Broad sarbecovirus neutralization by combined memory B cell antibodies to ancestral SARS-CoV-2.

Antibodies play a pivotal role in protecting from SARS-CoV-2 infection, but their efficacy is challenged by the continuous emergence of viral variants. In this study, we describe two broadly neutralizing antibodies cloned from the memory B cells of a single convalescent individual after infection with ancestral SARS-CoV-2. Cv2.3194, a resilient class 1 anti-RBD antibody, remains active against Omicron sub-variants up to BA.2.86. Cv2.3132, a near pan-Sarbecovirus neutralizer, targets the heptad repeat 2 membrane proximal region. When combined, Cv2.3194 and Cv2.3132 form a complementary SARS-CoV-2 neutralizing antibody cocktail exhibiting a local dose-dependent synergy. Thus, remarkably robust neutralizing memory B cell antibodies elicited in response to ancestral SARS-CoV-2 infection can withstand viral evolution and immune escape. The cooperative effect of such antibody combination may confer a certain level of protection against the latest SARS-CoV-2 variants.

SLC38A9 regulates SARS-CoV-2 viral entry.

SARS-CoV-2 viral entry into host cells depends on the cleavage of spike (S) protein into S1 and S2 proteins. Such proteolytic cleavage by furin results in the exposure of a multibasic motif on S1, which is critical for SARS-CoV-2 viral infection and transmission; however, how such a multibasic motif contributes to the infection of SARS-CoV-2 remains elusive. Here, we demonstrate that the multibasic motif on S1 is critical for its interaction with SLC38A9, an endolysosome-resident arginine sensor. SLC38A9 knockdown prevents S1-induced endolysosome de-acidification and blocks the S protein-mediated entry of pseudo-SARS-CoV-2 in Calu-3, U87MG, Caco-2, and A549 cells. Our findings provide a novel mechanism in regulating SARS-CoV-2 viral entry; S1 present in endolysosome lumen could interact with SLC38A9, which mediates S1-induced endolysosome de-acidification and dysfunction, facilitating the escape of SARS-CoV-2 from endolysosomes and enhancing viral entry.

The alarmin IL-33 exacerbates pulmonary inflammation and immune dysfunction in SARS-CoV-2 infection.

Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In this study, we observed that IL-33, a damage-associated molecular pattern molecule, is significantly increased in COVID-19 patients and in SARS-CoV-2-infected mice. Using IL-33-/- mice, we demonstrated that IL-33 deficiency resulted in significant decreases in bodyweight loss, tissue viral burdens, and lung pathology. These improved outcomes in IL-33-/- mice also correlated with a reduction in innate immune cell infiltrates, i.e., neutrophils, macrophages, natural killer cells, and activated T cells in inflamed lungs. Lung RNA-seq results revealed that IL-33 signaling enhances activation of inflammatory pathways, including interferon signaling, pathogen phagocytosis, macrophage activation, and cytokine/chemokine signals. Overall, these findings demonstrate that the alarmin IL-33 plays a pathogenic role in SARS-CoV-2 infection and provides new insights that will inform the development of effective therapeutic strategies for COVID-19.

An HIV-1 CRISPR-Cas9 membrane trafficking screen reveals a role for PICALM intersecting endolysosomes and immunity.

HIV-1 hijacks host proteins involved in membrane trafficking, endocytosis, and autophagy that are critical for virus replication. Molecular details are lacking but are essential to inform on the development of alternative antiviral strategies. Despite their potential as clinical targets, only a few membrane trafficking proteins have been functionally characterized in HIV-1 replication. To further elucidate roles in HIV-1 replication, we performed a CRISPR-Cas9 screen on 140 membrane trafficking proteins. We identified phosphatidylinositol-binding clathrin assembly protein (PICALM) that influences not only infection dynamics but also CD4+ SupT1 biology. The knockout (KO) of PICALM inhibited viral entry. In CD4+ SupT1 T cells, KO cells exhibited defects in intracellular trafficking and increased abundance of intracellular Gag and significant alterations in autophagy, immune checkpoint PD-1 levels, and differentiation markers. Thus, PICALM modulates a variety of pathways that ultimately affect HIV-1 replication, underscoring the potential of PICALM as a future target to control HIV-1.

