COVID-19 Nose-dive (Part 2): Biology & Medical Outcomes

In Part 2 of our Nose-dive series, we will examine the key biology actors of COVID-19 focused especially in the nasal passage—point of first contact between SARS-CoV-2 and human body. We will also evaluate these scientific findings’ conformity with real-world data from the pandemic.

The key biological processes of COVID-19 infection and disease progression are shown below. The two main players in the cell entry process are ACE2 (receptor for binding to the spike protein of the virus) and TMPRSS2 (activating protease for virus uptake into the cell). Downstream in the disease progression, ACE2 down-regulation (or decreased presence) occurs leading to a wide range of conditions from anosmia (loss of smell) and dyspnea (shortness of breath) to ARDS (acute respiratory distress syndrome), thrombosis (blood clotting), and cytokine storm (excessive inflammation) to acute death. ACE2 down-regulation is the main culprit for severe outcomes.

*In the assays and surveys described below, “+” indicates presence and “-“ indicates absence.*

ACE2 and TMPRSS2

ACE2 and TMPRSS2 are the two main biology actors in the viral entry process. In their study, Halwani et al. [i] reported both nasal and bronchial airway expressions of ACE2 and TMPRSS2 are lower in children compared with those in adults. They also found the lung airway expression of ACE2 and TMPRSS2 are significantly higher in smokers (vs. non-smokers) and in patients with COPD (vs. healthy subjects).

In a more detailed analysis of ACE2 nasal expression, Bunyavanich et al.[ii] found the lowest expression in children <10, followed by adolescent 10-17, young adults 18-24, and adults ≥25.

KEY TAKEWAYS

• With less nasal expression of both ACE2 and TMPRSS2, children (especially young children) have less “doors” for SARS-CoV-2 to enter.

Adaptive Immunity: T- & B-Cells

The human immune system has two fundamental defenses—innate immunity and adaptive immunity—for fighting a pathogen. Innate immunity is the first and “general” response initiated quickly after infection and retains no “memory”. Adaptive immunity is the second and “specific” response within three or more days of infection and retains “memory”; this immune “memory” enables a more rapid and efficient immune response upon subsequent re-exposure to the pathogen. Within adaptive immunity, the two defense units are T- and B-cells. T-cells (CD4+ and CD8+) drive cell-mediated immunity, whereas B-cells drive humoral (bodily fluid) immunity through antibody secretion. CD4+ “helper” T-cells coordinate immune response and enable B-cells to produce antibodies; CD8+ “cytotoxic” T-cells eliminate virus-infected cells. Within antibody classes, IgM is the first antibody made in bodily fluids and IgG is the second made, longer-lasting, and greatest in abundance; IgA is found in mucosa.

One of the most exciting developments in COVID-19 research is the discovery of pre-existing immunity, from T- and B- cells, found in unexposed individuals. In their T-cell study, Sette et al.[iii] found the below key points:

1. SARS-CoV-2-specific CD4+ and CD8+ T-cells were identified in 100% and 70%, respectively, of COVID-19 patients.
2. CD4+ T-cell responses against S protein were robust and correlated with SARS-CoV-2 IgG and IgA amounts.
3. CD4+ T-cell responses were also present against M and N proteins along with non-structural proteins (nsps).
4. CD8+ T-cell responses against S and M proteins were recognized as the dominant responses compared to other virus proteins.
5. SARS-CoV-2-specific CD4+ T-cells were detected in 40-60% of unexposed individuals, suggesting T-cell cross-reactivity among circulating common cold coronaviruses (HCoVs) and SARS-CoV-2.

In another T-cell study, Walz et al.[iv] examined SARS-CoV-2-specific and cross-reactive HLA class I and HLA-DR T-cell epitopes in COVID-19 patients and unexposed individuals and their relevance for immunity and disease outcome. They found the following key points:

1. SARS-CoV-2-specific T-cell epitopes enabled detection of post-infection T-cell immunity even in seronegative COVID-19 patients.
2. Cross-reactive SARS-CoV-2 T-cell epitopes revealed pre-existing T-cell immunity in 81% of unexposed individuals and validated cross-reactivity with common cold coronaviruses.
3. Anti-SARS-CoV-2 antibody levels were associated with severity of symptoms in COVID-19 patients but intensity of T-cell responses did not negatively affect COVID-19 severity.
4. Diversity of SARS-CoV-2 T-cell responses was increased in mild COVID-19 cases providing evidence that immunity requires recognition of multiple SARS-CoV-2 epitopes.

