Ronapreve (REGN-CoV; casirivimab and imdevimab) reduces the viral burden and alters the pulmonary response to the SARS-CoV-2 Delta variant (B.1.617.2) in K18-hACE2 mice using an experimental design reflective of a treatment use case.

With some exceptions, global policymakers have recommended against the use of existing monoclonal antibodies in COVID-19 due to loss of neutralization of newer variants. The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication using paradigms for susceptible and insusceptible variants. Virological efficacy and impact on pathogenicity was assessed in K18-hACE2 mice inoculated with either the Delta or BA.1 Omicron variants. Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post-infection, for the Delta variant but not the Omicron variant. It also blocked brain infection, which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a multifocal granulomatous inflammation in which the virus appeared to be confined. The current study provides evidence of an altered tissue response to SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data demonstrate that experimental designs that reflect treatment use cases are achievable in animal models for monoclonal antibodies. Extreme caution should be taken when interpreting prophylactic experimental designs that may not be representative of treatment.IMPORTANCEFollowing the emergence of the SARS-CoV-2 Omicron variant, the WHO recommended against the use of Ronapreve in its COVID-19 treatment guidelines due to a lack of efficacy based on current pharmacokinetic-pharmacodynamic understanding. However, the continued use of Ronapreve, specifically in vulnerable patients, was advocated by some based on in vitro neutralization data. Here, the virological efficacy of Ronapreve was demonstrated in both the lung and brain compartments using Delta as a paradigm for a susceptible variant. Conversely, a lack of virological efficacy was demonstrated for the Omicron variant. Comparable concentrations of both monoclonal antibodies were observed in the plasma of Delta- and Omicron-infected mice. This study made use of a reliable murine model for SARS-CoV-2 infection, an experimental design reflective of treatment, and demonstrated the utility of this approach when assessing the effectiveness of monoclonal antibodies.

Sequential Infection with Influenza A Virus Followed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads to More Severe Disease and Encephalitis in a Mouse Model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection ('twinfection') is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.

Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2.

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the 'arms race' with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.

Liquid biopsy for molecular characterization of diffuse large B-cell lymphoma and early assessment of minimal residual disease.

Circulating tumour DNA (ctDNA) allows genotyping and minimal residual disease (MRD) detection in lymphomas. Using a next-generation sequencing (NGS) approach (EuroClonality-NDC), we evaluated the clinical and prognostic value of ctDNA in a series of R-CHOP-treated diffuse large B-cell lymphoma (DLBCL) patients at baseline (n = 68) and after two cycles (n = 59), monitored by metabolic imaging (positron emission tomography combined with computed tomography [PET/CT]). A molecular marker was identified in 61/68 (90%) ctDNA samples at diagnosis. Pretreatment high ctDNA levels significantly correlated with elevated lactate dehydrogenase, advanced stage, high-risk International Prognostic Index and a trend to shorter 2-year progression-free survival (PFS). Valuable NGS data after two cycles of treatment were obtained in 44 cases, and 38 achieved major molecular response (MMR; 2.5-log drop in ctDNA). PFS curves displayed statistically significant differences among those achieving MMR versus those not achieving MMR (2-year PFS of 76% vs. 0%, p < 0.001). Similarly, more than 66% reduction in ΔSUVmax by PET/CT identified two subgroups with different prognosis (2-year PFS of 83% vs. 38%; p < 0.001). Combining both approaches MMR and ΔSUVmax reduction, a better stratification was observed (2-year PFS of 84% vs. 17% vs. 0%, p < 0.001). EuroClonality-NDC panel allows the detection of a molecular marker in the ctDNA in 90% of DLBCL. ctDNA reduction at two cycles and its combination with interim PET results improve patient prognosis stratification.

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection.

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in severe models of COVID-19.

Objectives: Immunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. Methods: Mice were immunosuppressed using cyclophosphamide and infected with a B lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed. Results: Treatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of A>G and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir.Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs). Conclusions: Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression.

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.

A novel strategy for an anti-idiotype vaccine: nanobody mimicking neutralization epitope of porcine circovirus type 2.

Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses.

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1-11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely.