Restrained memory CD8+ T cell responses favors viral persistence and elevated IgG responses in patients with severe Long COVID. (preprint)

During the COVID-19 pandemic it was widely described that certain individuals infected by SARS-CoV-2 experience persistent disease signs and symptoms, Long COVID, which in some cases is very severe with life changing consequences. To maximize our chances of identifying the underpinnings of this illness, we have focused on 121 of the most severe cases from >1000 patients screened in specialized clinics in Sweden and Belgium. We restricted this study to subjects with objective measures of organ damage or dysfunction, >3 months following a verified, but mild-to-moderate SARS-CoV-2 infection. By performing systems-level immunological testing and comparisons to controls fully convalescent following a similar mild/moderate COVID-19 episode, we identify elevated serological responses to SARS-CoV-2 in severe Long COVID suggestive of chronic antigen stimulation. Persistent viral reservoirs have been proposed in Long COVID and using multiple orthogonal methods for detection of SARS-CoV-2 RNA and protein in plasma we identify a subset of patients with detectable antigens, but with minimal overlap across assays, and no correlation to symptoms or immune measurements. Elevated serologic responses to SARS-CoV-2 on the other hand were inversely correlated with clonally expanded memory CD8+ T cells, indicating that restrained clonal expansion enables viral persistence, chronic antigen exposure and elevated IgG responses, even if antigen-detection in blood is not universally possible.

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.

Heterologous inactivated virus/mRNA vaccination response to BF.7, BQ.1.1, and XBB.1 (preprint)

The emergence of highly immune-escape Omicron variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as BQ and XBB, has led to concerns about the efficacy of vaccines. Using lentivirus-based pseudovirus neutralizing assay, we showed that heterologous vaccination involving parental mRNA vaccine as a booster or second booster in individuals that received two or three doses of inactivated vaccines strongly augments the neutralizing activity against emerging Omicron subvariants, including BF.7, BQ.1.1, and XBB.1, by 4.3- to 219-folds. Therefore, a heterologous boosting strategy with mRNA-based vaccines should be considered in populations where inactivated vaccines were primarily used.

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with critical COVID-19 (preprint)

Background We previously reported inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity in 1-5% of unvaccinated patients with life-threatening COVID-19, and autoantibodies against type I IFN in another 15-20% of cases. Methods We report here a genome-wide rare variant burden association analysis in 3,269 unvaccinated patients with life-threatening COVID-19 (1,301 previously reported and 1,968 new patients), and 1,373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. A quarter of the patients tested had antibodies against type I IFN (234 of 928) and were excluded from the analysis. Results No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI:1.5-528.7, P=1.1x10-4), in analyses restricted to biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70 [95%CI:1.3-8.2], P=2.1x10-4). Adding the recently reported TYK2 COVID-19 locus strengthened this enrichment, particularly under a recessive model (OR=19.65 [95%CI:2.1-2635.4]; P=3.4x10-3). When these 14 loci and TLR7 were considered, all individuals hemizygous (n=20) or homozygous (n=5) for pLOF or bLOF variants were patients (OR=39.19 [95%CI:5.2-5037.0], P=4.7x10-7), who also showed an enrichment in heterozygous variants (OR=2.36 [95%CI:1.0-5.9], P=0.02). Finally, the patients 13 with pLOF or bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10-5). Conclusions Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie lifethreatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.

Design, structure and plasma binding of ancestral β-CoV scaffold antigens (preprint)

The pandemic caused by Severe acute respiratory syndrome coronavirus 2 has had devastating consequences on global health and economy. Despite the success of vaccination campaigns emerging variants are of concern and novel viruses with the potential to drive future pandemics are circulating in nature. Development of vaccines can be challenging, as key viral protein antigens can be unstable or aggregate. In this study, we present the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interacted with plasma of patients recovered from COVID-19 but did not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yielded high resolution structures (2.6-2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. This captured closed state is stabilised by an intricate hydrogen bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together, which determines their relative spatial orientation. Finally, we show how AnSAs are potent scaffolds by replacing the ancestral RBD with the Wuhan wild-type sequence, which restored ACE2 binding and increased the interaction with convalescent plasma. In contrast to rational antigen design depending on prior structural knowledge, our work highlights how stable and potentially interesting antigens can be generated using exclusively available sequence information.

Genome-wide detection of human variants that disrupt intronic branchpoints (preprint)

ABSTRACT Pre-mRNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Fifty-six rare variants in 44 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach has been available to efficiently detect such variants in next-generation sequencing (NGS) data. We established a comprehensive human genome-wide BP database by integrating existing BP data, and by generating new BP data from RNA-seq of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We in-depth characterize multiple features of BP in major and minor introns, and find that BP and BP-2 (two-nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, whilst being comparable to the exonic background. We develop BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition in NGS data. BPHunter retrospectively identifies 48 of the 56 known pathogenic BP mutations in which we summarize a strategy for prioritizing BP mutation candidates, and the remaining 8 all create AG dinucleotides between BP and acceptor site which is probably the reason for mis-splicing. We demonstrate the utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia, and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which we validate experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter .

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies (preprint)

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-β are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

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.

Persistence of SARS-CoV-2 specific B- and T-cell responses in convalescent COVID-19 patients 6-8 months after the infection (preprint)

Background: The longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. Methods: A cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. Results: Anti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. Conclusions: Although the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection).

Differing impacts of global and regional responses on SARS-CoV-2 transmission cluster dynamics (preprint)

Although the global response to COVID-19 has not been entirely unified, the opportunity arises to assess the impact of regional public health interventions and to classify strategies according to their outcome. Analysis of genetic sequence data gathered over the course of the pandemic allows us to link the dynamics associated with networks of connected individuals with specific interventions. In this study, clusters of transmission were inferred from a phylogenetic tree representing the relationships of patient sequences sampled from December 30, 2019 to April 17, 2020. Metadata comprising sampling time and location were used to define the global behavior of transmission over this earlier sampling period, but also the involvement of individual regions in transmission cluster dynamics. Results demonstrate a positive impact of international travel restrictions and nationwide lockdowns on global cluster dynamics. However, residual, localized clusters displayed a wide range of estimated initial secondary infection rates, for which uniform public health interventions are unlikely to have sustainable effects. Our findings highlight the presence of so-called "super-spreaders", with the propensity to infect a larger-than-average number of people, in countries, such as the USA, for which additional mitigation efforts targeting events surrounding this type of spread are urgently needed to curb further dissemination of SARS-CoV-2.

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago (preprint)

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.