Respiratory viral infection promotes the awakening and outgrowth of dormant metastatic breast cancer cells in lungs (preprint)

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.

Immune imprinting revealed by SARS-CoV-2 Omicron infection prior to vaccination (preprint)

Immune imprinting or original antigenic sin (OAS) originally referred to a phenomenon of suboptimal immune response to a repeat exposure to a virus that was antigenically distinct from the original virus infection. OAS has been implicated in higher mortality in young people during the 2009-10 H1N1 pandemic where the elderly (H1N1 exposure in childhood) appeared relatively well protected compared to younger individuals whose first influenza infection was not H1N1. Immune imprinting is part of a rapid recall system and is highly effective against a slowly evolving virus (drifting) but not antigenically shifting viruses such as influenza and SARS CoV-2. As predicted by OAS, suboptimal neutralization responses to the highly divergent SARS-COV-2 lineage Omicron have been observed in animal models and individuals previously vaccinated with primary course of ancestral (Wu-1) vaccine. Due to the rapid scale up of vaccine before emergence of the antigenically distinct Omicron variant, it is unknown whether immunological imprinting for occurs in the context of SARS-COV-2 infection itself. We longitudinally assessed humoral responses to primary two dose Ad26.COV2.S Wu-hu-1 based vaccination in a Nigerian population following the global emergence of Omicron. At study entry in Jan 2023, we found 93% and 58% of pre-vaccination participants previously exposed to ancestral Wu-1 and Omicron virus respectively by anti-N IgG and anti-receptor binding domain (RBD) IgG Wu-1 and Omicron -specific antibodies. In participants with no evidence of prior exposure to Omicron, neutralisation against Wu-1 was significantly higher than Omicron variants as expected. However, serum neutralisation titres in participants who were anti-RBD Omicron IgG positive were paradoxically 2-fold lower for Omicron BA.1 as compared to Wu-1. This is clear evidence for imprinted immunity from the ancestral pre-omicron lineage viruses, and remarkably these old responses to Wu-1 were able to dominate over more recent, likely multiple, Omicron lineage infections. Furthermore, in these participants with prior exposure to Omicron and evidence of imprinting, we observed that further Omicron infection and Wu-1 based vaccine was associated with boosting of responses across variants with equalisation of neutralisation titres for Wu-1 and Omicron variants. However, omicron responses did not surpass ancestral responses, suggesting persistence of imprinting and only partial mitigation. Although neutralization responses at high titres were observed post dose 1 vaccination against ancestral and Omicron variants BA.1, BA.2, BA.4 in nearly all participants, neutralisation against the highly immune evasive XBB recombinant variant remained substantially lower, with a second vaccine dose providing very modest boosting. These data highlight immune imprinting against SARS-CoV-2 prior to vaccination and its persistence thereafter. In present day unvaccinated populations where serum neutralisation responses to pre-Omicron variants dominate, use of an omicron variant based vaccine should be used in preference to Wu-1 based vaccine to override imprinting and provide broader protection for vulnerable populations such as the elderly or those with compromised immunity.

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome (preprint)

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage (preprint)

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spikes interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Inhibitory Efficacy of Main Components of Scutellaria baicalensis on the Interaction between Spike Protein of SARS-CoV-2 and Human Angiotensin Converting Enzyme II (preprint)

Blocking the interaction of the SARS-CoV-2 spike protein with the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim in this study was to explore the efficacy of TCM treatments for SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. The antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis was found to be effective against SARS-CoV-2 infection through affecting the interaction of viral spike protein with the hACE2 protein. The active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside, respectively, found in S. baicalensis, exhibited strong inhibitory activities, targeting the viral spike protein or hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors of the interaction of the Omicron spike protein and hACE2 receptor from S. baicalensis. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed.

Single-Cell RNA Sequencing Reveals Heterogeneity of ALI Model and Epithelial Cell Alterations after Exposure to Electronic Cigarette Vapor (preprint)

Background Electronic cigarettes (e-cigarettes) have been advertised as a healthier alternative to traditional cigarettes; however, their exact effects on the bronchial epithelium are poorly understood. Air-liquid interface (ALI) culture allows human primary bronchial epithelial cells to differentiate into bronchial epithelium (ALI-HBE), providing an in vitro model that simulates the biological characteristics of normal bronchial epithelium.Methods Single-cell RNA sequencing of ALI-HBE was used to reveal previously unrecognized transcriptional heterogeneity within the human bronchial epithelium and cell type–specific responses to acute exposure to e-cigarette vapor (e-vapor) containing distinct components (nicotine and/or flavoring).Results Acute exposure to e-vapor containing nicotine affected gene expression related to secretory function and basal-to-secretory transformation. In addition, acute exposure to e-vapor containing flavoring might promote susceptibility to virus infection and activate epithelial-to-mesenchymal transition.Conclusion The ALI-HBE model recapitulates the heterogeneity and transcriptional characteristics of human bronchial epithelium. Single-cell sequencing data provided high-resolution insights into e-vapor–induced remodeling of bronchial epithelium. The data also indicate factors on bronchial epithelial cells that may promote SARS-CoV-2 infection and suggest therapeutic targets for restoring normal bronchial epithelium function after e-cigarette use.

