Second Boost of Omicron SARS-CoV-2 S1 Subunit Vaccine Induced Broad Humoral Immune Responses in Elderly Mice (preprint)

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death from the disease. However, repeated homologous boosters, while considered a solution for severe forms of the disease caused by new SARS-CoV-2 variants in elderly individuals and immunocompromised patients, cannot provide complete protection against breakthrough infections. This highlights the need for alternative platforms for booster vaccines. In our previous study, we assessed the boost effect of the SARS-CoV-2 Beta S1 recombinant protein subunit vaccine (rS1Beta) in aged mice primed with an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) via subcutaneous injection or intranasal delivery, which induced robust humoral immune responses (1). In this follow-up study, we demonstrated that a second booster dose of a non-adjuvanted recombinant Omicron (BA.1) S1 subunit vaccine with Toll-like receptor 4 (TLR4) agonist RS09 (rS1RS09OM) was effective in stimulating strong S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants in 100-week-old mice. Importantly, the second booster dose elicits cross-reactive antibody responses, resulting in ACE2 binding inhibition against the spike protein of SARS-CoV-2 variants, including Omicron (BA.1) and its subvariants. Interestingly, the levels of IgG and neutralizing antibodies correlated with the level of ACE2 inhibition in the booster serum samples, although Omicron S1-specific IgG level showed a weaker correlation compared to Wuhan S1-specific IgG level. Furthermore, we compared the immunogenic properties of the rS1 subunit vaccine in young, middle-aged, and elderly mice, resulting in reduced immunogenicity with age, especially an impaired Th1-biased immune response in aged mice. Our findings demonstrate that the new variant of concern (VOC) rS1 subunit vaccine as a second booster has the potential to offer cross-neutralization against a broad range of variants and to improve vaccine effectiveness against newly emerging breakthrough SARS-CoV-2 variants in elderly individuals who were previously primed with the authorized vaccines.

Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice (preprint)

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skin's immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages (preprint)

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal IgA response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal sIgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies targeting the receptor-binding domain of the spike protein (01A05, rmAb23, DXP-604 and XG014). Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2 and BA.4/5 with a 50-150-fold increase in potency, reaching the level of the most potent monoclonal antibodies described till date. In hACE2 transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Conversion of IgA and dimerization further enhanced or restored the neutralizing ability against the emerging Omicron sub-variants (DXP-604 for BQ.1, BQ.1.1 and BA2.75; 01A05 for BA2.75, BA.2.75.2 and XBB.1). Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by subvariants and, potentially, future VOCs.

Tetravalent SARS-CoV-2 S1 Subunit Protein Vaccination Elicits Robust Humoral and Cellular Immune Responses in SIV-Infected Rhesus Macaque Controllers (preprint)

The COVID-19 pandemic has highlighted the need for safe and effective vaccines to be rapidly developed and distributed worldwide, especially considering the emergence of new SARS-CoV-2 variants. Protein subunit vaccines have emerged as a promising approach due to their proven safety record and ability to elicit robust immune responses. In this study, we evaluated the immunogenicity and efficacy of an adjuvanted tetravalent S1 subunit protein COVID-19 vaccine candidate composed of the Wuhan, B.1.1.7 variant, B.1.351 variant, and P.1 variant spike proteins in a nonhuman primate model with controlled SIVsab infection. The vaccine candidate induced both humoral and cellular immune responses, with T- and B cell responses mainly peaking post-boost immunization. The vaccine also elicited neutralizing and cross-reactive antibodies, ACE2 blocking antibodies, and T-cell responses, including spike specific CD4+ T cells. Importantly, the vaccine candidate was able to generate Omicron variant spike binding and ACE2 blocking antibodies without specifically vaccinating with Omicron, suggesting potential broad protection against emerging variants. The tetravalent composition of the vaccine candidate has significant implications for COVID-19 vaccine development and implementation, providing broad antibody responses against numerous SARS-CoV-2 variants.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2 (preprint)

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters (preprint)

Immunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines. Here we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (ID, n=25) diseases. We show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to both virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus. Hence, additional booster doses are recommended to frail patients.

