Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

Misinformation Caused Increased Urban Mobility and the End of Social Confinement Before the Second Wave of COVID-19 in Amazonia.

Tendentious projections about COVID-19 in Brazil provided an appealing excuse for individuals and decision-makers to justify poor choices during a critical phase of the pandemic. The erroneous results likely contributed to premature resumption of in-person school classes and easing of restrictions on social contact, favoring the resurgence of COVID-19. In Manaus, the largest city in the Amazon region, the COVID-19 pandemic did not end in 2020 of its own accord, but rather rebounded in a disastrous second wave of the disease.

NVX-CoV2373-induced cellular and humoral immunity towards parental SARS-CoV-2 and VOCs compared to BNT162b2 and mRNA-1273-regimens.

BACKGROUND: The NVX-CoV2373-vaccine has recently been licensed, although knowledge on vaccine-induced humoral and cellular immunity towards the parental strain and variants of concern (VOCs) in comparison to mRNA-regimens is limited. METHODS: In this observational study, 66 individuals were recruited to compare immunogenicity and reactogenicity of NVX-CoV2373 with BNT162b2 or mRNA-1273. Vaccine-induced antibodies were analyzed using ELISA and neutralization assays, specific CD4 and CD8 T-cells were characterized based on intracellular cytokine staining using flow-cytometry after antigen-specific stimulation with parental spike or VOCs. RESULTS: Two doses of NVX-CoV2373 strongly induced anti-spike IgG, although IgG-levels were lower than after vaccination with BNT162b2 or mRNA-1273 (p = 0.006). Regardless of the vaccine and despite different IgG-levels, neutralizing activity towards VOCs was highest for Delta, followed by BA.2 and BA.1. The protein-based vaccine failed to induce any spike-specific CD8 T-cells which were detectable in 3/22 (14%) individuals only. In contrast, spike-specific CD4 T-cells were induced in 18/22 (82%) individuals, although their levels were lower (p<0.001), had lower CTLA-4 expression (p<0.0001) and comprised less multifunctional cells co-expressing IFNγ, TNFα and IL-2 (p = 0.0007). Unlike neutralizing antibodies, NVX-CoV2373-induced CD4 T-cells equally recognized all tested VOCs from Alpha to Omicron. In individuals with a history of infection, one dose of NVX-CoV2373 had similar immunogenicity as two doses in non-infected individuals. The vaccine was overall well tolerated. CONCLUSION: NVX-CoV2373 strongly induced spike-specific antibodies and CD4 T-cells, albeit at lower levels as mRNA-regimens. Cross-reactivity of CD4 T-cells towards the parental strain and all tested VOCs may hold promise to protect from severe disease.

Molecular Analyses of Clinical Isolates and Recombinant SARS-CoV-2 Carrying B.1 and B.1.617.2 Spike Mutations Suggest a Potential Role of Non-Spike Mutations in Infection Kinetics.

Some of the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are less susceptible to neutralization with post-vaccine sera and monoclonal antibodies targeting the viral spike glycoprotein. This raises concerns of disease control, transmissibility, and severity. Numerous substitutions have been identified to increase viral fitness within the nucleocapsid and nonstructural proteins, in addition to spike mutations. Therefore, we sought to generate infectious viruses carrying only the variant-specific spike mutations in an identical backbone to evaluate the impact of spike and non-spike mutations in the virus life cycle. We used en passant mutagenesis to generate recombinant viruses carrying spike mutations of B.1 and B.1.617.2 variants using SARS-CoV-2- bacterial artificial chromosome (BAC). Neutralization assays using clinical sera yielded comparable results between recombinant viruses and corresponding clinical isolates. Non-spike mutations for both variants neither seemed to effect neutralization efficiencies with monoclonal antibodies nor the response to treatment with inhibitors. However, live-cell imaging and microscopy revealed differences, such as persisting syncytia and pronounced cytopathic effect formation, as well as their progression between BAC-derived viruses and clinical isolates in human lung epithelial cell lines and primary bronchial epithelial cells. Complementary RNA analyses further suggested a potential role of non-spike mutations in infection kinetics.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Limited neutralisation of the SARS-CoV-2 Omicron subvariants BA.1 and BA.2 by convalescent and vaccine serum and monoclonal antibodies.

