4 Dec Episode 91 B and T immunity in asymptomatic, mild and severe COVID

Episode 91: B and T immunity in asymptomatic, mild and severe COVID

Dear colleagues,

Today’s episode focuses on the question if we can correlate different clinical presentation and evolution with differences in T and B cell phenotypes and function, assessed from peripheral blood.   The underlying question is whether this can give clues to pathogenetic or protective immune responses.

Some of these papers are “hard core immunology”, which is probably not directly understandable by those of you, who have limited background in that field, but I tried to emphasize, explain and interpret the main findings….  

1. Elevated Exhaustion Levels of Natural Killer (NK) and CD8+ T Cells as Indicators for Progression and Prognosis of COVID-19 Disease (Mingyue Li Front Immun Oct 2020)

A study on lymphocyte subsets and serum markers, that is useful as an introduction but that  remains limited because mild versus severe patients are only compared at admission, so purely trans-sectional and associative.  The main findings:

• In slide 2 Table 2 you find a number of biochemical markers, well known to be different between mild and severe disease.
• While all classical serum inflammatory markers (IL-6, IL-10, TNF) are higher in severe cases, specific cytotoxic molecules (granzyme A and Perforin) are up in mild but down in severe cases
• Slide 3 shows neutrophils up and lymphocytes down in severe cases, which is well known. Amongst the lymphocytes NK cells, CD4+ T cells, CD8+ T cells, B cells  and NKT cells are significantly reduced in severe vs mild patients
• The exhaustion markers CD244 (4B4) and PD-1 are increased on NK, CD8 and CD8 T cells of mild patients, but do not consistently increase from mild to severe cases (slide 3 and 4).
• Within NK cells cytotoxic CD3-CD56dimCD16+ cell cytotoxic population significantly decreased, while the “immature” CD3-CD56dimCD16- part increased in severe patients (slide 4)
• Nice summary in slide 5.
• Statistical analysis (LASSO?) revealed that the most discriminatory markers for severe disease were level of leukocytes, neutrophils, IL-6 and CD3-CD56dimCD16- cells. So, clearly, the exhaustion markers are interesting from a pathogenic point of view, but are not the main discriminating factor (as the title suggests….). Nevertheless the differences between the typical rise of inflammatory serum markers (IL-6, TNF) and decrease of some key cytotoxic molecules (granzyme A and perforin ) in severe versus mild cases, indeed points to “exhaustion” of this line of viral defence.

2. Humoral and circulating follicular helper T cell responses in recovered patients (Juno Nat Med Sept 2020): mild-to-moderate patients investigated about 1 Mo after symptoms and arranged according to plasma neut activity (slide 6 f) to facilitate reading and interpreting:

• Plasma Ab profile (Slide 6) a) high titers against Spike (S), b) lower against SARS-CoV-2 receptor binding domain (RBD-, c) weak against SARS-CoV-1 RDB; d) apparently strong cross-reactivity HKU1; f) range of neutralization,  e) poorly correlated with ACE2 inhibiting Ab
• Circulating B cell and follicular T helper (cTFH) cell reactivity: mainly against SARS-CoV-2 S outside RBD, but cTFH against HKU1 also expanded (Silde 7).
• Neutralization activity positively correlated with S and RBD antibody titers (slide 8), S- and RBD-specific B cell frequencies and S-specific cTFH (not shown) but the correlation  is not very strong.  
• Remarkably: all those parameters also associated with patients reporting greater symptom severity (slide 9)!

Interpretation according to authors:

• Disconnect between plasma neutralizing titers and anti-RBD immunity in many individuals suggests that sufficient non-RBD epitope targets exist to constitute an alternative pathway to similar virus neutralization outcomes.
• All aspects of humoral immunity stronger in patients with severe disease!

3. Functional SARS-CoV-2-specific immune memory persists after mild COVID-19 (Rodda medRxiv Aug 2020): In this study only mild patients were studied, for the first time around 35 days after infection (corresponding to the single time point in the previous paper) and again 51 days later (= 3 mo after infection).

• SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells not only persisted, but in some cases increased numerically over three months following symptom onset (slide 10).
• Memory CD4 as well as CD8 T cells secreted IFN-γ. and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies (slide 11).

4. Longitudinal analysis of the humoral response to SARS-CoV-2 spike RBD in convalescent plasma donors ( Perreault bioRxiv) In all donors, the level of antibodies remained relatively stable up to about 76 days after symptoms onset but then started to decrease more rapidly to reach, in some convalescent donors, a seronegative status within 100-110 days after symptoms onset (slide 12)

5. Evidence for sustained mucosal and systemic antibody responses to SARSCoV-2 (Isho medRxiv Aug 2020): IgA and IgG antibodies to the SARS-CoV-2 Spike and RBD domain (RBD) in serum and saliva.  Whereas IgA  rapidly decayed, IgG antibodies remained relatively stable up to 115 days post symptom onset (PSO) in both biofluids (slide 13).

6. All these findings are confirmed and to some extent further explained by the paper: Evolution 1 of Antibody Immunity to SARS-CoV-2 (Gaebler medRxiv Nov 2020), which was already briefly discussed in episode 84.  Main findings:

• IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected (slide 14 top).
• Neutralizing activity in plasma decreases by five-fold in pseudotype virus assays (slide 14 bottom).
• In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response (slide 15 for analysis of monoclonal Abs).
• Importantly: Analysis of intestinal biopsies obtained from asymptomatic individuals 3 months after COVID-19 onset, using immunofluorescence (slide 15), electron tomography or polymerase chain reaction, revealed persistence of SARS-CoV-2 in the small bowel of 7 out of 14 volunteers (example in slide 16).

We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

7. Clinical and immunological assessment of asymptomatic vs symptomatic COVID patients (Long Nat Med Aug 2020): Very remarkable results:

• The initial viral load is remarkably similar, but viral shedding takes longer  in non-symptomatic patients (slide 17 left).
• The specific IgG and IgM responses are lower in non-symptomatic subjects 3-4 weeks after infection (slide 17 right Fig 3 a)  and 40 % becomes negative within 3-4 months (Slide 17 Fig 3 c and e). 
• Pseudovirus neutralization is slightly higher in asymptomatics  at baseline (with a wider spread in symptomatics) and decreased in both groups (Slide 17 Fig 3 d).
• All pro- and anti-inflammatory cytokines were higher in the symptomatic group in acute phase (slide 18).

8. Different innate and adaptive immune response to SARS-CoV-2 infection of asymptomatic, mild and severe cases (Carsetti medRxiv Sept 2020):

• Asymptomatics similar to controls with regard to monocytes/lymphocytes ratio (MLR), NK cells, monocytes (M) and M/NK ratio. NK decrease and Mo increase with severity of disease. M/NK ratio in severe disease 10 X asy ! (Slide 19)
• T cell activation with disease progression: HLA-DR expression CD8 T more than CD4 T
• Influx of “fresh T cells” strong decrease of recent thymic emigrants in CD4 T cells (Slide 20)
• Asymptomatic subjects show early increase of IgA, IgG and IgM against S protein, but final levels are higher in severe disease (Slide 21).  

The authors interpretation:  

• Early burst of IgA in asymptomatics may rapidly and effectively eliminate the virus in the respiratory mucosa and prevent the development of a full adaptive immune reaction.
• The slightly slower IgG and IgA production that persist in time suggests that the adaptive immune response is triggered in mild disease and may be able to generate immunological memory.
• A long and severe disease fully activates the adaptive immune response and is associated with the production of anti-SARS-CoV-2 antibodies, plasma B cells and memory B cells.
• Low level of antibodies in the first two weeks after diagnosis and increase of the M/NK ratio may indicate patients at risk for increased severity

9. Pre-existing T cell memory as a risk factor for severe 1 COVID-19 in the elderly (Bacher medRxiv Sept 2020): This is a very interesting paper (already briefly discussed in Episode 76). The main message is:  that pre-existing memory T cells are present in all unexposed donors and increased in the elderly, not primarily driven by Common CoVs but reactive to SARS-CoV-2 itself .

