Mapping the Antibody Repertoires in Ferrets with Repeated Influenza A/H3 Infections: Is Original Antigenic Sin Really "Sinful"?

The influenza-specific antibody repertoire is continuously reshaped by infection and vaccination. The host immune response to contemporary viruses can be redirected to preferentially boost antibodies specific for viruses encountered early in life, a phenomenon called original antigenic sin (OAS) that is suggested to be responsible for diminished vaccine effectiveness after repeated seasonal vaccination. Using a new computational tool called Neutralization Landscapes, we tracked the progression of hemagglutination inhibition antibodies within ferret antisera elicited by repeated influenza A/H3 infections and deciphered the influence of prior exposures on the de novo antibody response to evolved viruses. The results indicate that a broadly neutralizing antibody signature can nevertheless be induced by repeated exposures despite OAS induction. Our study offers a new way to visualize how immune history shapes individual antibodies within a repertoire, which may help to inform future universal influenza vaccine design.

Evaluation and limitations of different approaches among COVID-19 fatal cases using whole-exome sequencing data.

BACKGROUND: COVID-19 caused by the SARS-CoV-2 infection may result in various disease symptoms and severity, ranging from asymptomatic, through mildly symptomatic, up to very severe and even fatal cases. Although environmental, clinical, and social factors play important roles in both susceptibility to the SARS-CoV-2 infection and progress of COVID-19 disease, it is becoming evident that both pathogen and host genetic factors are important too. In this study, we report findings from whole-exome sequencing (WES) of 27 individuals who died due to COVID-19, especially focusing on frequencies of DNA variants in genes previously associated with the SARS-CoV-2 infection and the severity of COVID-19. RESULTS: We selected the risk DNA variants/alleles or target genes using four different approaches: 1) aggregated GWAS results from the GWAS Catalog; 2) selected publications from PubMed; 3) the aggregated results of the Host Genetics Initiative database; and 4) a commercial DNA variant annotation/interpretation tool providing its own knowledgebase. We divided these variants/genes into those reported to influence the susceptibility to the SARS-CoV-2 infection and those influencing the severity of COVID-19. Based on the above, we compared the frequencies of alleles found in the fatal COVID-19 cases to the frequencies identified in two population control datasets (non-Finnish European population from the gnomAD database and genomic frequencies specific for the Slovak population from our own database). When compared to both control population datasets, our analyses indicated a trend of higher frequencies of severe COVID-19 associated risk alleles among fatal COVID-19 cases. This trend reached statistical significance specifically when using the HGI-derived variant list. We also analysed other approaches to WES data evaluation, demonstrating its utility as well as limitations. CONCLUSIONS: Although our results proved the likely involvement of host genetic factors pointed out by previous studies looking into severity of COVID-19 disease, careful considerations of the molecular-testing strategies and the evaluated genomic positions may have a strong impact on the utility of genomic testing.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Systematic Genomic Surveillance of SARS-CoV-2 Virus on Illumina Sequencing Platforms in the Slovak Republic-One Year Experience.

To explore a genomic pool of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the pandemic, the Ministry of Health of the Slovak Republic formed a genomics surveillance workgroup, and the Public Health Authority of the Slovak Republic launched a systematic national epidemiological surveillance using whole-genome sequencing (WGS). Six out of seven genomic centers implementing Illumina sequencing technology were involved in the national SARS-CoV-2 virus sequencing program. Here we analyze a total of 33,024 SARS-CoV-2 isolates collected from the Slovak population from 1 March 2021, to 31 March 2022, that were sequenced and analyzed in a consistent manner. Overall, 28,005 out of 30,793 successfully sequenced samples met the criteria to be deposited in the global GISAID database. During this period, we identified four variants of concern (VOC)-Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529). In detail, we observed 165 lineages in our dataset, with dominating Alpha, Delta and Omicron in three major consecutive incidence waves. This study aims to describe the results of a routine but high-level SARS-CoV-2 genomic surveillance program. Our study of SARS-CoV-2 genomes in collaboration with the Public Health Authority of the Slovak Republic also helped to inform the public about the epidemiological situation during the pandemic.

Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection.

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

Functional and structural modifications of influenza antibodies during pregnancy.

