Protective antibodies and T cell responses to Omicron variant three months after the booster dose of BNT162b2 vaccine (preprint)

The high number of mutations in the Omicron variant of SARS-CoV-2 cause its immune escape when compared to the earlier variants of concern (VOC). At least three vaccine doses are required for the induction of Omicron neutralizing antibodies and further reducing the risk for hospitalization. However, most of the studies have focused on the immediate response after the booster vaccination while the duration of immune response is less known. We here studied longitudinal serum samples from the vaccinated individuals up to three months after their third dose of the BNT162b2 vaccine for their capacity to produce protective antibodies and T cell responses to Wuhan and Omicron variants. After the second dose, the antibody levels to the unmutated spike protein were significantly decreased at three months, and only 4% of the individuals were able to inhibit Omicron spike interaction compared to 47%, 38%, and 14% of individuals inhibiting wild-type, delta, and beta variants spike protein. Nine months after the second vaccination, the antibody levels were similar to the levels before the first dose and none of the sera inhibited SARS-CoV-2 wild-type or any of the three VOCs. The booster dose remarkably increased antibody levels and their ability to inhibit all variants. Three months after the booster the antibody levels and the inhibition activity were trending lower but still up and not significantly different from their peak values at two weeks after the third dose. Although responsiveness towards mutated spike peptides was lost in less than 20 % of vaccinated individuals, the wild-type spike-specific CD4+ and CD8+ memory T cells were still present at three months after the booster vaccination in the majority of studied individuals. Our data show that two doses of the BNT62b2 vaccine are not sufficient to protect against the Omicron variant, however, the spike-specific antibodies and T cell responses are strongly elicited and well maintained three months after the third vaccination dose.

Bovine Colostrum Derived Antibodies Against SARS-CoV-2 Show Great Potential to Serve as a Prophylactic Agent (preprint)

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) until now imposes a serious burden to health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is still ongoing. One of the mechanisms how neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2 specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum just before calving. Here we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein, and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persists on the nasal mucosa for at least 4 hours as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for a rapid and versatile adaption for preparing prophylactic treatments against other diseases by using the defined characteristics of antibody movement into the colostrum.

Declined Antibody Responses to COVID-19 mRNA Vaccine within First Three Months (preprint)

Background: Although the SARS-CoV-2 mRNA vaccines have proven high efficacy, limited data exists on the duration of immune responses and their relation to age and side effects.Methods: We studied the antibody and memory T cell responses to Spike protein after the two-dose Comirnaty mRNA vaccine in 122 volunteers up to 3 months and correlated the findings with age and side effects.Findings: We found a robust antibody response after the second vaccination dose. However, the antibody levels declined at 6 and 12 weeks postvaccination, indicating a waning of the immune response. Regardless, the average levels remained higher compared to pre-vaccination or in COVID-19 convalescent individuals. The antibodies efficiently blocked ACE2 receptor binding to Spike protein of four variants of concern at one week but this was decreased at three months, in particular with B.1.351 and P1 isolates. 87% of individuals developed Spike-specific memory T cell responses, which were lower in individuals with increased proportions of immunosenescent CD8+ TEMRA cells. We found a decreased vaccination efficacy but fewer adverse events in older individuals, suggesting a detrimental impact of age on outcome.Interpretation: The mRNA vaccine induces a strong antibody response to four variants at 1 week postvaccination but decreases thereafter, in particular among older individuals. T cell responses, although detectable in the majority, were lower in individuals with immunosenescence. The deterioration of vaccine response needs to be monitored to define the optimal time for the revaccination. Funding: The Estonian Research Council, Icosagen Cell Factory, and SYNLAB Estonia.Declaration of Interests: None to declare. Ethics Approval Statement: The study has been approved by the Research Ethics Committee of the University of Tartu on February 15, 2021 (No 335/T-21). Patients signed informed consent before recruitment into the study.

Declined antibody responses to COVID-19 mRNA vaccine within first three months (preprint)