Isogenic iPSC-derived proximal and distal lung-on-chip models: Tissue- and virus-specific immune responses in human lungs (preprint)

Micro-physiological systems (MPS) are set to play a vital role in preclinical studies, particularly in the context of future viral pandemics. Nonetheless, the development of MPS is often impeded by the scarcity of reliable cell sources, especially when seeking various organs or tissues from a single patient for comparative analysis of the host immune response. Herein, we developed human airway-on-chip and alveolus-on-chip models using induced pluripotent stem cell (iPSC)-derived isogenic lung progenitor cells. Both models demonstrated the replication of two different respiratory viruses, namely SARS-CoV-2 and Influenza, as well as related cellular damage and innate immune responses-on-chip. Our findings reveal distinct immune responses to SARS-CoV-2 in the proximal and distal lung-on-chip models. The airway chips exhibited a robust interferon (IFN)-dependent immune response, whereas the alveolus chips exhibited dysregulated IFN activation but a significantly upregulated chemokine pathway. In contrast, Influenza virus infection induced a more pronounced immune response and cellular damage in both chip models compared to SARS-CoV-2. Thus, iPSC-derived lung-on-chip models may aid in quickly gaining insights into viral pathology and screening potential drugs for future pandemics.

Simulation of COVID-19 Epidemic from Potential Viral Loads in Saudi Arabian Wastewater Treatment Plants (preprint)

SARS-CoV-2 is a contagious respiratory virus that has been discovered in sewage, human waste, and wastewater treatment facilities. Wastewater surveillance has been considered one of the lowest-cost means of testing for tracking the COVID-19 outbreak in communities. This paper highlights the dynamics of the virus's infection, persistence, and occurrence in wastewater treatment plants. Our aim is to develop and implement a mathematical model to infer the epidemic dynamics from the possible density of SARS-CoV-2 viral load in wastewater. We present a long-normal model and fractional order of susceptible-exposed-infected-recovery (SEIR) epidemic model for predicting the spread of the COVID-19 disease from the wastewater data. We study the dynamic properties of the fractional order SEIR model with respect to the fractional ordered values. The model is used to comprehend how the coronavirus spreads through wastewater treatment plants in Saudi Arabia. Our modeling approach can help with wastewater surveillance for early prediction and cost-effective monitoring of the epidemic outbreak in a situation of low testing capacity.

Growth media affects susceptibility of air-lifted human nasal epithelial cell cultures to SARS-CoV2, but not Influenza A, virus infection. (preprint)

Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus- grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.

Megakaryocyte infection by SARS-CoV-2 drives the formation of pathogenic afucosylated IgG antibodies in mice (preprint)

Despite more than 90% of total plasma fucosylated IgG, specific IgGs with low core fucosylation are found sporadically in response to enveloped virus infections and to alloantigens on blood cells. IgG responses with low core fucosylation are directly pathogenic in SARS-CoV-2 and dengue infections. In COVID-19, formation of IgG with low core fucosylation (afucosylated IgG) against spike protein (S) predicts and directly mediates disease progression to severe form. Low fucosylation of IgG causes increased antibody-dependent cellular toxicity mediated by intense Fc{gamma}R-mediated stimulation of platelets, monocytes, macrophages, and natural killer cells. The mechanism of afucosylated IgG formation has remained elusive thus far in COVID-19, dengue infection, and other disorders. This study demonstrates that infection of megakaryocytes by SARS-CoV-2 drives the formation of pathogenic anti-S afucosylated IgGs, causing pulmonary vascular thrombosis, acute lung injury, and death in Fc{gamma}-expressing mice.