In a T-cell study correlating clinical outcomes (phenotypes), Buggert et. al[v] systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T-cell responses in unexposed individuals, exposed family members, and patients with acute or convalescent COVID-19. They found the following key points:

1. CD4+ and CD8+ T-cells were unphysiologically low in acute moderate (AM) or severe (AS) COVID-19 patients compared with those in unexposed individuals who donated blood during the pandemic (2020 BD).
2. Memory CD8+ T-cells from AM or AS expressed a distinct cluster of markers associated with activation and the cell cycle including CD38, HLA-DR, Ki-67, and PD-1; a similar pattern was observed for memory CD4+ T-cells from AM or AS (MC=mild convalescent COVID-19 patients).
3. Acute-phase SARS-CoV-2-specific CD8+ T-cells were characterized by expression of immune activation molecules (CD38, HLA-DR, and Ki-67), inhibitory receptors (PD-1 and TIM-3), and cytotoxic molecules (granzyme B and perforin); convalescent-phase SARS-COV-2-specific CD8+ T-cells were characterized by an early differentiated memory (CCR7+ CD127+ CD45 -/+ TCF-1+) phenotype.
4. Expression frequencies of CCR7 and CD45RA were directly correlated with the number of symptom-free days after infection, whereas that of granzyme B was inversely correlated with the number of symptom-free days after infection.
5. Cross-reactive T-cell responses against S and/or M proteins were detected in 28% of healthy individuals who donated blood before the pandemic (2019 BD); N cross-reactivity was not observed in this group. The highest T-cell response against any of these three virus proteins (N or S or M) was observed in severe convalescent (SC, 100%) followed by, in descending order, mild convalescent (MC, 87%), exposed family members (Exp, 67%), and unexposed individuals who donated blood during the pandemic (2020 BD, 46%).
6. SARS-CoV-2-specific CD4+ T-cells predominantly expressed INF-γ, IL-2, and TNF, whereas SARS-CoV-2-specific CD8+ T-cells predominantly expressed INF-γ and CD107a.
7. SARS-CoV-2-specific CD4+ T-cell responses were proportionately larger overall than the corresponding SARS-CoV-2-specific CD8+ T cell-responses (Exp = 1.8-fold, MC = 1.4-fold, SC = 1.8-fold).
8. Most IFN-γ+ SARS-CoV-2-specific CD4+ T-cells produced TNF, whereas most IFN-γ+ SARS-CoV-2-specific CD8+ T-cells produced granzyme B and perforin.
9. SARS-CoV-2-specific CD4+ and CD8+ T-cell responses were present in seronegative individuals, although at lower frequencies compared with seropositive individuals (41% vs. 99% respectively). Higher antibody responses were correlated with the severity of COVID-19, whereas there was minimal difference among T-cell responses from different COVID-19 clinical states.

Collectively, the functional and phenotypic mapping of SARS-CoV-2-specific T-cell immunity across the full spectrum of exposure, infection, and disease is shown below.

In a B-cell study, Kassiotis et al.[vi] observed pre-existing humoral immunity in uninfected and unexposed individuals. They found the below key points:

1. The presence of SARS-CoV-2 S-reactive antibodies of all three Ig classes (IgM, IgG, and IgA) effectively distinguished COVID-19 patients. A proportion of SARS-CoV-2 uninfected people had SARS-CoV-2 S-specific antibodies comprising predominantly of lower levels of IgG and no IgM or IgA.
2. The S protein has two subunits—S1 and S2—mediating cell attachment and entry, respectively. S2 exhibits a higher degree of homology among coronaviruses than S1. Sera from both COVID-19 and HCoV patients react comparably in a SARS-CoV-2 S-coated ELISA, whereas only those from COVID-19 patients reacted in a SARS-CoV-2 S1-coated ELISA.
3. 13% (12/95) of uninfected serum samples exhibited IgG antibodies cross-reactive with conserved epitopes in SARS-CoV-2 S2 and N proteins.
4. 83% (5/6) of sera from SARS-CoV-2 uninfected with common cold infection (SARS-CoV-2 – HCoV +) with detected S-reactive antibodies (Ab +) neutralized SARS-CoV-2, on average, less potently than COVID-19 sera (SARS-CoV-2 + Ab +). None of the sera from SARS-CoV-2 uninfected with common cold infection and without detected S-reactive antibodies (Ab -) did neutralize SARS-CoV-2.
5. The frequency of infection with each HCoV type displays a characteristic age distribution with children and adolescents having the highest overall infection frequency. In a study with younger SARS-CoV-2 uninfected (age 1-16), 44% (21/48) had detectable SARS-CoV-2 S-reactive IgG antibodies. The prevalence of SARS-CoV-2 S-reactive IgG antibodies increased with age, peaking at 62% after 6 years of age. The cross-reactive seroprevalence in children were significantly higher than those in adults.
6. IgG reactivity against each HCoV type was independently correlated with the presence of cross-reactive antibodies against SARS-CoV-2; the presence of IgG memory reactive with HCoVs as well as SARS-CoV-2 was observed.

