—have not been observed in other major variants → may be more important to explain enhanced delta infectivity.

Summary of their findings:

1. Using a series of human monoclonal antibodies, derived from patients, infected with the 2020 wild-type (WT) SARS-CoV-2, they show in Fig 1:
   • Delta is equally sensitive as to neutralizing Ab against RBD (Fig 1C)
   • Delta is no longer sensitive to neutralizing Ab against NTD (Fig 1B), but is more susceptible to enhancing anti-NTD (Fig 1 D)

As a consequence: there is a possibility that the Delta variant maintains the infectivity in the presence of anti-RBD neutralizing antibodies as a result of NTD enhancing antibodies.  

But, this is not really shown.

2. Using sera from Pfizer-vaccinated individuals:

Decreased neutralizing activity against delta virus, both RBD and NTD (Fig 2 and 3)

3. Into the RBD of delta (with L452R and T478) additional mutations, associated with resistance in other VOC such as K417N (beta), N439K (see Ep 167-11), E484K (beta and gamma) and N501Y (alpha, beta, gamma).  The complete (artificial) delta mutant is called delta 4+.  Two different version: of delta 4+ were made: with either WT NTD or delta  NTD.

Effect of sera from Pfizer vaccinees (Fig 6)  

• • Lower sensitivity of delta 4+ to neut by Pfizer sera
  • Tendency to enhancement of infection, but this is dependent on the delta NTD: no enhancement, but still neut if NTD is derived from WT

→ Hence neut by Pfizer sera mainly oriented towards RBD, while NTD can contribute to infectivity enhancement by Ab if NTD contains delta mutations.

4. Mouse immunization studies: (Fig 7)

Fig 7 B/C:            After WT vaccination → strong neut against WT; weak against delta

                               After delta vaccine → strong neut against both WT and delta

Fig 7 D/E Effect on delta + 4:      after WT vaccine: sera rather enhance infection

                                                               After delta vaccine: sera still neutralize fully

Delta vaccination is superior: sera will neutralize WT, delta and delta+4

WT vaccination will induce strong neut against WT, (very) weak neut against delta

But may enhance infection with delta+ 4   

Cave: this is an artificial system with “pseudoviruses”: SARS-CoV-2 spike, used as an “envelope” for vesiculo-stomatitis virus (VSV).  Hence all other structural (e.g. capsid) and non-structural (e.g. polymerase) proteins are derived from VSV.  So, it is not sure whether which of these mutations would work in the context of viable SARS-CoV-2.   

My preliminary general conclusions:

At this moment:  universal vaccination with WT-based vaccines remains indicated

• Moderna > Pfizer > Adeno > inactivated
• With “weaker” vaccines, heterologous prime boost maybe indicated: e.g. Adeno + mRNA or Adeno + inactivated  

In view of higher infectivity of delta, more chances on breakthrough and “cryptic” asymptomatic transmission, which may become obvious only when it hits vulnerable people, who may become seriously ill, even if they are vaccinated (immune-suppressed elderly > 90 yrs old) :

• Vaccinate > 90 % of the total population to reduce viral circulation to the maximum: Need to vaccinate children?  Need for mandatory vaccination for HCW and in areas where voluntary vaccination doesn’t succeed (e.g. Brussels; Southern States of US…) ?  
• Avoid high risk on transmission: e.g. wearing masks, especially when people come together in close contact and if some are not vaccinated (e.g. schools…) and/ or remain vulnerable despite vaccination (e.g. hospitals, residential care …) ?

Future:

1. SARS-CoV-2 will remain a “moving target”: the evolution of delta towards “superbug” is already happening by acquiring additional “immune escape spike mutations”. 

• Most attention is focused on RBD of S1: K417N is already being acquired and E4848K, N501Y etc may follow  (see Ep 167-10). 
• The role of the N-terminal domain needs further study (see Ep 167-8 and -10).  Also the S1-S2 cleavage site.
• For real viruses, however, the pathway to acquisition is much more complex than manipulating the spike in the lab. Additional mutations in other parts of S (e.g. S2) and other viral proteins (polymerase, nucleoprotein, non-structural proteins) may be required to have a viable and very fit SARS-CoV-2.

So, it may take time and we must use that (presumably short) “breather pause” for 2) and 3)  

2. Vaccinate the world as quickly and completely as possible: the best chances for further evolution towards “super-SARS-CoV-2” is when the virus is confronted with a “mixed” population: some vaccinated, others not. Just like antibiotics resistance: it is only emerging if treatments are incomplete, drugs “circulate” in suboptimal concentrations in (animal) reservoirs etc.

3. More “universal” (variant-proof) vaccines should be designed:
   • Inclusion of all relevant mutations in spike (potentially “mosaic” mixtures of VOC)
   • Induction of T cell immunity against more conserved parts of various viral proteins?
   • Mucosal vaccines? Attenuated live vaccines?

A lot of work to do!

Best wishes,

Guido