SARS-CoV-2 CQ.1 and CQ.1.1 lineage isolates: 100% synonymous base substitution at furin arginine CGG–CGG codons (preprint)

The SARS‐CoV2 spike glycoprotin S1/S2 junction not only has no analogy in other becoronaviruses, including neither the Laos closest known relatives, but also it has two arginine codons CGG–CGG, whose usage is extremely rare in coronaviruses. The 12-nt SARS-CoV-2 S gene insert having that CGG–CGG genetic footprint 100% match to several human mRNA RefSeq transcripts which are located in exons of ubiquitous and highly expressed genes or specific genes of target human tissues of virus infection. An hypothesis for the probable human origin of the S1/S2 junction polybasic motif (originally PRRA) includes that it has been acquired by recombination between the genome of a SARS-CoV-2 progenitor and mRNA transcripts within human infected cells. Since the furin arginine pair is essential for virus infection to human cells and arginine has six codons SARS-CoV-2 can optimize the CGG–CGG codons. Here I show that in a sample of the available GISAID SARS-CoV-2 CQ.1 (516) and CQ.1.1 (117) lineage isolates the 100% of them showed synonymous base substitution at this position. The CGG–CGG footrprint to have changed to CGT–CGG. The location of the CQ.1 lineage isolates was: Asia 8.30% (45 isolates), Europe 76.57% (415), North America 6.64% (36), Oceania 8.30% (45) and South America 0.18% (1). That of CQ.1.1 lineage isolates was: Europe 45.6% (57) and North America 54.4 (68). Based on NCBI Virus database SARS-CoV-2 lineages associated to spike glycoprotein sequences I created a SARS-CoV-2 lineage ranking the from earliest CQ.1 date (August 30, 2022) up to date. Of 418,257 records, there were 1,165 distinct lineages. Ranking positions for CQ.1 and CQ.1.1 lineages were 582 and 506, respectivaly, both in the second quartile. These results coincide with those obtained previosly in SARS-CoV-2 CQ.2 and EE.2 lineage isolates. Everything points to that through evolution SARS-CoV-2 adapts the extremely rare CGG–CGG footprint to its own genomic parameters, thereby also erasing an evidence on its origin.

SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity (preprint)

The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.

Analyzing a SEIR-Type Mathematical Model of SARS-COVID-19 Using Piecewise Fractional Order Operators (preprint)

The continuing public health issue known as COVID-19 (the 2019 Novel Corona virus infection) has a global emphasis. Despite (or perhaps because of) the fact that there are significant gaps in our understanding of COVID-19 epidemiology, transmission dynamics, research methods, and management breakout poses a new kind of global hazard. The good news is that there is currently enough knowledge about the epidemic process to allow for the creation of mathematical forecasting models. We modify a conventional SEIR epidemic model to the unique dynamic compartments and epidemic features of COVID 19 as it spreads in a population with a diverse age structure. Although many US states and other nations around the world followed lockdown and reopening processes, we perform some analysis on using some techniques of the epidemic course. A new perspective of fractional calculus known as piecewise derivatives of fractional order is used to study the proposed model. Sufficient conditions are established to show the existence theory. In addition, a numerical scheme based on Newton’s polynomials is established to simulate the approximate solutions of the proposed model by using various fractional orders. Some real data results are also shown with comparison of the numerical results.

Impact of COVID-19 on gamete quality and embryo development in patients with COVID-19 positive undergoing ART (preprint)

Backgroud:COVID-19 was recognized a public health issue and SARS-CoV-2 was assumed to infect human ovary and cross the blood–testis barrier. Method:To explore the infection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients’ semen and follicular fluid and its potential clinical outcome. Ten female patients with an average age of 30.7±4.11years and eight male patients with an average age of 32.13±4.45 years diagnosed with COVID-19 and their spouses were negative to COVID-19 were included. Results: None of ten male and 8 female patients with COVID-19 affecting was absence of SARS-CoV-2 RNA in semen and follicular fluid. The sperm parameters, the rates of oocytes maturation, fertilization, cleavage and blastulation between the control and test group was not statistically significant (P>0.05),but the sperm quality, the oocyte maturation and fertilization, the blastulation showed a decline tendency in COVID-19 affected patients. Conclusion: COVID-19 affection may have an uncertian negative influence on the gamete quality and embryo development. Our new knowledge will help to evaluate the impact of COVID-19 on fertility in virus infected patients.