Establishment of a multisite umbrella cohort study protocol to describe the epidemiology and aetiologies of acute undifferentiated febrile illness in Latin America.

INTRODUCTION: Acute undifferentiated febrile illnesses (AUFIs) impose a large burden in the tropics. Understanding of AUFI's epidemiology is limited. Insufficient diagnostic capacity hinders the detection of outbreaks. The lack of interconnection in healthcare systems hinders timely response. We describe a protocol to study the epidemiology and aetiologies of AUFI and pathogen discovery in strategic areas of Latin America (LA). METHODS AND ANALYSIS: Global Infectious Diseases Network investigators comprising institutions in Colombia, Dominican Republic, México, Perú and the USA, developed a common cohort study protocol. The primary objective is to determine the aetiologies of AUFI at healthcare facilities in high-risk areas. Data collection and laboratory testing for viral, bacterial and parasitic agents are performed in rural and urban healthcare facilities and partner laboratories. Centralised laboratory and data management cores deploy diagnostic tests and data management tools. Subjects >6 years with fever for <8 days without localised infection are included in the cohort. They are evaluated during the acute and convalescent phases of illness. Study personnel collect clinical and epidemiological information. Blood, urine, nasal or pharyngeal swabs and saliva are collected in the acute phase and blood in convalescent phase. Specimens are banked at -80°C. Malaria, dengue and COVID-19 are tested onsite in the acute phase. The acute-phase serum is PCR tested for dengue, chikungunya, Venezuelan equine encephalitis, Mayaro, Oropouche, Zika, and yellow fever viruses. Paired convalescent and acute serum antibody titters are tested for arbovirus, Leptospira spp, and Rickettsia spp. Serum is used for viral cultures and next-generation sequencing for pathogen discovery. Analysis includes variable distributions, risk factors and regression models. Laboratory results are shared with health authorities and network members. ETHICS AND DISSEMINATION: The protocol was approved by local ethics committees and health authorities. The results will be published in peer-reviewed journals. All study results are shared with local and regional health authorities.

Reduced anti-viral IgG repertoire in HIV-exposed but uninfected infants compared to HIV-unexposed infants.

Infants who are HIV exposed but uninfected (iHEU) have higher risk of viral infections compared to infants who are HIV unexposed (iHUU). We explored the effect of intrauterine HIV exposure on the infant antibody repertoire by quantifying plasma immunoglobulin (Ig) G against 206 eukaryote-infecting viruses using phage immunoprecipitation sequencing (PhiPSeq) in iHEU and iHUU at birth and 36 weeks of life. Maternal HIV infection altered the infant IgG repertoire against eukaryote-infecting viruses at birth, resulting in significantly lower antibody breadth and diversity among iHEU compared to iHUU. Neonatal anti-viral IgG repertoire was dominated by antibodies against viruses belonging to the Herpesviridae family, although, by 36 weeks, this had shifted toward antibodies against enteroviruses, likely due to waning of maternal-derived antibodies and polio vaccine-induced antibody responses as expected. The observed reduced anti-viral IgG repertoire breadth and diversity acquired at birth in iHEU might contribute to the increased rates of viral infections among iHEU during early life.

Humoral and cellular immune responses following Omicron BA.2.2 breakthrough infection and Omicron BA.5 reinfection.

The emergence of novel Omicron subvariants has raised concerns regarding the efficacy of immunity induced by prior Omicron subvariants breakthrough infection (BTI) or reinfection against current circulating Omicron subvariants. Here, we prospectively investigated the durability of antibody and T cell responses in individuals post Omicron BA.2.2 BTI, with or without subsequent Omicron BA.5 reinfection. Our findings reveal that the emerging Omicron subvariants, including CH.1.1, XBB, and JN.1, exhibit extensive immune evasion induced by previous infections. Notably, the level of IgG and neutralizing antibodies were found to correlate with subsequent Omicron BA.5 reinfection. Fortunately, T cell responses recognizing both Omicron BA.2 and CH.1.1 peptides were observed. Furthermore, Omicron BA.5 reinfection may alleviate immune imprinting induced by WT-vaccination, bolster virus-specific ICS+ T cell responses, and promote the phenotypic differentiation of virus-specific memory CD8+ T cells. Antigen-updated or T cell-conserved vaccines are needed to control the transmission of diverse emerging SARS-CoV-2 variants.

