Patients’ population characteristics according to vaccination doses and age
The study cohort included 47 patients hospitalized for COVID-19 pneumonia resulting from SARS-CoV-2 infection, during Delta and Omicron waves. Patients’ characteristics are shown in Table 1. Overall, median age was 78.41 years [IQR 68 – 84], 22/47 (47%) were female, and 12/47 (29%) had history of cancer. Based on the WHO clinical progression scale [22], 25/47 (57%) patients were classified as moderate (score 4 and 5, s≤5) and 19/47 (43%) as severe patients (score 6, s>5). Patients that appeared critically ill at admission and needed ICU were not included in the study. Apart from 2 patients (age>80 years), who experienced a negative progression of the disease and died (at admission s >5; at death s=10), all the other patients achieved a full remission.
At hospital admission, 17/47 (36%) individuals were not vaccinated (VACno), whilst the remaining 30/47 (64%) had received 2 doses (18/30, 60%, VAC2) or 3 doses (12/30, 40%, VAC3) of anti-SARS-CoV-2 vaccine, designed versus (vs) the original Wuhan strain. Comparing general characteristics of vaccinated and unvaccinated patients, the ratio male/female was similar in the two groups (VACno vs VAC2+3), while the VAC2+3 one was relatively older than the VACno (medians years 80 vs 71, respectively). The older group (VAC2+3) was more likely to have experienced some comorbidities compared to the younger one (VACno), including obesity (19% vs 0%), chronic obstructive pulmonary disease (COPD, 4% vs 0), diabetes (12% vs 6%), cancer (31% vs 25%), or other diseases (31% vs 13%, specified in table 1), albeit none of the difference was statistically significant. Both VAC2+3 and VACno experienced pulmonary arterial hypertension (PAH) with similar frequency (39% vs 38%). Within VAC2+3 group, individuals who received 3 vaccination doses were less likely to have experienced PAH (10% of cases in VAC3 vs 56% in VAC2). With regards to the percentage of severe patients (s>5), this was lower in VAC2+3 group (39%) than in VACno (50%) and among the vaccinated, those with three doses were less likely to have experienced severe symptoms (30% of s>5 in VAC3 vs 44% in VAC2) (Table 1).
Anti-SARS-CoV-2 response in elderly versus aged patients
Further, we explored the impact of age on the disease outcome and immune response in the context of COVID-19 pneumonia in presence or absence of vaccination. We thus divided the population into two strata: one below 70 years of age (≤70y, n=14) and one over 70 (>70y, n=33) and the characteristics of these 2 groups are provided in Supplementary Table 2. As it could be expected, the individuals>70y were more likely to have experienced comorbidities associated with aging such as PAH (29% in ≤70y vs 36% in >70y), diabetes (absent in ≤70 vs 12% in >70y), cancer (21% in ≤70y vs 27% in >70y) and other diseases (14% in ≤70 vs 24% in >70y). Lack of vaccination was more frequent in younger individuals, with 50% of ≤70y and 30% of >70y subjects being VACno. Of note, in the elderly group, administration of three doses of vaccination resulted in a lower proportion of severe cases (14% severe cases in VAC3 vs 43% in VAC2 and 44% in VACno). We further run a multivariable regression model comparing subjects ≤ 70 yrs and > 70 yrs of age adjusted for vaccine doses and gender (Table 2). We found that elderly had an overall lower anti-SARS-CoV-2 humoral response (IgG-RBD-S) with an expansion of CD28null CD4 populations. Of note, none of the individuals who received 3 doses experienced death, whilst the two people who died were both >70y: one was VAC2 and the other was VACno (Supplementary Table 2).
Vaccination was associated with increased anti-SARS-CoV-2 humoral response and neutralizing activity
Humoral response was evaluated by measuring circulating IgG-N, IgM-S, IgG-RBD-S Antibodies (Ab) (Table 3, Figure 1). Overall, IgG-N were detectable in 33/47 (70%), IgM-S in 26/47 (55%) and IgG-RBD-S in 30/47 (81%). By linear regression models adjusted for gender, age and cancer, comparing individuals that received or not the vaccine, we reported that IgG-RBD-S Ab levels were higher in VAC2+3 compared to VACno (p=0.0026, Table 3, Figure 1) conversely to what was observed for IgG-N Ab levels which were lower in (VAC2+3 compared to VACno (p=0.0408, Table 3, Figure 1. IgM-S levels did not vary across the groups.
In a separate regression model, using the same adjustments described above, we evaluated the impact of one or two doses of vaccine, and we compared the humoral response in VACno vs VAC2 or VAC3 (Supplementary Table 3). We observed that both VAC2 and VAC3 had higher levels of IgG-RBD-S compared to VACno, but this was only significant for VAC2 (p=0.0001). On the other hand, anti-N IgG levels decrease with the number of vaccine doses, with the highest level detected in VACno group (p=0.0014 compared with VAC3), as showed in Figure 1 and Table 3.