The association of gene expressions of MAP3K7, NF2, STK3 and STK4 therewith Quantum Resonance Magnetic Analyzer (QRMA) in COVID-19 patients (preprint)

Objectives COVID-19 is initiated by coronavirus infections and it can principally damage the cell’s function of the lung. Assessment of some important genes may capability more operational administration in patients. Therefore, in the recent study, we investigated mRNA and protein expression of MAP3K7, NF2, STK3 and STK4 in virus-infected patients compared with the control group. In addition, we used Quantum Resonance Magnetic Analyzer (QRMA) in order to analyze blood lipids, basic physical quality, lung function and immune system which are divided into twenty-five different variables in patients compared to healthy individuals.Materials and Methods The statistical population was divided into two different groups: patients and healthy individuals. The Expression of the mRNA level of objective genes was assessed with the SYBR Green Real-time Polymerase Chain Reaction method. The protein level expression of target genes was calculated by Enzyme-linked Immunosorbent Assay. Furthermore, in this study, we use QRMA to analyze some quantities such as blood lipids, basic physical quality, lung function and immune system in patients compared to healthy individuals. Statistical analyses were accomplished with SPSS software (version, 18) and Graph-Pad Prism software (Graph-Pad Prism, version 8.0.1).Results The mRNA and protein expressions level of NF2, MAP3K7, STK3 and STK4 genes in the group of patients decreased significantly compared to healthy individuals. The evaluation of blood lipids, basic physical quality, lung function and immune system in the study by QRMA showed that the Arterial oxygen content Paco2, low-density lipoprotein-C, tonsil immune index and immunoglobulin index were significantly higher in patients group compared to healthy individuals. In contrast, the amount of High-density lipoprotein-C, bone marrow index, respiratory immune index and gastrointestinal immune index were significantly lower in patients group compared to healthy individuals.Conclusions Compared to healthy individuals, the amount of mRNA and protein expressed by NF2, MAP3K7, STK3 and STK4 genes was reduced in COVID-19 patients. In truth, these genes have some critical function in some cellular and sub-cellular pathways. Accordingly, when the expression of genes decreased maybe it can increase the influence of coronavirus. Various mechanisms are involved in COVID-19, the increasing of the amount of the arterial oxygen content Paco2, low-density lipoprotein-C, tonsil immune index, immunoglobulin index and decreasing the amount of the High-density lipoprotein-C, bone marrow index, respiratory immune index and gastrointestinal immune index in COVID-19 patients, can be involved in the effects of coronavirus. Therefore, a deep perception of the relationship between these objective genes and QRMA therewith pathogenesis of COVID-19 infections in a larger population with longer supplements is required.

Seroprevalence of SARS-CoV-2 and humoral immune responses to mRNA vaccines among people who use drugs - In the light of tailored mitigating strategies (preprint)

Background People who use drugs (PWUD) have increased risk of acquiring SARS-CoV-2 and having severe courses of COVID-19. However, during the first wave of the pandemic, surprisingly few PWUD tested positive for SARS-CoV-2 in Oslo. Aims: To investigate the seroprevalence of SARS-CoV-2, the antibody responses to virus infections and SARS-CoV-2 vaccines, and the vaccination rate among PWUD compared to the general population. Methods: Design: A prospective cohort study. Setting: Data was collected from residents at six institutions for homeless PWUD and users of a low-threshold clinic for opioid agonist treatment. Data was collected at baseline (N=99) and follow-up (N=25) and consisted of questionnaires and blood samples. Data on vaccination was collected from the National Vaccine Register. Serologic methods included detection of antibodies to different virus proteins, detection of neutralizing antibodies to SARS-CoV-2, and antibodies to Spike-FL, receptor-binding domain of the Spike protein and nucleocapsid from SARS-CoV-2. Results Antibodies to SARS-CoV-2 were detected in 4/99 samples from PWUD in the months before vaccines were available. The corresponding frequency for population-based screening was 2.8%.  The levels of serum antibodies to seasonal coronaviruses and EBV in PWUD, were also similar to those measured in population-based screening. The levels of binding and neutralizing antibodies to SARS-CoV-2 measured in samples obtained from PWUD (N=25) after the second vaccine dose were comparable to those observed in healthy controls. Concerning humoral immune responses to COVID-19 vaccination, there was no difference between PWUD and healthy individuals. Eighty-four and eighty-nine per cent had received at least one dose of corona vaccine among PWUD and the general population, respectively. Conclusion Results showed that PWUD did not have increased seroprevalence of SARS-CoV-2 and did not have increased serum antibodies to seasonal coronaviruses and EBV. Vaccine responses were not different from controls demonstrating that vaccination is a viable strategy to confer protection against SARS-CoV-2 in PWUD

Comprehensive analysis of nasal IgA antibodies induced by intranasal administration of the SARS-CoV-2 spike protein (preprint)