Pregnancy represents a unique tolerogenic immune state which may alter susceptibility to infection and vaccine response. Here, we characterized humoral immunity to seasonal influenza vaccine strains in pregnant and non-pregnant women. Although serological responses to influenza remained largely intact during late pregnancy, distinct modifications were observed. Pregnant women had reduced hemagglutinin subtype-1 (H1)- IgG, IgG1, IgG2, and IgG3, hemagglutination inhibition, and group 1 and 2 stem IgG titers. Intriguingly, H1-specific avidity and FcγR1 binding increased, and influenza antibodies had distinct Fc and Fab glycans characterized by increased di-galactosylation and di-sialylation. H1-specific Fc-functionality (i.e. monocyte phagocytosis and complement deposition) was moderately reduced in pregnancy. Multivariate antibody analysis revealed two distinct populations (pregnant vs. non-pregnant) segregated by H1 FcγR1 binding, H1-IgG levels, and Fab and Fc glycosylation. Our results demonstrated a structural and functional modulation of influenza humoral immunity during pregnancy that was antigen-specific and consistent with reduced inflammation and efficient placental transport.

SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains.

SARS-CoV-2 variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta) show increased transmissibility and enhanced antibody neutralization resistance. Here we demonstrate in K18-hACE2 transgenic mice that B.1.1.7 and B.1.351 are 100-fold more lethal than the original SARS-CoV-2 bearing 614D. B.1.1.7 and B.1.351 cause more severe organ lesions in K18-hACE2 mice than early SARS-CoV-2 strains bearing 614D or 614G, with B.1.1.7 and B.1.351 infection resulting in distinct tissue-specific cytokine signatures, significant D-dimer depositions in vital organs and less pulmonary hypoxia signaling before death. However, K18-hACE2 mice with prior infection of early SARS-CoV-2 strains or intramuscular immunization of viral spike or receptor binding domain are resistant to the lethal reinfection of B.1.1.7 or B.1.351, despite having reduced neutralization titers against these VOC than early strains. Our results thus distinguish pathogenic patterns in K18-hACE2 mice caused by B.1.1.7 and B.1.351 infection from those induced by early SARS-CoV-2 strains, and help inform potential medical interventions for combating COVID-19.

Highly pathogenic avian influenza A/Guangdong/17SF003/2016 is immunogenic and induces cross-protection against antigenically divergent H7N9 viruses.

Avian influenza A(H7N9) epidemics have a fatality rate of approximately 40%. Previous studies reported that low pathogenic avian influenza (LPAI)-derived candidate vaccine viruses (CVVs) are poorly immunogenic. Here, we assess the immunogenicity and efficacy of a highly pathogenic avian influenza (HPAI) A/Guangdong/17SF003/2016 (GD/16)-extracted hemagglutinin (eHA) vaccine. GD/16 eHA induces robust H7-specific antibody responses in mice with a marked adjuvant antigen-sparing effect. Mice immunized with adjuvanted GD/16 eHA are protected from the lethal LPAI and HPAI H7N9 challenges, in stark contrast to low antibody titers and high mortality in mice receiving adjuvanted LPAI H7 eHAs. The protection correlates well with the magnitude of the H7-specific antibody response (IgG and microneutralization) or HA group 2 stem-specific IgG. Inclusion of adjuvanted GD/16 eHA in heterologous prime-boost improves the immunogenicity and protection of LPAI H7 HAs in mice. Our findings support the inclusion of GD/16-derived CVV in the pandemic preparedness vaccine stockpile.

Reduced Influenza B-Specific Postvaccination Antibody Cross-reactivity in the B/Victoria Lineage-Predominant 2019/20 Season.

BACKGROUND: The influenza activity of the 2019/20 season remained high and widespread in the United States with type B viruses predominating the early season. The majority of B viruses characterized belonged to B/Victoria (B/Vic) lineage and contained a triple deletion of amino acid (aa) 162-164 in hemagglutinin (3DEL). These 3DEL viruses are antigenically distinct from B/Colorado/06/2017 (CO/06)-the B/Vic vaccine component of the 2018/19 and 2019/20 seasons representing the viruses with a double deletion of aa 162-163 in hemagglutinin (2DEL). METHODS: We performed molecular characterization and phylogenetic analysis of circulating B/Vic viruses. We also conducted hemagglutination inhibition (HAI) assay using archived human postvaccination sera collected from healthy subjects administered with different types of 2018/19 or 2019/20 seasonal vaccines. Their HAI cross-reactivity to representative 3DEL viruses was analyzed. RESULTS: The CO/06-specific human postvaccination sera, after being adjusted for vaccine type, had significantly reduced HAI cross-reactivity toward representative 3DEL viruses, especially the 136E+150K subgroup. The geometric mean titers against 3DEL viruses containing 136E+150K mutations were 1.6-fold lower in all populations (P = .051) and 1.9-fold lower in adults (P = .016) compared with those against the 136E+150N viruses. CONCLUSIONS: Our results indicate that postvaccination antibodies induced by the B/Vic vaccine component of the 2019/20 influenza season had reduced HAI cross-reactivity toward predominant 3DEL viruses in the United States. A close monitoring of the 3DEL 136E+150K subgroup is warranted should this subgroup return and predominate the 2020/21 influenza season.

