17 Jan 2021 Episode 101 Mutants and Variabts

Dear colleagues,

After my last mail, I received and found some additional recent info on COVID in Africa.

• Nzoghe et al (Ap 100- 24) confirm the finding of For Yue Tso (Ep 100-9) on 20% of pre-existing cross-reactivity towards SARS-Cov-2 Nucleoprotein, this time in Gabon, while Tso made the same observation in Zambia and Tanzania.  As discussed, the (protective or pathological) significance of these antibodies is unclear.
• According to a report from KEMRI (Ep 101-25) the seroprevalence of SARS-CoV-2 has more than doubled in blood donors between Marc-June as compared to July-Sept: from an average of 5.5 to 13.3% , with large differences between rural and urban areas (for details see slide 2). HCW and attendants of the antenatal clinic Kenyatta National Hospital go over 40 %.  It is not clear which test has been used, but the seroprevalence of pre-pandemic sera was less than 1 %, so presumable Spike specific.   There is no increase in hospital deaths.
• A very similar picture in Cape Town South-Africa around 40 % seroprevalence in ante-natal attendants and also in HIV(+) patients (Ep 100-26 and Slide 3).    

Now let’s have a look at the wealth of recent papers on potential escape  mutations from neutralizing antibodies  and the upcoming UK, South-African and Brazilian infectious variants. 

1. First a few papers on mutations in spike that confer resistance to antibodies:

1. 1.  The Nature paper by Lun (Ep 100-1) shows the overall structure of SARS-CoV-2 spike and the interaction with the ACE-2 receptor.  This is important background to understand the possible significance of mutations with regard to infectivity. (slides 4,5,6)

1. 2. The very recent bioRxiv preprint by Greaney (Ep 100-2) identifies mutations that affect the neutralization by polyclonal serum from convalescent patients, because these mutations could also become “escape” after vaccination? 

First they proof that most of the neutralizing activity of serum is focused on the receptor-binding-domain (RBD). See slide 7

Then they show that mutations that strongly reduced binding of polyclonal sera fell in one of three discrete regions of the RBD (slide 8):

(1) the receptor-binding ridge within the receptor-binding motif (RBM), including L455,F456, I472, Y473, E484, G485, F488

(2) a loop in the RBM opposite the ridge (spanning all sites 443–450, and the structurally adjacent sites at 494–501), 

(3) a surface patch in the core RBD (including Y365, Y369, S383 and P384)

As can be seen in slide 8, these are the positions, where mutations change the binding of serum antibodies from convalescent patients. 

Fig 6B in slide 9 illustrates that the four mutations that have the highest frequency among sequenced viruses (S477N, N439K, N501Y, and Y453F) do not strongly affect serum antibody binding by any samples tested in this paper. 

Nevertheless the N501Y mutation is just adjacent to the 443-450 RBM loop and is common to the UK and South-African variant.  It is thought to rather increase the binding to ACE-2 than to induce escape from neut Ab.

By contrast E484Q that is present in the South-African variant can be considered as an escape mutation, according to the findings in this paper, but NOT so much the co-occurring K417N in the South-African variant.

The authors conclude that E484 in the “ridge of RBM” is the site of most concern for viral mutations that impact binding and neutralization by polyclonal serum antibodies targeting the RBD. However, mutations at the other serum antibody epitopes (e.g., the 443–450 loop and residues around 484 such as 455, 456, 485, 486, and 490) are also worth monitoring, since they also have antigenic impacts.

1. 3. These conclusions are largely consistent with the study by Barnes in Nature (Ep 100-3), who studies human monoclonal neutralizing antibodies, derived from convalescent patients and distinguishes 3 classes of antibodies, 2 of which bind to very similar residues in the RBD, while the third binds outside.  As can be seen in slide 10 (showing the “footprint” of class 1 and 2 antibodies), however, the K417 is also involved in this case 

1. 4.  As a reminder, in the paper by Weisblum (Ep 98-13) found escape mutations in N-Terminal domain (NTD) for plasma COV47 and in the 443-450 loop for COV NY, but for the monoclonals from these patients, the mutations were rather in 484 and the 494–501 and the 346 position (slide 11).

1. 5. Similarly, for the Andreoni paper (Ep 98-12) on induction of resistance by a polyclonal serum, the 484 position comes up, together with alterations in the NTD (slide 13)

1. 6. As a final reminder the resistance profile of the Regeneron and Eli Lilly monoclonal antibodies (Ep 98-7): you recognize the same pattern as for the study by Greaney: the RBM (including 484), the 443-451 loop and the  adjacent site  (494-500) and also the  417 position  for the Eli, Lilly monoclonal (slide 14).

