Achieving complete metabolic response in stage IV lung adenocarcinoma with chemotherapy, nivolumab, ipilimumab, and salvage SBRT: A case report.

Oncogene-negative, PDL1-negative metastatic non-small cell lung cancer (NSCLC) presents significant treatment challenges due to its complexity and resistance to conventional therapies. The case report presented addresses a 55-year-old male patient with oncogene-negative, PDL1-negative stage IV lung adenocarcinoma, showcasing an exceptional complete metabolic response to a multimodality treatment combining double immune checkpoint inhibition (ICI) and chemotherapy, followed by salvage stereotactic body radiotherapy (SBRT). The patient underwent a treatment regimen incorporating two cycles of carboplatin, pemetrexed, nivolumab, and ipilimumab followed by nivolumab, and ipilimumab maintenance. After a partial response, SBRT was applied to persistent lesions, achieving a complete metabolic response. This case highlights the potential of combining dual ICI with chemotherapy and SBRT in treating oncogene-negative, PDL1-negative NSCLC underscoring the importance of multimodality treatment strategies.

Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient virion production.

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV. IMPORTANCE: Arenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection.

Comprehensive profiling of neutralizing polyclonal sera targeting coxsackievirus B3.

Despite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus, coxsackievirus B3. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predictive of escape, assess epitope dominance at the population level, and reveal a need for at least two mutations to achieve significant escape from multiple sera. Overall, our data provide a comprehensive analysis of how polyclonal sera target a non-enveloped viral capsid and help define both immune dominance and escape at the population level.

Antibody-dependent NK-cell and neutralizing antibody responses against the Spike protein of Wuhan-Hu-1 and Omicron BA.1 SARS-CoV-2 variants in vaccinated experienced and vaccinated naïve individuals.

Antibodies triggering Fc-mediated NK cell activity may contribute to protection against disease caused by SARS-CoV-2 infection in humans. However, how these Fc-mediated humoral responses compare between individuals displaying hybrid immunity (Vac-ex) and those fully vaccinated with no history of SARS-CoV-2 infection (Vac-n) and whether they correlate with neutralizing antibody (NtAb) responses remains largely undetermined. In this retrospective study serum samples from 50 individuals (median age, 44.5 years; range, 11-85; 25 males), 25 Vac-ex and 25 Vac-n were studied. A flow-cytometry-based antibody-mediated NK-cell activation assay was used to quantitate effector NK-cells stimulated to express LAMP1 (lysosomal associated membrane protein 1), MIP1 (Macrophage inflammatory protein 1), and interferon-γ (IFNγ); NK cells isolated from two donors (D1 and D2) were used. NtAb levels targeting the Spike protein of Wuhan-Hu-1 and Omicron BA.1 SARS-CoV-2 variants were quantitated using a SARS-CoV-2 S pseudotyped neutralization assay. Regardless of the SARS-CoV-2 variant S antigen used in the NK-cell activation assay, the frequency of NK cells stimulated to express LAMP-1, MIP1ß, and IFNγ was higher in Vac-ex compared with Vac-n (p values ranging from 0.07 to 0.006) for D1; this was only seen for BA.1 when NK cells from D2 were employed. The frequency of functional NK cells activated by antibody binding to either Wuhan-Hu-1 or Omicron BA.1 S protein was not significantly different for both VAC-ex and VAC-n. In contrast, NtAb titers against BA.1 were around 10-fold lower than that against Wuhan-Hu-1. Vac-ex displayed higher NtAb titers against both (sub)variants than Vac-n. NK-cell responses correlated poorly with NtAb titers (ρ ≤ 0.30). The data demonstrate higher cross-reactivity across variants of concern for antibodies triggering Fc-mediated NK cell than for NtAb. Moreover, Vac-Ex seemed to display more robust functional antibody responses as compared with Vac-n.

A machine learning model for predicting serum neutralizing activity against Omicron SARS-CoV-2 BA.2 and BA.4/5 sublineages in the general population.