Background Although the SARS-CoV-2 mRNA vaccines have proven high efficacy, limited data exists on the duration of immune responses and their relation to age and side effects. Methods We studied the antibody and memory T cell responses to Spike protein after the two-dose Comirnaty mRNA vaccine in 122 volunteers up to 3 months and correlated the findings with age and side effects. Findings We found a robust antibody response after the second vaccination dose. However, the antibody levels declined at 6 and 12 weeks postvaccination, indicating a waning of the immune response. Regardless, the average levels remained higher compared to pre-vaccination or in COVID-19 convalescent individuals. The antibodies efficiently blocked ACE2 receptor binding to Spike protein of four variants of concern at one week but this was decreased at three months, in particular with B.1.351 and P1 isolates. 87% of individuals developed Spike-specific memory T cell responses, which were lower in individuals with increased proportions of immunosenescent CD8+ TEMRA cells. We found a decreased vaccination efficacy but fewer adverse events in older individuals, suggesting a detrimental impact of age on outcome. Interpretation The mRNA vaccine induces a strong antibody response to four variants at 1 week postvaccination but decreases thereafter, in particular among older individuals. T cell responses, although detectable in the majority, were lower in individuals with immunosenescence. The deterioration of vaccine response needs to be monitored to define the optimal time for the revaccination. Funding The Estonian Research Council, Icosagen Cell Factory, and SYNLAB Estonia. Research in context Evidence before this study The first studies addressing the immune responses in older individuals after the administration of SARS-CoV-2 mRNA vaccines have been published. We searched PubMed and medRxiv for publications on the immune response of SARS-CoV-2-mRNA vaccines, published in English, using the search terms “SARS-CoV-2”, “COVID-19”, “vaccine response”, “mRNA vaccine”, up to May 20th, 2021. To date, most mRNA vaccine response studies have not been peer-reviewed, and data on the dynamics of antibody response, role of age and side effects on SARS-CoV-2-mRNA vaccines in real vaccination situations is limited. Some studies have found a weaker immune response in older individuals after the first dose and these have been measured at a relatively short period (within one to two weeks) after the first dose but little longer-term evidence exists on the postvaccination antibody persistence. Added value of this study In this study, we assessed the antibody response up to three months after the full vaccination with Pfizer-BioNTech Comirnaty mRNA vaccine in 122 individuals. Our findings show strong Spike RBD antibody responses one week after the second dose with the capacity to block ACE2-Spike protein interaction, however, the antibodies declined significantly at three months after the second dose. The inhibition of ACE2-Spike interaction was weaker with South African (B.1.351) and Brazilian (P.1) than with Wuhan and UK (B.1.1.7) SARS-CoV-2 isolates. At three months 87% of vaccinated individuals developed either CD4+ or CD8+ T cell responses. Those negative for Spike-specific T cell response also tended to have lower Spike-specific antibody levels. In addition, CD4+ T cell response was decreased among vaccinated individuals with elevated levels of senescent CD8+ TEMRA cells. We found a weaker antibody response and faster waning of antibodies in older vaccinated individuals, which correlated with fewer side effects at the time of vaccinations. Implications of all the available evidence Our results show that two doses of Pfizer-BioNTech Comirnaty mRNA vaccine induce a strong antibody and T cell responses to Spike RBD region but the antibody levels are declined at three months after the second dose. Nevertheless, even at three months, the anti-Spike RBD antibody levels stay significantly higher than at prevaccination, after the first dose of vaccine, or in Covid-19 postinfection. Our findings implicate older individuals to have fewer vaccination adverse effects and weaker immune response after the vaccination and point to the need for more individualized vaccination protocols, in particular among older people.

ACE2 peptide fragment interacts with several sites on the SARS-CoV-2 spike protein S1 (preprint)

The influence of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2) was studied to model the interaction of the virus with its host cell. This peptide corresponds to the sequence 24–42 of the ACE2 α1 domain, which is the binding site for the S1 protein. The on-rate and off-rate of S1-ACE2 complex formation were measured in the presence of various peptide concentrations using Bio-Layer Interferometry (BLI). The formation of the S1-ACE2 complex was inhibited when the S1 protein was preincubated with the peptide, however, no significant inhibitory effect was observed in the absence of preincubation. Dissociation kinetics revealed that the peptide remained bound to the S1-ACE2 complex and stabilized this complex. This suggestion was confirmed by computational mapping of the S1 protein surface for peptide binding that revealed two additional sites, located at some distance from the receptor binding domain of S1. These additional binding sites may affect the interaction between the peptide, the S1 protein, and ACE2.

ACE2 peptide fragment interacts with several sites on the SARS-CoV-2 spike protein S1 (preprint)

The influence of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2(ACE2) was studied to model the interaction of the virus with its host cell. This peptide corresponds to the sequence 24-42 of the ACE2 1 domain, which is the binding site for the S1 protein. The on-rate and off-rate of S1-ACE2 complex formation were measured in the presence of various peptide concentrations using Bio-Layer Interferometry (BLI). The formation of the S1-ACE2 complex was inhibited when the S1 protein was preincubated with the peptide, however, no significant inhibitory effect was observed in the absence of preincubation. Dissociation kinetics revealed that the peptide remained bound to the S1-ACE2 complex and stabilized this complex. Computational mapping of the S1 protein surface for peptide binding revealed two additional sites, located at some distance from the receptor binding domain (RBD) of S1. These additional binding sites affect the interaction between the peptide, the S1 protein, and ACE2.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations (preprint)

Recombinant neutralizing antibodies (nAbs) derived from recovered patients have proven to be effective therapeutics for COVID-19. Here, we describe the use of advanced protein engineering and modular design principles to develop tetravalent synthetic nAbs that mimic the multi-valency exhibited by IgA molecules, which are especially effective natural inhibitors of viral disease. At the same time, these nAbs display high affinity and modularity typical of IgG molecules, which are the preferred format for drugs. We show that highly specific tetravalent nAbs can be produced at large scale and possess stability and specificity comparable to approved antibody drugs. Moreover, structural studies reveal that the best nAb targets the host receptor binding site of the virus spike protein, and thus, its tetravalent version can block virus infection with a potency that exceeds that of the bivalent IgG by an order of magnitude. Design principles defined here can be readily applied to any antibody drug, including IgGs that are showing efficacy in clinical trials. Thus, our results present a general framework to develop potent antiviral therapies against COVID-19, and the strategy can be readily deployed in response to future pathogenic threats.

Identification of novel antiviral drug combinations in vitro and tracking their development (preprint)

Combination therapies have become a standard for the treatment for HIV and HCV infections. They are advantageous over monotherapies due to better efficacy and reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify several new synergistic combinations against emerging and re-emerging viral infections in vitro. We observed synergistic activity of nelfinavir with investigational drug EIDD-2801 and convalescent serum against SARS-CoV-2 infection in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of vemurafenib combination with emetine, homoharringtonine, gemcitabine, or obatoclax against echovirus 1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar and niclosamide were synergistic against HCV infection in hepatocyte derived Huh-7.5 cells, whereas combinations of monensin with lamivudine and tenofovir were synergistic against HIV-1 infection in human cervical TZM-bl cells. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status. Overall, the development of combinational therapies could have a global impact improving the preparedness and protection of the general population from emerging and re-emerging viral threats.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells (preprint)

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. (preprint)

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune {gamma}-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.