BACKGROUND: In recent months, Omicron variants of SARS-CoV-2 have become dominant in many regions of the world, and case numbers with Omicron subvariants BA.1 and BA.2 continue to increase. Due to numerous mutations in the spike protein, the efficacy of currently available vaccines, which are based on Wuhan-Hu 1 isolate of SARS-CoV-2, is reduced, leading to breakthrough infections. Efficacy of monoclonal antibody therapy is also likely impaired. METHODS: In our in vitro study using A549-AT cells constitutively expressing ACE2 and TMPRSS2, we determined and compared the neutralizing capacity of vaccine-elicited sera, convalescent sera and monoclonal antibodies against authentic SARS-CoV-2 Omicron BA.1 and BA.2 compared with Delta. FINDINGS: Almost no neutralisation of Omicron BA.1 and BA.2 was observed using sera from individuals vaccinated with two doses 6 months earlier, regardless of the type of vaccine taken. Shortly after the booster dose, most sera from triple BNT162b2-vaccinated individuals were able to neutralise both Omicron variants. In line with waning antibody levels three months after the booster, only weak residual neutralisation was observed for BA.1 (26%, n = 34, 0 median NT50) and BA.2 (44%, n = 34, 0 median NT50). In addition, BA.1 but not BA.2 was resistant to the neutralising monoclonal antibodies casirivimab/imdevimab, while BA.2 exhibited almost a complete evasion from the neutralisation induced by sotrovimab. INTERPRETATION: Both SARS-CoV-2 Omicron subvariants BA.1 and BA.2 escape antibody-mediated neutralisation elicited by vaccination, previous infection with SARS-CoV-2, and monoclonal antibodies. Waning immunity renders the majority of tested sera obtained three months after booster vaccination negative in BA.1 and BA.2 neutralisation. Omicron subvariant specific resistance to the monoclonal antibodies casirivimab/imdevimab and sotrovimab emphasizes the importance of genotype-surveillance and guided application. FUNDING: This study was supported in part by the Goethe-Corona-Fund of the Goethe University Frankfurt (M.W.) and the Federal Ministry of Education and Research (COVIDready; grant 02WRS1621C (M.W.).

Characterization of the Antibody and Interferon-Gamma Release Response after a Second COVID-19 Booster Vaccination.

The emergence of SARS-CoV-2 Omicron subvariants prompted countries to call for accelerated booster vaccinations to limit disease and transmission. Here, we characterized correlates of protection over time after the second booster or after Omicron BA.1 infection comparing variants of concern (VOCs). Sera from subjects before and two and seven weeks after the second booster or after Omicron infection were examined for the level of Spike receptor-binding-domain (RBD)-specific antibodies. Furthermore, neutralizing antibodies (nABs) were characterized in in vitro neutralization assays comparing the variants of concern Alpha, Beta, Delta, and Omicron BA.1 and BA.2 against the ancestral strain B.1. Here, the second booster resulted in an increase in anti-RBD-IgG-antibodies, remaining nearly constant over time, accompanied by an increase in nABs against B.1 and the VOCs Alpha, Beta, Delta, and Omicron BA.1 and BA.2. However, compared to B.1, the neutralizing capacity against the Omicron subvariants remained low and was limited after the second booster vaccination. This indicates that antibody-mediated protection against infection with this VOC is unlikely, as evidenced by the fact that three individuals of our study cohort became infected with Omicron BA.1 after the second booster. T cell activation was measured by interferon-gamma release assays in a subgroup of subjects and was increased in all subjects tested after the second booster vaccination, correlating with the amount of Spike-specific antibodies. In subjects with Omicron BA.1 breakthrough infection, a significant increase in nABs to all VOCs studied was observed independently of booster vaccinations. Taken together, our data indicate that a second booster or Omicron BA.1 infection mediate a substantial increase in anti-Spike IgG antibodies; however, infection with Omicron BA.1 induced a stronger increase in neutralizing antibodies against the different VOCs.

Wastewater surveillance allows early detection of SARS-CoV-2 omicron in North Rhine-Westphalia, Germany.

Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany). Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021. In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.

Detection and Quantification of SARS-CoV-2 by Real-Time RT-PCR Assay.

The pandemic coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread and high mortality rate. Detection and quantification of the (+) ssRNA virus, which has a genome size of 29,903 nucleotides, is commonly performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting conserved sequences. Here, we describe a one-step RT-qPCR protocol for the quantitative detection of SARS-CoV-2 genomic RNA targeting M and RdRP genes, respectively, as well as active virus replication detecting subgenomic RNAs (sgRNA 4 and 8) that are formed by discontinuous transcription of the viral genome. Concomitantly, an input control targeting the human RNaseP gene (RPP30) was used in multiplex PCR to monitor the input of human nucleic acids. In vitro-transcribed RNA harboring the amplicon regions for M and RdRP regions served to set up a standard curve for absolute quantification.In conclusion, the method described here allows for the detection and quantification of SARS-CoV-2 RNA isoforms for research by both using a probe-based or SYBR Green-based approach, but is also suitable for diagnostic purposes.

Dynamics of COVID-19 in Amazonia: A history of government denialism and the risk of a third wave.