The observation of higher antibody responses in severe COVID-19 patients is a very important subject for therapeutic and vaccine development. We need to develop a full understanding of the antibody response so that avoidable “cytokine storms” (excessive immune response/inflammation) could be mitigated. In their study, den Dunnen et al.[vii] found anti-Spike IgG from severe COVID-19 patients promotes macrophage hyper-inflammatory responses, leading to endothelial barrier disruption and microvascular thrombosis. Specifically, they observed the below key points:

1. Sera of non-COVID-19 patients and anti-Spike IgG-negative COVID-19 patients exhibited no up-regulation (increased presence) of pro-inflammatory species (IL-1β, IL-6, IL-8, and TNF) when stimulated by PolyIC, whereas up-regulation of those species was observed in sera of anti-Spike IgG-positive COVID-19 patients when stimulated by PolyIC. RNAseq analysis of macrophage stimulated with sera from anti-Spike IgG-positive COVID-19 patients also showed clear induction of pro-inflammatory response as illustrated by induction of TNF, interleukins, chemokines, and macrophage differentiation factors.
2. Higher anti-Spike IgG titers were correlated with higher cytokine responses.
3. Co-stimulation of macrophages with spike protein and serum of severe COVID-19 patients (Serum) produced long-lasting endothelial barrier disruption (higher endothelial permeability and less resistance). In addition, platelet adhesion to endothelium was increased. In parallel, von Willebrand Factor (vWF) release from endothelium was also increased, indicative of an active pro-coagulant state leading to thrombosis.
4. Decreased fucosylation and increased galactosylation of anti-Spike IgG were observed when compared to total IgG within tested patients. An inverse correlation between IgG fucosylation and production of IL-1β, IL-6, and TNF was observed. Anti-Spike IgG from severe COVID-19 patients has an aberrant glycosylation pattern (decreased fucosylation and increased galactosylation) that makes these antibodies more inflammatory than “common” antibodies from greater production of pro-inflammatory species.
5. Of noteworthy interest, recombinant anti-Spike IgG immune complexes generated substantially less pro-inflammatory IL-1β, IL-6, and TNF than anti-Spike IgG immune complexes from COVID-19 serum. There was no difference in the generation of the anti-inflammatory IL-10. This finding could be pivotal in treating COVID-19 with recombinant antibodies in the right therapeutic window.

KEY TAKEAWAYS

• Pre-existing adaptive immunity, T-cell and B-cell immunity/antibodies, from prior exposure to common cold HCoVs is found up to 80% of general population. This is consistent with the finding from the Princess Diamond cruise ship at the beginning of the pandemic where only 20% of the ship population were infected. Only pre-existing immunity could account for not a greater spread in a confined environment.
• Children aged 6-16 possess more “cross-reactive” S-reactive antibodies against SARS-CoV-2 than other age groups, peaking at 60% seroprevalence.
• Current testing measures do not provide precise indication of community/herd immunity because they do not take into account the diverse epitopes present on the SARS-CoV-2 proteins (S, N, M, E, and nsps) as well as “cross-reactive” epitopes from the four common cold HCoVs.
• High T-cell responses were found comparably among COVID-19 exposed individuals, mild and severe patients, whereas high antibody responses were found in COVID-19 severe patients. High antibody responses are associated with a pro-inflammatory state (vs. anti-inflammatory state) leading to severe “cytokine storm” outcomes.
• Recombinant antibodies targeting SARS-CoV-2 spike protein were found to be less inflammatory (vs. anti-spike antibodies from COVID-19 patients) and could potentially provide effective treatment in the right therapeutic window.

RAS Balance

Up to this juncture, the discussion has centered on viral infection and replication. Our attention now turns to disease progression and clinical outcome in this section. As described previously in “COVID-19: The Infectious Chronic Disease”, COVID-19 is as much an accelerated chronic disease as it is an infectious disease because of the receptor (ACE2) and disease progression (ACE2 down-regulation) involved. Because chronic diseases (sometimes referred to “diseases of aging”) have a strong vascular component, Santulli et al.[viii] conducted a comprehensive review on the model of COVID-19 as an endothelial/vascular disease.

Besides ACE2 and TMPRSS2, other biology actors such as sialic acid receptor, CD147, and Cathepsin B/L have been shown to be “entry” factors for SARS-CoV-2. All of these factors are known to be expressed by endothelial cells. Endothelial dysfunction is a common feature of the clinical manifestations observed in COVID-19 patients including hypertension, diabetes mellitus, neurologic disorders, kidney disease, and thrombosis.