Anti-SARS-CoV-2 antibodies in a nasal spray efficiently block viral transmission between ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread in the population. We recently reported the production of bovine colostrum-derived antibodies that can neutralize the virus. These have been formulated into a nasal spray. The immunoglobulin preparation is capable of blocking interaction of the trimeric spike protein (Tri S) of SARS-CoV-2 with the cellular receptor angiotensin-converting enzyme 2 (ACE2), entry of a pseudovirus carrying the Tri S into ACE2 over-expressing human embryonic kidney (HEK) cells, and entry of the virus into live Vero E6 cells. Using an ELISA assay, we demonstrate here that this holds true for different SARS-CoV-2 variants of concern. Using the ferret transmission model, we show that the nasal spray formulation of anti-SARS-CoV-2 immunoglobulins efficiently blocks transmission of SARS-CoV-2 from infected to uninfected ferrets. The results indicate that the use of the nasal spray in humans can add an effective additional layer of protection against the virus, and might be applicable for other viruses of the upper respiratory tract.

Inhibiting retinoic acid signaling in dendritic cells suppresses respiratory syncytial virus infection through enhanced antiviral immunity.

Retinoic acid (RA), controls the immunoregulatory functions of many immune cells, including dendritic cells (DCs), and is important for mucosal immunity. In DCs, RA regulates the expression of pattern recognition receptors and stimulates interferon production. Here, we investigated the role of RA in DCs in mounting immunity to respiratory syncytial virus (RSV). To abolish RA signaling in DCs, we used mice expressing a dominant negative form of retinoic acid receptor-α (RARα) under the CD11c promoter (CD11c-dnRARα). Paradoxically, upon RSV challenge, these animals had lower viral burden, reduced pathology, and greater Th1 polarized immunity than wild-type (WT) mice. Moreover, CD11c-dnRARα DCs infected with RSV showed enhancement in innate and adaptive immunity genes, while genes associated with viral replication were downregulated. These findings suggest that the absence of RA signaling in DCs enhances innate immunity against RSV infection leading to decreased viral load and reduced pathogenicity.

Alphaherpesvirus manipulates retinoic acid metabolism for optimal replication.

Retinoic acid (RA), derived from retinol (ROL), is integral to cell growth, differentiation, and organogenesis. It is known that RA can inhibit herpes simplex virus (HSV) replication, but the interactions between alphaherpesviruses and RA metabolism are unclear. Our present study revealed that alphaherpesvirus (HSV-1 and Pseudorabies virus, PRV) infections suppressed RA synthesis from ROL by activating P53, which increased retinol reductase 3 (DHRS3) expression-an enzyme that converts retinaldehyde back to ROL. This process depended on the virus-triggered DNA damage response, the degradation of class I histone deacetylases, and the subsequent hyperacetylation of histones H3 and H4. Counteracting DHRS3 or P53 enabled higher RA synthesis and reduced viral growth. RA enhanced antiviral defenses by promoting ABCA1- and ABCG1-mediated lipid efflux. Treatment with the retinoic acid receptor (RAR) agonist palovarotene protected mice from HSV-1 infection, thus providing a potential therapeutic strategy against viral infections.

Evaluation of an RBD-nucleocapsid fusion protein as a booster candidate for COVID-19 vaccine.