Intranasal vaccination is an attractive strategy for preventing COVID-19 disease as it stimulates the production of multimeric secretory immunoglobulin A (IgAs), the predominant antibody isotype in the mucosal immune system, at the target site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. Currently, the evaluation of intranasal vaccine efficacy is based on the measurement of polyclonal antibody titers in nasal lavage fluid. However, how individual multimeric secretory IgA protects the mucosa from SARS-CoV-2 infection remains to be elucidated. To understand the precise contribution and molecular nature of multimeric secretory IgAs induced by intranasal vaccines, we developed 99 monoclonal IgAs from nasal mucosa and 114 monoclonal IgAs or IgGs from nonmucosal tissues of mice that were intranasally immunized with the SARS-CoV-2 spike protein. The nonmucosal IgAs exhibited shared origins and both common and unique somatic mutations with the related nasal IgA clones, indicating that the antigen-specific plasma cells in the nonmucosal tissues originated from B cells stimulated at the nasal mucosa. Comparing the spike protein binding reactivity, angiotensin-converting enzyme-2-blocking and SARS-CoV-2 virus neutralization of monomeric and multimeric IgA pairs recognizing different epitopes showed that even nonneutralizing monomeric IgA, which represents 70% of the nasal IgA repertoire, can protect against SARS-CoV-2 infection when expressed as multimeric secretory IgAs. Our investigation is the first to demonstrate the function of nasal IgAs at the monoclonal level, showing that nasal immunization can provide effective immunity against SARS-CoV-2 by inducing multimeric secretory IgAs at the target site of virus infection.

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine (preprint)

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions.  Outer membrane vesicles (OMV) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMV can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies (nAbs). The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induced nAbs against Omicron BA.1 and BA.5, as judged by pseudovirus infectivity assay. Importantly, we show that the RBM 438-509 ancestral-OMVs elicited antibodies which efficiently neutralized in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants, suggesting its potential use as a pan Coronavirus vaccine.  Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Longitudinal SARS-CoV-2 neutralization of Omicron BA.1, BA.5 and BQ.1.1 after four vaccinations and the impact of break-through infections in hemodialysis patients (preprint)

Background Individuals on hemodialysis are more vulnerable to SARS-CoV-2 infection than the general population due to end-stage kidney disease-induced immunosuppression. Methods 26 hemodialysis patients experiencing SARS-CoV-2 infection after 3rd vaccination were matched 1:1 to 26 out of 92 SARS-CoV-2 naives by age, sex, dialysis vintage and immunosuppressive drugs receiving a 4th vaccination with an mRNA-based vaccine. A competitive surrogate neutralization assay was used to monitor vaccination success. To determine infection neutralization titers, Vero-E6 cells were infected with SARS-CoV-2 variants of concern (VoC), Omicron sub-lineage BA.1, BA.5, and BQ.1.1. 50% inhibitory concentration (IC50, serum dilution factor 1:x) was determined before, four weeks after and 6 months after the 4th vaccination. Results 52 hemodialysis patients received four COVID-19 vaccinations and were followed up for a median of 6.3 months. Patient characteristics did not differ between the matched cohorts. Patients without a SARS-CoV-2 infection had a significant reduction of real virus neutralization capacity for all Omicron sub-lineages after six months (p<0.001 each). Those patients with a virus infection did not experience a reduction of real virus neutralization capacity after six months. Compared to the other Omicron VoC the BQ.1.1 sub-lineage had the lowest virus neutralization capacity. Conclusions SARS-CoV-2-naive hemodialysis patients had significantly decreased virus neutralization capacity six months after the 4th vaccination whereas patients with a SARS-CoV-2 infection had no change in neutralization capacity. This was independent of age, sex, dialysis vintage and immunosuppression. Therefore, in infection-naive hemodialysis patients a fifth COVID-19 vaccination might be reasonable 6 months after the 4th vaccination.

Impaired potency of neutralizing antibodies against cell-cell fusion mediated by SARS-CoV-2 (preprint)

The SARS-CoV-2 Omicron subvariants have dominated the pandemic due to their high transmissibility and immune evasion conferred by the spike mutations. The Omicron subvariants can spread by cell-free virus infection and cell-cell fusion, the latter of which is more effective but has not been extensively investigated. In this study, we developed a simple and high-throughput assay that provides a rapid readout to quantify cell-cell fusion mediated by the SARS-CoV-2 spike proteins without using live or pseudotyped virus. This assay can be used to identify variants of concern and to screen for prophylactic and therapeutic agents. We further evaluated a panel of monoclonal antibodies (mAbs) and vaccinee sera against D614G and Omicron subvariants, finding that cell-cell fusion is substantially more resistant to mAb and serum inhibition than cell-free virus infection. Such results have important implications for the development of vaccines and antiviral antibody drugs against cell-cell fusion induced by SARS-CoV-2 spikes.