Conclusion: In all these studies, a number of critical positions for resistance/escape to monoclonal and polyclonal antibodies (i.e. convalescent) are evident: the 484 position is very dominant, the 443-450 loop and 494–501 are often involved, but also 417, 346 and several positions in NTD

2. The hot topic on new more infectious UK, South-African and Brazilian variants

2.1. The UK variant, B117 or Variant of Concern (VOC) 202012/01 or 20B/501Y.V1

2.1.1  Leung describes the history and puts the lineage into perspective (Ep 100-4)

In fact two independent N501 Y variants have arisen in UK:

• The first 501Y lineage (501Y Variant 1) appeared in Wales in early Sept and persisted till Nov.
• The second 501Y lineage (501Y Variant 2, also named B.1.1.7, 20B/501Y.V1 and VOC-202012/01) appeared in England in late September and largely expanded to become the predominant lineage in the region since late November. (slide 15)

In addition, globally, two other lineages with 501Y (without Δ69/Δ70) have been detected in Australia, circulating from June to July and in South-Africa from October 2020 on. (slide 16)

2.1.2. Davies (Ep 100-5) describes it as follows: 17 mutations (14 non-synonymous mutations and 3 deletions (see slide 15). There are 8 in Spike:  (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H) Of these:

- N501Y: enhanced binding to ACE-2 receptor→ increased infectivity

- P681H: adjacent to furin cleavage site →increased infectivity?

- del 69-70:     Found as immune escape in immune deficient individuals

                               Increased infectivity?

Responsible for the diagnostic sign of S-drop-out:  loss of S signal, but preserved (even enhanced) ORF1 and N signal in TaqsMan PCR (loss of primer binding)

The proportion of COVID-19 cases caused by VOC 202012/01 is growing rapidly in the South East, East of England and London regions ( Fig. 1A ) and is associated with an increase in the estimated reproduction number R t ( Fig. 1B ).  Slide 17

According to their modeling, increased infectiousness is the key factor for rapid spread; much more likely than immune escape; increased susceptibility among children; and shorter generation time. Slide 18

Regardless of control measures simulated, all NHS regions are projected to experience a subsequent wave of COVID-19 cases and deaths, peaking in spring 2021 for London, South East and East of England, and in summer 2021 for the rest of England ( Fig. 4 ). Slide 19

In the absence of substantial vaccine roll-out, cases, hospitalisations, ICU admissions and deaths in 2021 may exceed those in 2020 ( Table 1 ).

2.1.3. Kidd (Ep 100-6) shows that S gene dropout samples have significantly higher viral loads

Figure 2 showed a high proportion of S-gene negative samples; note the number having an S-gene undetectable profile (178 of 641; 27.7%) at far right, compared with ORF and N-gene undetectable positive profiles (both 13 of 641; 2.0%). Slide 20 left.

These S dropout samples had an increased viral load in N and ORF (up to 10,000 x higher), implying increased infectiousness.  Slide 20 right.

2.1.4. Volz (Ep 100-7) uses the S Gene Target Failure (SGTF) to study the epidemiology of B117 or VOC and concludes that:

• • Changes in VOC frequency inferred from genetic data correspond closely to changes inferred by S-gene target failures (SGTF)
  • Genetic diversity of this lineage has changed in a manner consistent with exponential growth
  • VOC has higher transmissibility than non-VOC lineages
  • Available SGTF data indicate a shift in the age composition of reported cases, with a larger share of under 20 year olds
  • Reproduction rate under strict lockdown 40-80 % higher for VOV as compared to non-VOC

See slide 21

2.2. South African variant  501Y.V2: Tegaly medRxiv Dec 2020 (Ep 100-8)

Evolution of mutational profile:

• On 15 October: D80A, D215G, E484K, N501Y and A701V
• End Nov: L18F, R246I and K417N
• Potential deletion of three amino acids at L242_244L

Clearly: changes in NTD (L18F, D80A, D215G and R246I + del L242-244L)); changes in RBD (K417N, E484K and N501Y) and near S1-S2 split (A701V)

See slide 22

The three changes in RBD are suspected to increase affinity for ACE-2 and/or escape from (polyclonal) neutralizing antibodies.

                Coincides with start of second wave:

• • Originated in Nelson Mandela Bay, spread over Western and Eastern Cape and has replaced other strains.  See slide 23

2.3. Brazilian variant

2.3.1. According to Voloch in medRxiv on 26 Dec (Ep 100-9), this new variant  was discovered as a cluster in the originally subdominant B.1.1.28 lineage. It is characterized by the following mutations:

• • Two variants mapped the 5’ and 3’ end (C100U and C29754U) respectively, a synonymous C28253U (F120F) in ORF8, and two missense G28628U (A119S) and G28975U (M234I) in N protein.
  • Importantly the E484K in S protein
  • Some other mutations in S and M, not consistently present  

This variant is becoming dominant between Sept and Nov (slide 24).

It is still mainly present in the capital, but gets dispersed in the state (slide 25)

Clearly, in this report, the 417 and 501 mutations are not mentioned.

2.3.2. A case report on 6 January by Vasques Nonaka provides the first evidence of reinfection with this new variant in Brazil, far away from Rio.  They also claim that the variant has spread to UK, USA, Portugal and Australia (Ep 100-10) Slide 26

2.3.3.  According to a report from the National Institute of Infectious Diseases on 12 January (Ep 100-11), another variant of the B.1.1.248  lineage was isolated from a returning traveler in the Amazon, with 12 mutations in Spike, which include not only E484K, but also N501Y and N417T  See Slide 27. (which also provides a nice comparison with the UK and SA variants)

That’s it for today…. Enjoy.

Guido