Imprinting of Repeated Influenza A/H3 Exposures on Antibody Quantity and Antibody Quality: Implications for Seasonal Vaccine Strain Selection and Vaccine Performance.

Background: Reduced seasonal influenza vaccine effectiveness (VE) was observed in individuals who received repeated annual vaccinations. Preexisting influenza antibody levels were also found inversely correlated with postvaccination titers. These reports suggest that preexisting immunity may affect contemporary seasonal vaccine performance. Methods: Influenza A/H3 specific cross-reactivity of postvaccination sera from humans with or without preexisting immunity was assessed by hemagglutination inhibition (HAI) assay. Ferret antisera induced by repeated H3 exposures were also subjected to HAI, antibody affinity, and antibody avidity analyses. Results: Human postvaccination sera derived from subjects with or without preexisting immunity showed different cross-reactivity against H3 variant viruses. Similarly, the breadth of cross-reactive ferret antibodies induced by repeated H3 exposures was also broadened. Antigenic differences between H3 viruses characterized by ferret antisera became smaller as the number of exposures increased. Although repeated H3 exposures induced "original antigenic sin" phenomena in HAI titers against later exposed viruses, resultant ferret antibodies showed gradually enhanced avidity for different H3/hemagglutinin. Increased antibody avidity was found to be inversely correlated with decreased antigenic differences among H3 viruses characterized. Conclusions: Our results suggest that repeated H3 exposures imprinted not only antibody quantity but also antibody quality. The "naive" ferret model currently used for vaccine strain selection does not recapitulate the complexity of human preexisting immunity. Vaccine strains identified hereby may not provide coverage sufficient for those who were frequently infected and/or vaccinated, leading to the reduced VE observed.

Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

Cancer-associated S100P protein binds and inactivates p53, permits therapy-induced senescence and supports chemoresistance.

S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression.

The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration.

Carbonic anhydrase IX is a hypoxia-induced transmembrane enzyme linked with solid tumors. It catalyzes the reversible hydration of CO2 providing bicarbonate ions for intracellular neutralization and protons for extracellular acidosis, thereby supporting tumor cell survival and invasiveness. CA IX is the only human CA isoform containing the proteoglycan (PG) domain in its extracellular part. The PG domain appears to enhance the catalytic activity of CA IX and mediate its binding to the extracellular matrix. Moreover, manipulation of the CA IX level by siRNA or overexpression modulates cell adhesion pathway so that in the presence of CA IX, cells display an increased rate of adhesion and spreading. Here we show that deletion of the PG domain as well as treatment with the PG-binding monoclonal antibody M75 can impair this CA IX effect. Accordingly, CA IX-expressing cells show more prominent and elongated maturing paxillin-stained focal contacts (FC) than CA IX-negative controls, proving the role of CA IX in cell spreading. However, during active cell movement, CA IX is relocalized to lamellipodia and improves migration via its catalytic domain. Thus, we examined the influence of CA IX on FC turnover in these structures. While the lamellipodial regions lacking CA IX display dash-like adhesions, the CA IX-enriched neighboring regions exhibit dynamic dot-like FCs. These results suggest that CA IX can promote initial adhesion through its PG domain, but at the same time it facilitates formation of nascent adhesions at the leading edge of moving cells. Thereby it may allow for transmission of large forces and enhanced migration rate, presumably through catalytic activity and impact of pHe on FC dynamics. Thus, we provide the first evidence that CA IX protein localizes directly in focal adhesion (FA) structures and propose its functional relationship with the proteins involved in the regulation of FC turnover and maturation.

Suppression of carbonic anhydrase IX leads to aberrant focal adhesion and decreased invasion of tumor cells.

Carbonic anhydrase IX (CA IX) is a well-recognized hypoxia marker with promising diagnostic and therapeutic value. CA IX regulates the pH in hypoxic tumor cells and, thereby, contributes to microenvironmental acidosis and cell migration. To gain a better insight into the molecular processes driven by CA IX, we performed gene expression profiling of HT-1080 fibrosarcoma cells subjected to CA IX depletion by shRNA silencing. We identified the focal adhesion pathway as being significantly inhibited in the absence of CA IX and confirmed this finding by functional assays. Thus, we obtained the first direct evidence for the role of CA IX in focal adhesion.