Supervised machine learning (ML) methods have been used to predict antibody responses elicited by COVID-19 vaccines in a variety of clinical settings. Here, we explored the reliability of a ML approach to predict the presence of detectable neutralizing antibody responses (NtAb) against Omicron BA.2 and BA.4/5 sublineages in the general population. Anti-SARS-CoV-2 receptor-binding domain (RBD) total antibodies were measured by the Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics) in all participants. NtAbs against Omicron BA.2 and BA4/5 were measured using a SARS-CoV-2 S pseudotyped neutralization assay in 100 randomly selected sera. A ML model was built using the variables of age, vaccination (number of doses) and SARS-CoV-2 infection status. The model was trained in a cohort (TC) comprising 931 participants and validated in an external cohort (VC) including 787 individuals. Receiver operating characteristics analysis indicated that an anti-SARS-CoV-2 RBD total antibody threshold of 2300 BAU/mL best discriminated between participants either exhibiting or not detectable Omicron BA.2 and Omicron BA.4/5-Spike targeted NtAb responses (87% and 84% precision, respectively). The ML model correctly classified 88% (793/901) of participants in the TC: 717/749 (95.7%) of those displaying ≥2300 BAU/mL and 76/152 (50%) of those exhibiting antibody levels <2300 BAU/mL. The model performed better in vaccinated participants, either with or without prior SARS-CoV-2 infection. The overall accuracy of the ML model in the VC was comparable. Our ML model, based upon a few easily collected parameters for predicting neutralizing activity against Omicron BA.2 and BA.4/5 (sub)variants circumvents the need to perform not only neutralization assays, but also anti-S serological tests, thus potentially saving costs in the setting of large seroprevalence studies.

A performance comparison of two (electro) chemiluminescence immunoassays for detection and quantitation of serum anti-spike antibodies according to SARS-CoV-2 vaccination and infections status.

The information provided by SARS-CoV-2 spike (S)-targeting immunoassays can be instrumental in clinical-decision making. We compared the performance of the Elecsys® Anti-SARS-CoV-2 S assay (Roche Diagnostics) and the LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin) using a total of 1176 sera from 797 individuals, of which 286 were from vaccinated-SARS-CoV-2/experienced (Vac-Ex), 581 from vaccinated/naïve (Vac-N), 147 from unvaccinated/experienced (Unvac-Ex), and 162 from unvaccinated/naïve (Unvac-N) individuals. The Roche assay returned a higher number of positive results (907 vs. 790; p = 0.45; overall sensitivity: 89.3% vs. 77.6%). The concordance between results provided by the two immunoassays was higher for sera from Vac-N (Ï°: 0.58; interquartile ranges [IQR]: 0.50-0.65) than for sera from Vac-Ex (Ï°: 0.19; IQR: -0.14 to 0.52) or Unvac-Ex (Ï°: 0.18; IQR: 0.06-0.30). Discordant results occurred more frequently among sera from Unvac-Ex (34.7%) followed by Vac-N (14.6%) and Vac-Ex (2.7%). Antibody levels quantified by both immunoassays were not significantly different when <250 (p = 0.87) or <1000 BAU/ml (p = 0.13); in contrast, for sera ≥1000 BAU/ml, the Roche assay returned significantly higher values than the DiaSorin assay (p < 0.008). Neutralizing antibody titers (NtAb) were measured in 127 sera from Vac-Ex or Vac-N using a S-pseudotyped virus neutralization assay of Wuhan-Hu-1, Omicron BA.1, and Omicron BA.2. The correlation between antibody levels and NtAb titers was higher for sera from Vac-N than those from Vac-Ex, irrespective of the (sub)variant considered. In conclusion, neither qualitative nor quantitative results returned by both immunoassays are interchangeable. The performance of both assays was found to be greatly influenced by the vaccination and SARS-CoV-2 infection status of individuals.

Cumulative incidence of SARS-CoV-2 infection in the general population of the Valencian Community (Spain) after the surge of the Omicron BA.1 variant.

Studies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision-making. A cross-sectional study on 935 participants, conducted in the Valencian Community (VC), measuring anti-SARS-CoV-2-receptor binding domain-RBD-total antibodies and anti-Nucleocapsid (N)-IgGs via electrochemiluminescence assays. Quantitation of neutralizing antibodies (NtAb) against ancestral and Omicron BA.1 and BA.2 variants and enumeration of SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells was performed in 100 and 137 participants, respectively. The weighted cumulative incidence was 51.9% (95% confidence interval [CI]: 48.7-55.1) and was inversely related to age. Anti-RBD total antibodies were detected in 97% of participants; vaccinated and SARS-CoV-2-experienced (VAC-ex; n = 442) presented higher levels (p < 0.001) than vaccinated/naïve (VAC-n; n = 472) and nonvaccinated/experienced (UNVAC-ex; n = 63) subjects. Antibody levels correlated inversely with time elapsed since last vaccine dose in VAC-n (Rho, -0.52; p < 0.001) but not in VAC-ex (rho -0.02; p = 0.57). Heterologous booster shots resulted in increased anti-RBD antibody levels compared with homologous schedules in VAC-n, but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75%, and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively. For Omicron BA.2, the figures were 97%, 84%, and 40%, respectively. SARS-CoV-2-S-reactive IFN-γ T cells were detected in 73%, 75%, and 64% of VAC-ex, VAC-n and UNVAC-ex, respectively. Median frequencies for both T-cell subsets were comparable across groups. In summary, by April 2022, around half of the VC population had been infected with SARS-CoV-2 and, due to extensive vaccination, displayed hybrid immunity.