Further, we explored the impact of vaccination on the Ab neutralization activity during natural infection driving pneumonia. We tested neutralizing antibodies against both circulating variants Delta, BA.1 and BA.4/5 and human seasonal coronaviruses (HCOVs, 229E, HKU1, NL63). Overall, individuals who received vaccination (VAC2+3) showed significantly higher levels of neutralizing activity against the circulating variants compared to VACno (p=0.0.34 Delta; p=0.044 BA.1 and p=0.038 BA.4/5; Table 3). Of note, this difference was mainly driven by VAC2, rather than VAC3 (Supplementary Table 3). Activity versus seasonal coronaviruses was not different between the groups.
Cellular immune response was elevated in individuals who received vaccination, regardless to age, gender or cancer history
At admission, extensive phenotypic profiling was also performed to evaluate the immune activation in the B and T (CD4, CD8) cell compartments. All the cellular subpopulations were included in the linear regression models and reported in Table 3 and Supplementary Table 3.
With regards to B-cells, we observed only that the total B count was higher in VAC2+3 compared to VACno (p=0.0079), meanwhile none of the activated populations were different (Table 3 and Supplementary Table 3). This data was probably driven mostly by the comparison VAC2 vs VAC0 (Supplementary Table 3, p=0.0028). When looking at the CD4 sub-populations in the three vaccination groups, levels of Th1 lymphocytes (CCR6-/CXCR3+) appeared to be the most abundant compared to the other Th subtypes (Th2, Th17-1, Th17) (Figure 2A-B, Supplementary Table 3). Of note, the proportion of the Th2 cell varied across the groups, with the VAC2+3 showing higher levels compared to VACno (p=0.009, Figure 2A; adjusted value in Table 3). This difference remained significant also when the number of vaccine doses was considered. Indeed, both VAC2 (p=0.0233) and VAC3 (p=0.0241) had higher levels compared to VACno (adjusted values in Supplementary Table 3, Figure 2B). We did not observe significant differences regarding the other CD4 populations, a part of an increase of the effector memory CD4 in the VAC3 compared to VACno (EM-CD4+, p= 0.0325, Supplementary Table 3).
Finally, we explored the CD8 population and we found an increase of the proportion of CD8 in VAC2+3 compared to VACno (p=0.008, Table 3) and this association persisted only when comparing separately VACno to VAC3 (p=0.0319, Supplementary Table 3); furthermore, individuals who received 3 vaccine doses also had higher total CD8 counts (p=0.0002) compared to unvaccinated (Supplementary Table 3). When looking at the CD4/CD8 lymphocytes ratio (Figure 2C), consistently with the multivariable adjusted analysis, we observed an expansion of the CD8 in VAC3 group.
Soluble cytokines levels during COVID-19 pneumonia varied according to vaccination doses
Alongside the characterization of humoral and cellular responses of our cohort, we also profiled the serum levels of cytokines and included the data within the multivariable linear regression models (Table 3, Supplementary Table 3). Probably in response to COVID-19 pneumonia and independently from vaccine administration, cytokines levels appeared overall strongly correlated with each other, in the three VAC groups (Figure 3). No statistically significant differences were observed between vaccinated and not vaccinated patients (VAC2+3 vs VACno). When considering the number of vaccination doses (Supplementary Table 3), we found higher levels of GM-CSF in VAC2 vs VACno (p=0.0250), meanwhile the pro-inflammatory cytokine IFN-α appeared to be reduced in VAC3 vs VACno (p=0.0388).
Both humoral and cellular immune response is influenced by the virus variants driving pneumonia
Overall, Delta variant was the most representative in VAC2 (n=13/18, 72%), detected in 41% of VAC0, whereases undetected in VAC3 (Table 1). We then evaluated the impact of the type of variants (Delta vs Omicron, Table 4) using a multiple regression adjusted for vaccine dose, age, gender and cancer. Delta infections were able to elicit a higher humoral response in terms of IgM-S (p=0.0301) and IC50 vs Delta (p=0.0123), with a trend for higher IgG-RBD-S (p=0.0715). Further, infections with Delta also increased pro-inflammatory cytokines, such as IFN-a (p=0.0463) and IL-6 (p=0.0010). Alongside a trend for higher IgG-RBD-S in Delta, we also observed an expansion in the B cells compartments, including resting B cells (CD27+IgD-CD21+, p=0.0400) and Switched B cells (CD27+IgD-IgM-, p= 0.0176). Together with an increase of pentamer a-specific IgM-S in Delta infections, we reported higher levels of the naïve CD4 T cells (p=0.0025) and a decrease of the CD27- (memory) CD4 T cells (p=0.0147). Helper CD4 and CD8 populations did not appear to be affected by type of variants.