SARS-CoV-2 S1 Subunit Booster Vaccination Elicits Robust Humoral Immune Responses in Aged Mice (preprint)

Currently approved COVID-19 vaccines prevent symptomatic infection, hospitalization, and death of the disease. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants raises concerns of reduced vaccine effectiveness and increased risk of infection. Repeated homologous booster in elderly individuals and immunocompromised patients is considered to solve severe form of disease caused by new SARS-CoV-2 variants but cannot protect completely against breakthrough infection. In our previous study we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2-S1 (Ad5.S1) in mice, resulting in that a single immunization with Ad5.S1, via subcutaneously injection or intranasal delivery, induced robust humoral and cellular immune responses. As a follow up study, here we showed that vaccinated mice had high titers of anti-S1 antibodies at one year after vaccination compared to PBS immunized mice. Furthermore, one booster dose of non-adjuvanted recombinant S1Beta (rS1Beta) subunit vaccine was effective in stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against the Wuhan, Beta, and Delta strain with 3.6- to 19.5-fold change increases. Importantly, the booster dose elicits cross-reactive antibody responses resulting in ACE2 binding inhibition against spike of SARS-CoV-2 variants (Wuhan, Alpha, Beta, Gamma, Delta, Zeta, Kappa, New York, India) as early as two-week post-boost injection, persisting over 28 weeks after a booster vaccination. Interestingly, levels of neutralizing antibodies were correlated with not only level of S1-binding IgG but also level of ACE2 inhibition in the before- and after-booster serum samples. Our findings show that S1 recombinant protein subunit vaccine candidate as a booster has potential to offer cross-neutralization against broad variants, and has important implications for vaccine control of new emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccine like AZD1222 and Ad26.COV2.S.

Association between Vitamin D Serum Levels and Immune Response to the BNT162b2 Vaccine for SARS-CoV-2.

The use of micronutrients such as vitamin D could improve the response to viral vaccines, particularly in immunosuppressed and immunosenescent subjects. Here, we analysed the association between serum 25-hydroxyvitamin D (25OHD) levels and the immune response elicited by the BNT162b2 vaccine in a cohort of 101 healthcare workers naïve for SARS-CoV-2 infection. We observed no significant differences in anti-spike (S) IgG and T-cell responses according to the 25OHD status at baseline. However, significant correlations between the 25OHD concentration at baseline and (i) the anti-S response (p < 0.020) and (ii) the neutralizing antibody (NT) titre (p = 0.040) at six months after the second dose were detected. We concluded that adequate levels of vitamin D may improve the immune response to mRNA vaccines such as BNT162b2, and that further larger studies are warranted in order to confirm these preliminary observations.

ORFeome Phage Display Reveals a Major Immunogenic Epitope on the S2 Subdomain of SARS-Cov-2 Spike Protein (preprint)

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of Spike and lost neutralization efficacy against continuously emerging SARS-CoV-2 variants, which especially mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralizing antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both, NGS and epitope mapping of a monoclonal antibody with known binding site. The most prominent epitope captured represented parts of Spike´s fusion peptide (FP). It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell and the serine protease TMPRSS2 cleaves Spike within this sequence. Blocking of this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence were rather rare among SARS-CoV-2 variants so far, this may be an advantage in the fight against future virus variants.

Adenovirus-Vectored SARS-CoV-2 Vaccine Expressing S1-N Fusion Protein (preprint)