The city of Manaus (the capital of Brazil's state of Amazonas) has become a key location for understanding the dynamics of the global pandemic of COVID-19. Different groups of scientists have foreseen different scenarios, such as the second wave or that Manaus could escape such a wave by having reached herd immunity. Here we test five hypotheses that explain the second wave of COVID-19 in Manaus: 1) The greater transmissibility of the Amazonian (gamma or P.1) variant is responsible for the second wave; 2) SARS-CoV-2 infection levels during the first wave were overestimated by those foreseeing herd immunity, and the population remained below this threshold when the second wave began at the beginning of December 2020; 3) Antibodies acquired from infection by one lineage do not confer immunity against other lineages; 4) Loss of immunity has generated a feedback phenomenon among infected people, which could generate future waves, and 5) A combination of the foregoing hypotheses. We also evaluated the possibility of a third wave in Manaus despite advances in vaccination, the new wave being due to the introduction of the delta variant in the region and the loss of immunity from natural contact with the virus. We developed a multi-strain SEIRS (Susceptible-Exposed-Infected-Removed-Susceptible) model and fed it with data for Manaus on mobility, COVID-19 hospitalizations, numbers of cases and deaths. Our model contemplated the current vaccination rates for all vaccines applied in Manaus and the individual protection rates already known for each vaccine. Our results indicate that the SARS-CoV-2 gamma (P.1) strain that originated in the Amazon region is not the cause of the second wave of COVID-19 in Manaus, but rather this strain originated during the second wave and became predominant in January 2021. Our multi-strain SEIRS model indicates that neither the doubled transmission rate of the gamma variant nor the loss of immunity alone is sufficient to explain the sudden rise of hospitalizations in late December 2020. Our results also indicate that the most plausible explanation for the current second wave is a SARS-CoV-2 infection level at around 50% of the population in early December 2020, together with loss of population immunity and early relaxation of restrictive measures. The most-plausible model indicates that contact with one strain does not provide protection against other strains and that the gamma variant has a transmissibility rate twice that of the original SARS-CoV-2 strain. Our model also shows that, despite the advance of vaccination, and even if future vaccination advances at a steady pace, the introduction of the delta variant or other new variants could cause a new wave of COVID-19.

Limited Neutralization of Authentic Severe Acute Respiratory Syndrome Coronavirus 2 Variants Carrying E484K In Vitro.

Whether monoclonal antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been investigated using pseudoviruses. In this study we show that bamlanivimab, casirivimab, and imdevimab efficiently neutralize authentic SARS-CoV-2, including variant B.1.1.7 (alpha), but variants B.1.351 (beta) and P.2 (zeta) were resistant against bamlanivimab and partially resistant to casirivimab. Whether antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variantshas been investigated using pseudoviruses. We show that authentic SARS-CoV-2 carrying E484K were resistant against bamlanivimab and less susceptible to casirivimab, convalescent and vaccine-elicited sera.

Brazil's COVID-19 Epicenter in Manaus: How Much of the Population Has Already Been Exposed and Are Vulnerable to SARS-CoV-2?

Is Brazil's COVID-19 epicenter really approaching herd immunity? A recent study estimated that in October 2020 three-quarters of the population of Manaus (the capital of the largest state in the Brazilian Amazon) had contact with SARS-CoV-2. We show that 46% of the Manaus population having had contact with SARS-CoV-2 at that time is a more plausible estimate, and that Amazonia is still far from herd immunity. The second wave of COVID-19 is now evident in Manaus. We predict that the pandemic of COVID-19 will continue throughout 2021, given the duration of naturally acquired immunity of only 240 days and the slow pace of vaccination. Manaus has a large percentage of the population that is susceptible (35 to 45% as of May 17, 2021). Against this backdrop, measures to restrict urban mobility and social isolation are still necessary, such as the closure of schools and universities, since the resumption of these activities in 2020 due to the low attack rates of SARS-CoV-2 was the main trigger for the second wave in Manaus.

How Brazil's President turned the country into a global epicenter of COVID-19.

In this manuscript, we point out that the federal government headed by President Bolsonaro has pursued a political agenda that contributed to the spread of COVID-19, transforming the country into a major repository for SARS-CoV-2 and its variants, thus representing a risk for worldwide containment efforts. Furthermore his actions are also weakening democratic institutions, which could counter his political agenda, effectively facilitating the spread of COVID-19. Thus, the perpetuation of the COVID-19 pandemic in Brazil is due to human behaviour factors, especially high-level public decision makers.

Antibody-Mediated Neutralization of Authentic SARS-CoV-2 B.1.617 Variants Harboring L452R and T478K/E484Q.

The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.

Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication.

The First Case of Immunity Loss and SARS-CoV-2 Reinfection by the Same Virus Lineage in Amazonia.

We report the first confirmed record of a SARS-CoV-2 immunity loss and reinfection for the Amazon region and for Brazil by the same virus lineage. The patient presented an asymptomatic condition the first time and an aggravated one after reinfection. We raise the possibility of a recessive genotype in the Amazonian population that does not generate an immune memory response to SARS-CoV-2.

Scenarios for the Spread of COVID-19 in Manaus, Northern Brazil.

We use a compartmental model to project scenarios for the spread of COVID-19 in the city of Manaus, with different degrees of social distancing and isolation of infectious individuals. In a scenario without any containment measures, our projections point to more than 50,000 (simultaneously) infectious individuals before the end of April 2020. Only in a scenario with strict and extensive social distancing (lockdown), implemented and observed as of March 23rd, 2020, would this number be less than 50,000 in the first half of June. Furthermore, an increase in the number of tests or in the collective awareness among the population regarding the importance of strict distancing measures would have only had a significant impact if implemented before the end of March.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.