These pre-existing memory T cells possess only low TCR avidity, suggesting impaired functionailty. This functional impairment is closely mirrored in T cells from severe COVID-19 patients in contrast to mild disease, suggesting that they may originate from pre-existing memory T cells.

Thus we suggest the immunological age as a potential risk factor for severe COVID-19.

• Fig 1 C shows low but clear frequency of SARS-CoV-2 memory T cells in all unexposed subjects. Fig 1 D: Memory T cells from unexposed recognize a more variable number of epitopes than is the case in COVID-19 patients, who focus on Spike, membrane and nucleocapsid (Slide 22)
• Fig 2 shows that SARS-CoV-2 specific CD4 T cells from COVID patients have functional TH1/T follicular profile: high production of IFN-g, IL-21 and PD1, hence good “help” for B cells (Slide 23).
• SARS-CoV-2 specific memory T cells in unexposed subjects

Frequencies correlated:

• • Immunological age (Fig 4 E slide 24))
  • Not with memory to Common CoV (Fig 4 G)

Low functional avidity and less focus on dominant epitopes

• Similarly, in severe (hospitalized) COVID patients functional avidity is lower than in  mild/moderate (non-hospitalized) subjects (Fig 6 slide 24))

These findings are clearly in line with Immune senescence: A Predisposing Risk Factor for the Development of COVID-19 (Hazeldine Front Imm Oct 2020): slide 25

Overall interpretation:

1. There is overwhelming evidence of activation of all aspects of innate, but also adaptive T and B cell  immunity in moderate and severe disease, with some evidence of NK and T cell cytotoxic exhaustion in severe cases, but with even higher antibody (and neut responses).
2. There is clear evidence that the immune activation in people with an “immune senescent profile” (elderly and/or comorbities) is linked to “dysfunction” of the system, with “over-activation”, causing tissue damage without efficient containment of the virus replication.  The hypothesis, proposed by Bacher, is attractive: because of a long history of many different immune challenges in these people, they have low affinity “memory-type” T and B cells that react to SARS-CoV-2 (and many other antigens, even if they have not seen them before).  Activation of these “low affinity” T/B cells gives rise to an inefficient type of over-inflammation….  
3. In the peripheral blood, we cannot pinpoint a particular parameter of innate or adaptive immune activation that is associated with protection (in asymptomatic or mild cases).  The claim by Carsetti of an earlier humoral response in asymp is not very convincing.
4. Clearly, however, the “real fight” against the virus is in the lungs and the parameters we find in the periphery could be either representative (overflow of cytokines and antibodies) or  rather the “mirror image” of what happens in the lungs, especially for the T cells.  It is quite possible for instance that high affinity T cells accumulate in the lungs, leaving the low affinity ones for the periphery….    
5. After recovery, specific antibodies tend to decrease, but not to the same extent in all subjects.  The findings of Gaebler et al on evolution of antibodies and viral persistence in the GI system is very intriguing in this regard.  This persistence could have various and even opposite consequences: give rise to recurrence of the evolved “escaping” virus in some cases or on the contrary, prevent re-infection, because the antibodies evolve to cover a wider range of evolved viruses….      
6. Based on all these data in adults, one could reason “ex absurdo” to explain the much more favorable outcome in children: they have a much more “fresh” immune system, where both the innate and adaptive arm are being “trained” to address new immune challenges rapidly and effectively all the time.  But clearly, this is an easy superficial speculation that does not explain the precise mechanisms.  Is it a more swift IFN response, more rapid NK cells, B or T cells….?

Best wishes,

Guido   

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