New perspectives in the management of small cell lung cancer.

The treatment of small cell lung cancer (SCLC) is a challenge for all specialists involved. New treatments have been added to the therapeutic armamentarium in recent months, but efforts must continue to improve both survival and quality of life. Advances in surgery and radiotherapy have resulted in prolonged survival times and fewer complications, while more careful patient selection has led to increased staging accuracy. Developments in the field of systemic therapy have resulted in changes to clinical guidelines and the management of patients with advanced disease, mainly with the introduction of immunotherapy. In this article, we describe recent improvements in the management of patients with SCLC, review current treatments, and discuss future lines of research.

The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.

The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. IMPORTANCE Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.

SARS-CoV-2 Omicron BA.1 variant breakthrough infections in nursing home residents after an homologous third dose of the Comirnaty® COVID-19 vaccine: Looking for correlates of protection.

We investigated whether peripheral blood levels of SARS-CoV-2 Spike (S) receptor binding domain antibodies (anti-RBD), neutralizing antibodies (NtAb) targeting Omicron S, and S-reactive-interferon (IFN)-γ-producing CD4+ and CD8+ T cells measured after a homologous booster dose (3D) with the Comirnaty® vaccine was associated with the likelihood of subsequent breakthrough infections due to the Omicron variant. An observational study including 146 nursing home residents (median age, 80 years; range, 66-99; 109 female) evaluated for an immunological response after 3D (at a median of 16 days). Anti-RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells were enumerated by whole-blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti-RBD antibody levels were comparable in infected and uninfected participants (21 123 vs. 24 723 BAU/ml; p = 0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p = 0.32) and frequency of virus-reactive CD4+ (p = 0.82) and CD8+ (p = 0.91) T cells were similar across participants in both groups. anti-RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445 vs. 4345 BAU/ml; p = 0.59 and 188.5 vs. 88.9; p = 0.70, respectively). Having detectable NtAb against Omicron or SARS-CoV-2-S-reactive-IFNγ-producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p = 0.54; OR, 0.0; p = 0.99 and OR 3.70; p = 0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID-19 due to the Omicron variant in nursing home residents.

Preparedness needs research: How fundamental science and international collaboration accelerated the response to COVID-19.

The first cluster of patients suffering from coronavirus disease 2019 (COVID-19) was identified on December 21, 2019, and as of July 29, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been linked with 664,333 deaths and number at least 16,932,996 worldwide. Unprecedented in global societal impact, the COVID-19 pandemic has tested local, national, and international preparedness for viral outbreaks to the limits. Just as it will be vital to identify missed opportunities and improve contingency planning for future outbreaks, we must also highlight key successes and build on them. Concomitant to the emergence of a novel viral disease, there is a 'research and development gap' that poses a threat to the overall pace and quality of outbreak response during its most crucial early phase. Here, we outline key components of an adequate research response to novel viral outbreaks using the example of SARS-CoV-2. We highlight the exceptional recent progress made in fundamental science, resulting in the fastest scientific response to a major infectious disease outbreak or pandemic. We underline the vital role of the international research community, from the implementation of diagnostics and contact tracing procedures to the collective search for vaccines and antiviral therapies, sustained by unique information sharing efforts.

Mutagenic Analysis of Hazara Nairovirus Nontranslated Regions during Single- and Multistep Growth Identifies both Attenuating and Functionally Critical Sequences for Virus Replication.

Hazara nairovirus (HAZV) is a member of the family Nairoviridae in the order Bunyavirales and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3' and 5' NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3' NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5' NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent cis-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed.IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3' and 5' ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3' and 5' NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera.

Recent advances in uveal melanoma treatment.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Recent advances in the understanding of molecular characteristics helped to determine which tumors are most likely to progress. About 50% of patients carrying genetic alterations such as chromosomal aberrations and mutations are at significant risk for metastatic disease of which the majority will succumb to UM within few months. Currently, there is no effective treatment for metastatic uveal melanoma, and we hope this review will encourage researchers and clinicians to work to find a better standard of care. In this article we provide a comprehensive overview of the molecular framework of UM, highlighting the most common mutations involved in this kind of cancer. It also covers the most recent treatments from basic research to clinical trials, including small molecules, nucleic acids or immunotherapy, among others. It is intended to serve as a key reference for clinicians and researchers working in this field.