Illustratively, hypertension is the most common comorbidity observed in COVID-19 patients and is evidenced in patients with worse prognosis and higher mortality.

ACE2 is an important actor of RAS (Renin-Angiotensin-System) that governs cardio-pulmonary and vascular health. Specifically, ACE2 is a key component of the “protective” axis counter-balancing the “disease” axis.

Before the pandemic, there were over 1,900 publications on ACE2 in title/abstract field, documenting the extensive understanding on ACE2 biology. The effects of the main RAS actors, AngII from “disease” axis and ACE2 and Ang(1-7) from “protective” axis, on the major biological systems are summarized below.[ix]

As COVID-19 disease progresses, ACE2 down-regulation occurs and the “disease” axis is knocked downward. For the healthy individuals, their strong RAS balance could withstand this RAS knock-down; because children have pristine RAS balance, they are the most resistant to the COVID-19 induced RAS knock-down. For the vulnerable individuals with pre-existing conditions with RAS dysfunctions (e.g., hypertension, heart disease, lung disease, neurodegeneration, metabolic disorders), the COVID-19 induced RAS knock-down is on top of a knock-down state already, culminating in a double RAS knock-down with severe clinical outcomes and possibly acute death.

From the Santulli review, AngII level in the plasma of COVID-19 patients is markedly elevated and linearly associated with viral load and lung injury. AngII is known to increase microvascular permeability, to induce the transcription of tissue factor in endothelial cells, and to activate platelets, leading to thrombosis. Microvascular permeability in pulmonary endothelium leads to edema exacerbating hypoxia from lung infection. Furthermore, along with inflammation and thrombosis, AngII-induced vasoconstriction produces a hyper-hypoxic state. Endothelial cells represent one-third of the lung cell population and pulmonary endothelial damage is considered the hallmark of ARDS. ARDS is a critical clinical marker for severity of outcome in both SARS and COVID-19 patients. If ARDS is unchecked, then respiratory failure occurs. As noted previously, a dysregulated immune response is observed in the late stages of COVID-19 (severe cases). This cytokine storm could lead to overwhelming systemic inflammation, multiple organ failure, and death. The interplay of viral infection and disease progression/immune response with clinical outcomes and associated biomarkers are summarized below.

COVID-19 Biology Corroboration with Pandemic Human Data

The two most important pieces of information for COVID-19 is the patient survival rate (survived patients/infected patients) and the comorbidity prevalence in COVID-19 patients because comorbidity is the highest risk factor for COVID-19. The patient survival rate from CDC based on age groups (updated 9/10/20)[x] are shown in the below table.

The comorbidity prevalence from CDC (updated 10/7/20)[xi] was 94% with an average of 2.6 comorbidities per death. Also given in the table, the qualitative “protection” indices for ACE2 & TMPRSS2 (“Doors” for virus entry), Adaptive Immunity (“Borrowed Defense” against virus entry and replication from common cold cross-reactivity), and RAS Balance (“Key Defense” against disease progression) were ranked for each age group with 1 being the highest “protection”. Age serves as a “proxy” for pre-existing health conditions/comorbidities. Overall, the human data is consistent with the COVID-19 infection and disease progression model based on known science. For the healthy individuals, they could effectively clear the virus without any symptoms or mild symptoms before COVID-19 induced ACE2 down-regulation engenders severe clinical outcomes. For the vulnerable individuals with pre-existing conditions, they could not effectively clear the virus before COVID-19 induced ACE2 down-regulation engenders severe clinical outcomes including death. Simplistically, SARS-CoV-2 has a dual nature—it is common cold HCoV for the healthy individuals and it is SARS-CoV for the vulnerable individuals. For the continuum in-between, the clinical outcomes range from respiratory infection/pneumonia to accelerated onset and/or progression of chronic diseases.

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[i] doi: 10.1016/j.omtm.2020.05.013

[ii] doi:10.1001/jama.2020.8707

[iii] doi: 10.1016/j.cell.2020.05.015

[iv] doi: 10.21203/rs.3.rs-35331/v1

[v] doi: 10.1016/j.cell.2020.08.017

[vi] doi: 10.1101/2020.05.14.095414

[vii] doi: 10.1101/2020.07.13.190140

[viii] doi:10.3390/jcm9051417

[ix] doi: 10.1161/CIRCRESAHA.120.317015

[x] https://www.cdc.gov/coronavirus/2019-ncov/hcp/planning-scenarios.html

[xi] https://www.cdc.gov/nchs/nvss/vsrr/covid_weekly/index.htm#Comorbidities