Additional COVID-19 vaccines that are safe, easy to manufacture, and immunogenic are needed for global vaccine equity. Here, we developed a recombinant type 5 adenovirus vector encoding for the SARS-CoV-2 S1 subunit antigen and nucleocapsid as a fusion protein (Ad5.SARS-CoV-2-S1N) delivered to BALB/c mice through multiple vaccine administration routes. A single subcutaneous (S.C.) immunization with Ad5.SARS-CoV-2-S1N induced a similar humoral response, along with a significantly higher S1-specific cellular response, as a recombinant type 5 adenovirus vector encoding for S1 alone (Ad5.SARS-CoV-2-S1). Immunogenicity was improved by homologous prime boost strategies, using either S.C. or intranasal (I.N.) delivery of Ad5.SARS-CoV-2-S1N, and further improved through heterologous prime boost, with traditional intramuscular (I.M.) injection, using subunit recombinant S1 protein. Priming with low dose (1x1010 v.p.) of Ad5.SARS-CoV-2-S1N and boosting with either wildtype recombinant rS1 or B.1.351 recombinant rS1 induced a robust neutralizing response, that was sustained against immune evasive Beta and Gamma SARS-CoV-2 variants, along with a long-lived plasma cell response in the bone marrow 29 weeks post vaccination. This novel Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity in mice and supports the further development of COVID-19 based vaccines incorporating the nucleoprotein as a target antigen.

mRNA-COVID19 vaccination can be considered safe and tolerable for frail patients (preprint)

Background Frail patients are considered at relevant risk of complications due to COVID-19 infection and, for this reason, are prioritized candidates for vaccination. As these patients were originally not included in the registration trials, fear related to vaccine side-effects and disease worsening was one of the reasons for vaccine hesitancy. Herein we report the safety profile of the prospective, multicenter, national VAX4FRAIL study (NCT04848493) to evaluate vaccines in a large trans-disease cohort of patients with solid or hematological malignancies, neurological and rheumatological diseases. Methods Between March 3rd and September 2nd, 2021, 566 patients were evaluable for safety endpoint: 105 received the mRNA-1273 vaccine and 461 the BNT162b2 vaccine. Frail patients were defined per protocol as patients under treatment with hematological malignancies (131), solid tumors (191), immune-rheumatological diseases (86), and neurological diseases (158), including multiple sclerosis and generalized myasthenia. The impact of the vaccination on the health status of patients was assessed through a questionnaire focused on the first week after each vaccine dose. Results The most frequently reported moderate-severe adverse events were pain at the injection site (60.3% after the first dose, 55.4% after the second), fatigue (30.1% - 41.7%), bone pain (27.4% - 27.2%) and headache (11.8% - 18.9%). Risk factors associated with the occurrence of severe symptoms after vaccine administration were identified through a multivariate logistic regression analysis: age was associated with severe fever presentation (younger patients vs. middle-aged vs. older ones), females presented a higher probability of severe pain at the injection site, fatigue, headache, and bone pain; the mRNA-1237 vaccine was associated with a higher probability of severe pain at the injection site and fever. After the first dose, patients presenting a severe symptom were at a relevant risk of recurrence of the same severe symptom after the second one. Overall, 11 patients (1.9%) after the first dose and 7 (1.2%) after the second one required to postpone or suspend the disease-specific treatment. Finally, 2 fatal events occurred among our 566 patients. These two events were considered unrelated to the vaccine. Conclusions Our study reports that mRNA-COVID-19 vaccination is safe also in frail patients as expected side effects were manageable and had a minimum impact on patient care path.

Humoral and T-cell immune response after three doses of mRNA SARS-CoV-2 vaccines in fragile patients: the Italian VAX4FRAIL study (preprint)

BackgroundPatients with solid or hematological tumors, neurological and immune-inflammatory disorders represent potentially fragile subjects with increased risk to experience severe COVID-19 and inadequate response to SARS-CoV2 vaccination. MethodsWe designed a prospective Italian multicentric study to assess humoral and T-cell response to SARS-CoV2 vaccination in patients (n=378) with solid tumors (ST), hematological malignancies (HM), neurological (ND) and immuno-rheumatological diseases (ID). The immunogenicity of primary vaccination schedule and of the booster dose were analyzed. ResultsOverall, patient seroconversion rate after two doses was 62.1%. A significant lower rate was observed in HM (52.4%) and ID (51.9%) patients compared to ST (95.6%) and ND (70.7%); a lower median level of antibodies was detected in HM and ID versus the others (p<0.0001). A similar rate of patients with a positive SARS-CoV2 T-cell response was observed in all disease groups, with a higher level observed in the ND group. The booster dose improved humoral responses in all disease groups, although with a lower response in HM patients, while the T-cell response increased similarly in all groups. In the multivariable logistic model, the independent predictors for seroconversion were disease subgroups, type of therapies and age. Notably, the ongoing treatment known to affect the immune system was associated with the worst humoral response to vaccination (p<0.0001), but had no effects on the T-cell responses. ConclusionsImmunosuppressive treatment more than disease type per se is a risk factor for low humoral response after vaccination. The booster dose can improve both humoral and T-cell response. Articles main point- Lower rate of seroconversion was observed in fragile patients as compared to healthy controls - The booster dose improves humoral and T-cell response in all fragile patient groups - Immunosuppressive treatment was associated with the worst humoral response to vaccination, but had no effects on T-cell responses.