Despite successful vaccines and updates, constant mutations of SARS-CoV-2 makes necessary the search for new vaccines. We generated a chimeric protein that comprises the receptor-binding domain from spike and the nucleocapsid antigens (SpiN) from SARS-CoV-2. Once SpiN elicits a protective immune response in rodents, here we show that convalescent and previously vaccinated individuals respond to SpiN. CD4+ and CD8+ T cells from these individuals produced greater amounts of IFN-γ when stimulated with SpiN, compared to SARS-CoV-2 antigens. Also, B cells from these individuals were able to secrete antibodies that recognize SpiN. When administered as a boost dose in mice previously immunized with CoronaVac, ChAdOx1-S or BNT162b2, SpiN was able to induce a greater or equivalent immune response to homologous prime/boost. Our data reveal the ability of SpiN to induce cellular and humoral responses in vaccinated human donors, rendering it a promising candidate.

Immunosuppression-induced Zika virus reactivation causes brain inflammation and behavioral deficits in mice.

Zika virus (ZIKV) is a neurotropic flavivirus that can persist in several tissues. The late consequences of ZIKV persistence and whether new rounds of active replication can occur, remain unaddressed. Here, we investigated whether neonatally ZIKV-infected mice are susceptible to viral reactivation in adulthood. We found that when ZIKV-infected mice are treated with immunosuppressant drugs, they present increased susceptibility to chemically induced seizures. Levels of subgenomic flavivirus RNAs (sfRNAs) were increased, relative to the amounts of genomic RNAs, in the brains of mice following immunosuppression and were associated with changes in cytokine expression. We investigated the impact of immunosuppression on the testicles and found that ZIKV genomic RNA levels are increased in mice following immunosuppression, which also caused significant testicular damage. These findings suggest that ZIKV can establish new rounds of active replication long after acute stages of disease, so exposed patients should be monitored to ensure complete viral eradication.

Cytokine-like-Vago-mediated antiviral response in Penaeus monodon via IKK-NF-κB signaling pathway.

Interferon (IFN) system is the primary mechanism of innate antiviral defense in immune response. To date, limited studies of IFN system were conducted in crustaceans. Previous report in Penaeus monodon demonstrated the interconnection of cytokine-like molecule Vago and inhibitor of kappa B kinase-nuclear factor κB (IKK-NF-κB) cascade against white spot syndrome virus (WSSV). This study further identified five different PmVago isoforms. Upon immune stimulation, PmVagos expressed against shrimp pathogens. PmVago1, PmVago4, and PmVago5 highly responded to WSSV, whereas, PmVago1 and PmVago4 RNAi exhibited a rapid mortality with elevated WSSV replication. Suppression of PmVago1 and PmVago4 negatively affected proPO system, genes in signal transduction, and AMPs. WSSV infection additionally induced PmVaog4 granule accumulation and cellular translocation to the area of cell membrane. More importantly, PmVago1 and PmVago4 promoters were stimulated by PmIKK overexpression; meanwhile, they further activated Dorsal and Relish promoter activities. These results suggested the possible roles of the cytokine-like PmVago via IKK-NF-κB cascade against WSSV infection.

Sirtuin 2 regulates neutrophil functions through NAD+ synthesis pathway in virus infection.

Neutrophils play an important role in antiviral immunity, but the underlying mechanisms remain unclear. Here, we found that SIRT2 deficiency inhibited the infiltration of neutrophils, as well as the secretion of inflammatory cytokines and the formation of neutrophil extracellular traps (NETs), ameliorating disease symptoms during acute respiratory virus infection. Mechanistically, SIRT2 deficiency upregulates quinolinic acid (QA)-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) and leads to expression of quinolinate phosphoribosyltransferase (QPRT), which promotes the synthesis of QA for NAD+ and limits viral infection when de novo NAD+ synthesis is blocked. Tryptophan-2,3-oxygenase expressed in epithelial cells metabolizes tryptophan to produce kynurenine and 3-hydroxyaminobenzoic acid, which is a source of intracellular QA in neutrophils. Thus, our findings reveal a previously unrecognized QPRT-mediated switch in NAD+ metabolism by exploiting neutrophil-derived QA as an alternative source of replenishing intracellular NAD+ pools induced by SIRT2 to regulate neutrophil functions during virus infection, with implications for future immunotherapy approaches.