Heterologous immunization with inactivated vaccine followed by mRNA booster elicits strong humoral and cellular immune responses against the SARS-CoV-2 Omicron variant (preprint)

Background There has been an unprecedented global effort to produce safe and effective vaccines against SARS-CoV-2. However, production challenges, supply shortages and unequal global reach, together with an increased number of breakthrough infections due to waning of immunity and the emergence of new variants of concern (VOC), have prolonged the pandemic. To boost the immune response, several heterologous vaccination regimes have been tested and have shown increased antibody responses compared to homologous vaccination. Here we evaluated the effect of mRNA vaccine booster on immunogenicity in individuals who had been vaccinated with two doses of inactivated vaccines. Methods The levels of specific antibodies against the receptor-binding domain (RBD) of the spike protein from wild-type virus and the Beta, Delta and Omicron variants were measured in healthy individuals who had received two doses of homologous inactivated (BBIBP-CorV or CoronoVac) or mRNA (BNT162b2 or mRNA-1273) vaccines, and in donors who were given an mRNA vaccine boost after two doses of either vaccine. Pre-vaccinated healthy donors, or individuals who had been infected and subsequently received the mRNA vaccine were also included as controls. In addition, specific memory B and T cell responses were measured in a subset of samples. Results A booster dose of an mRNA vaccine significantly increased the level of specific antibodies that bind to the RBD domain of the wild-type (6-fold) and VOCs including Delta (8-fold) and Omicron (14-fold), in individuals who had previously received two doses of inactivated vaccines. The level of specific antibodies in the heterologous vaccination group was furthermore similar to that in individuals receiving a third dose of homologous mRNA vaccines or boosted with mRNA vaccine after natural infection. Moreover, this heterologous vaccination regime significantly enhanced the specific memory B and T cell responses. Conclusions Heterologous prime-boost immunization with inactivated vaccine followed by an mRNA vaccine boost markedly increased the levels of specific antibodies and B and T cell responses and may thus increase protection against emerging SARS-CoV-2 variants including Omicron.

Human serum from SARS-CoV-2 vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant in comparison to the original Wuhan strain and the Beta and Delta variants (preprint)

Background The ongoing COVID-19 pandemic is caused by the beta coronavirus SARS-CoV-2. COVID-19 manifests itself from mild or even asymptomatic infections to severe forms of life-threatening pneumonia. At the end of November 2021, yet another novel SARS-CoV-2 variant named B.1.1.529 or Omicron was discovered and classified as a variant of concern (VoC) by the WHO. Omicron shows significantly more mutations in the amino acid (aa) sequence of its spike protein than any previous variant, with the majority of those concentrated in the receptor binding domain (RBD). In this work, the binding of the Omicron RBD to the human ACE2 receptor was experimentally analyzed in comparison to the original Wuhan SARS-CoV-2 virus, and the Beta and Delta variants. Moreover, we compared the ability of human sera from COVID-19 convalescent donors and persons fully vaccinated with BNT162b2 (Corminaty) or Ad26.COV2.S (Janssen COVID-19 vaccine) as well as individuals who had boost vaccine doses with BNT162b2 or mRNA-1273 (Spikevax) to bind the different RBDs variants. Methods The Omicron RBD with 15 aa mutations compared to the original Wuhan strain was produced baculovirus-free in insect cells. Binding of the produced Omicron RBD to hACE was analyzed by ELISA. Sera from 27 COVID-19 patients, of whom 21 were fully vaccinated and 16 booster recipients were titrated on the original Wuhan strain, Beta, Delta and Omicron RBD and compared to the first WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) using the original Wuhan strain as reference. Results The Omicron RBD showed a slightly reduced binding to ACE2 compared to the other RBDs. The serum of COVID-19 patients, BNT162b2 vaccinated and boost vaccinated persons showed a reduced binding to Omicron RBD in comparison to the original Wuhan strain, Beta und Delta RBDs. In this assay, the boost vaccination did not improve the RBD binding when compared to the BNT162b2 fully vaccinated group. The RBD binding of the Ad26.COV2.S serum group was lower at all compared to the other groups. Conclusions The reduced binding of human sera to Omicron RBD provides first hints that the current vaccinations using BNT162b2, mRNA-1273 and Ad26.COV2.S may be less efficient in preventing infections with the Omicron variant.