Ten-year retrospective data analysis reveals frequent respiratory co-infections in hospitalized patients in Augsburg.

Clinical data on the types of respiratory pathogens which are most frequently engaged in respiratory co-infections of children and adults are lacking. We analyzed 10 years of data on a total of over 15,000 tests for 16 viral and bacterial pathogens detected in clinical samples at the University Hospital of Augsburg, Germany. Co-infection frequencies and their seasonal patterns were examined using a proportional distribution model. Co-infections were detected in 7.3% of samples, with a higher incidence in children and males. The incidence of interbacterial and interviral co-infections was higher than expected, whereas bacterial-viral co-infections were less frequent. H. influenzae, S. pneumoniae, rhinovirus, and respiratory syncytial virus (RSV) were most frequently involved. Most co-infections occurred in winter, but distinct summer peaks were also observed, which occurred even in children, albeit less pronounced than in adults. Seasonality of respiratory (co-)infections decreased with age. Our results suggest to adjust existing testing strategies during high-incidence periods.

A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

Nudiviruses in free-living and parasitic arthropods: evolutionary taxonomy.

The nudiviruses (family: Nudiviridae) are large double-stranded DNA (dsDNA) viruses that infect insects and crustaceans, and have most recently been identified from ectoparasitic members (fleas and lice). This virus family was created in 2014 and has since been expanded via the discovery of multiple novel viral candidates or accepted members, sparking the need for a new taxonomic and evolutionary overview. Using current information (including data from public databases), we construct a new comprehensive phylogeny, encompassing 49 different nudiviruses. We use this novel phylogeny to propose a new taxonomic structure of the Nudiviridae by suggesting two new viral genera (Zetanudivirus and Etanudivirus), from ectoparasitic lice. We detail novel emerging relationships between nudiviruses and their hosts, considering their evolutionary history and ecological role.

Optimization data for an ARTIC-/Illumina-based whole-genome sequencing protocol and pipeline for SARS-CoV-2 analysis.

In January 2021, Germany commenced surveillance of SARS-CoV-2 variants under the Corona Surveillance Act, which ceased in July 2023. The objective was to bolster pandemic control, as specific alterations in amino acids, particularly within the spike protein, were linked to heightened transmission and decreased vaccine effectiveness. Consequently, our team conducted whole genome sequencing using the commercially accessible ARTIC protocol on Illumina's NextSeq500 platform and MiSeq for SARS-CoV-2 positive samples obtained from patients at Heidelberg University Hospital, affiliated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. Throughout the pandemic, we refined the existing ARTIC V4 protocol as well as our bioinformatics pipeline, the details of which are outlined in this report. This report reflects the protocol for the MiSeq analysis, the protocol for the NextSeq500 can be found in our previous publication.

A cryptic site in class 5 epitope of SARS-CoV-2 RBD maintains highly conservation across natural isolates.

The emergence of SARS-CoV-2 variants raises concerns about the efficacy of existing COVID-19 vaccines and therapeutics. Previously, we identified a conserved cryptic class 5 epitope of SARS-CoV-2 receptor binding domain (RBD) by two cross-neutralizing antibodies 7D6 and 6D6. Intriguingly, this site remains resistant to substantial mutations occurred in ever-changing SARS-CoV-2 subvariants. As compared to class 3 antibody S309, 6D6 maintains broad and consistent neutralizing activities against SARS-CoV-2 variants. Furthermore, 6D6 effectively protected hamster from the virulent Beta strain. Sequence alignment of approximately 6 million documented SARS-CoV-2 isolates revealed that 6D6 epitope maintains an exceptionally high conservation rate (99.92%). Structural analysis demonstrated that all 33 mutations accumulated in XBB.1.5 since the original strain do not perturb the binding 6D6 to RBD, in line with the sequence analysis throughout the antigenicity evolution of SARS-CoV-2. These findings suggest the potential of this epitope serving as a critical determinant for vaccines and therapeutic design.