Immunity to SARS-CoV-2 up to 15 months after infection (preprint)

Background: Information concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset. Methods: The levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison. Findings: Following a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months. Conclusion: The data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies. Funding: EU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.

SARS-CoV-2 vaccine breakthrough infections with the alpha variant are asymptomatic or mildly symptomatic among health care workers (preprint)

Vaccine breakthrough SARS-CoV-2 infection has been monitored in 3720 healthcare workers receiving 2 doses of BNT162b2. SARS-CoV-2 infection is detected in 33 subjects, with a 100-day cumulative incidence of 0.93%. Vaccine protection against acquisition of SARS-CoV-2 infection is 83% (95%CI: 58-93%) in the overall population and 93% (95%CI: 69-99%) in SARS-CoV-2-experienced subjects, when compared with a non-vaccinated control group from the same Institution, in which SARS-CoV-2 infection occurs in 20/346 subjects (100-day cumulative incidence: 5.78%). The infection is symptomatic in 16 (48%) vaccinated subjects vs 17 (85%) controls (p=0.001). All analyzed patients, in whom the amount of viral RNA was sufficient for genome sequencing, results infected by the alpha variant. Antibody and T-cell responses are not reduced in subjects with breakthrough infection. Evidence of virus transmission, determined by contact tracing, is observed in two (6.1%) cases. This real-world data support the protective effect of BNT162b2 vaccine. A triple antigenic exposure, such as two-dose vaccine schedule in experienced subjects, may confer a higher protection.

SARS-CoV-2 mRNA vaccine BNT162b2 elicited a robust humoral and cellular response against SARS-CoV-2 variants. (preprint)

BNT162b2 vaccine was introduced in Italy on 27th December 2020 and healthcare workers were rapidly vaccinated. In this study, we demonstrated that one vaccine dose was sufficient for eliciting a sustained humoral and cell-mediated response in SARS-CoV-2 experienced healthcare workers but had a lower effect in SARS-CoV-2 naïve subjects. However, 98% naïve subjects developed both neutralizing antibodies and Spike-specific T-cells after the second dose. Moreover, the antibody and T-cell responses were effective against viral variants since a partial reduction in antibody response was observed only against the South-African variant in SARS-CoV-2 naïve individuals, while the T-cell response was less affected.

Neutrophil extracellular traps induce the epithelial-mesenchymal transition: implications in post-COVID-19 fibrosis (preprint)

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Neutrophils activated with PMA (PMA-Neu), a stimulus known to induce NETs formation, induce both EMT and cell death in the lung epithelial cell line, A549. Notably, NETs isolated from PMA-Neu induce EMT without cell damage. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs. Thus, we tested in an in vitro alveolar model the hypothesis that virus-induced NET may drive EMT. Co-culturing A549 at air-liquid interface with alveolar macrophages, neutrophils and SARS-CoV2, we demonstrated a significant induction of the EMT in A549 together with high concentration of NETs, IL8 and IL1{beta}, best-known inducers of NETosis. Lung tissues of COVID-19 deceased patients showed that epithelial cells are characterized by increased mesenchymal markers. These results show for the first time that NETosis plays a major role in triggering lung fibrosis in COVID-19 patients.