Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis.

BACKGROUND: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). METHODS: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. FINDINGS: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. INTERPRETATION: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. FUNDING: CEPI.

Cytotoxicity of human antibodies targeting the circumsporozoite protein is amplified by 3D substrate and correlates with protection.

Human monoclonal antibodies (hmAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on the sporozoite surface are a promising tool for preventing malaria infection. However, their mechanisms of protection remain unclear. Here, using 13 distinctive PfCSP hmAbs, we provide a comprehensive view of how PfCSP hmAbs neutralize sporozoites in host tissues. Sporozoites are most vulnerable to hmAb-mediated neutralization in the skin. However, rare but potent hmAbs additionally neutralize sporozoites in the blood and liver. Efficient protection in tissues mainly associates with high-affinity and high-cytotoxicity hmAbs inducing rapid parasite loss-of-fitness in the absence of complement and host cells in vitro. A 3D-substrate assay greatly enhances hmAb cytotoxicity and mimics the skin-dependent protection, indicating that the physical stress imposed on motile sporozoites by the skin is crucial for unfolding the protective potential of hmAbs. This functional 3D cytotoxicity assay can thus be useful for downselecting potent anti-PfCSP hmAbs and vaccines.

Assessment of the Biological Impact of SARS-CoV-2 Genetic Variation Using an Authentic Virus Neutralisation Assay with Convalescent Plasma, Vaccinee Sera, and Standard Reagents.

In the summer of 2020, it became clear that the genetic composition of SARS-CoV-2 was changing rapidly. This was highlighted by the rapid emergence of the D614G mutation at that time. In the autumn of 2020, the project entitled "Agility" was initiated with funding from the Coalition for Epidemic Preparedness Innovations (CEPI) to assess new variants of SARS-CoV-2. The project was designed to reach out and intercept swabs containing live variant viruses in order to generate highly characterised master and working stocks, and to assess the biological consequences of the rapid genetic changes using both in vitro and in vivo approaches. Since November 2020, a total of 21 variants have been acquired and tested against either a panel of convalescent sera from early in the pandemic, and/or a panel of plasma from triple-vaccinated participants. A pattern of continuous evolution of SARS-CoV-2 has been revealed. Sequential characterisation of the most globally significant variants available to us, generated in real-time, indicated that the most recent Omicron variants appear to have evolved in a manner that avoids immunological recognition by convalescent plasma from the era of the ancestral virus when analysed in an authentic virus neutralisation assay.

Intrinsic host susceptibility among multiple species to intranasal SARS-CoV-2 identifies diverse virological, biodistribution and pathological outcomes.

SARS-CoV-2 exhibits a diverse host species range with variable outcomes, enabling differential host susceptibility studies to assess suitability for pre-clinical countermeasure and pathogenesis studies. Baseline virological, molecular and pathological outcomes were determined among multiple species-one Old World non-human primate (NHP) species (cynomolgus macaques), two New World NHP species (red-bellied tamarins; common marmosets) and Syrian hamsters-following single-dose, atraumatic intranasal administration of SARS-CoV-2/Victoria-01. After serial sacrifice 2, 10 and 28-days post-infection (dpi), hamsters and cynomolgus macaques displayed differential virus biodistribution across respiratory, gastrointestinal and cardiovascular systems. Uniquely, New World tamarins, unlike marmosets, exhibited high levels of acute upper airway infection, infectious virus recovery associated with mild lung pathology representing a host previously unrecognized as susceptible to SARS-CoV-2. Across all species, lung pathology was identified post-clearance of virus shedding (antigen/RNA), with an association of virus particles within replication organelles in lung sections analysed by electron microscopy. Disrupted cell ultrastructure and lung architecture, including abnormal morphology of mitochondria 10-28 dpi, represented on-going pathophysiological consequences of SARS-CoV-2 in predominantly asymptomatic hosts. Infection kinetics and host pathology comparators using standardized methodologies enables model selection to bridge differential outcomes within upper and lower respiratory tracts and elucidate longer-term consequences of asymptomatic SARS-CoV-2 infection.

Expanding the Malaria Antibody Toolkit: Development and Characterisation of Plasmodium falciparum RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies.

Malaria, an infection caused by apicomplexan parasites of the genus Plasmodium, continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines. Here we describe the development of recombinant human antibodies to three target proteins from Plasmodium falciparum: reticulocyte binding protein homologue 5 (PfRH5), cysteine-rich protective antigen (PfCyRPA), and circumsporozoite protein (PfCSP). All three proteins are key targets in the development of vaccines for blood-stage or pre-erythrocytic stage infections. We have developed potent anti-PfRH5, PfCyRPA and PfCSP monoclonal antibodies that will prove useful tools for the standardisation of assays in preclinical research and the assessment of these antigens in clinical trials. We have generated some very potent anti-PfRH5 and anti-PfCyRPA antibodies with some clones >200 times more potent than the polyclonal anti-AMA-1 antibodies used for the evaluation of blood stage antigens. While the monoclonal and polyclonal antibodies are not directly comparable, the data provide evidence that these new antibodies are very good at blocking invasion. These antibodies will therefore provide a valuable resource and have potential as biological standards to help harmonise pre-clinical malaria research.

Assessing the Variability of Cell-Associated HIV DNA Quantification through a Multicenter Collaborative Study.

Reliable and accurate quantification of cell-associated HIV DNA (CA HIV DNA) is critical for early infant diagnosis, clinical management of patients under therapy, and to inform new therapeutics efficacy. The present study assessed the variability of CA HIV DNA quantification obtained from various assays and the value of using reference materials to help harmonize the measurements. Using a common set of reagents, our multicenter collaborative study highlights significant variability of CA HIV DNA quantification and lower limit of quantification across assays. The quantification of CA HIV DNA from a panel of infected PBMCs can be harmonized through cross-subtype normalization but assay calibration with the commonly used 8E5 cell line failed to reduce quantification variability between assays, demonstrating the requirement to thoroughly evaluate reference material candidates to help improve the comparability of CA HIV DNA diagnostic assay performance. IMPORTANCE Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy. Our study demonstrates that HIV DNA measurement of the same samples can vary significantly from one laboratory to another, due to heterogeneity in the assay, protocol, and reagents used. We show that when carefully selected, reference materials can reduce measurement variability and harmonize HIV DNA quantification across laboratories, which will help contribute to improved diagnosis and clinical management of patients living with HIV.

Clinical validation of a quantitative HIV-1 DNA droplet digital PCR assay: Applications for detecting occult HIV-1 infection and monitoring cell-associated HIV-1 dynamics across different subtypes in HIV-1 prevention and cure trials.

BACKGROUND: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels, with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections. OBJECTIVES: We clinically validated an HIV-1 DNA quantitative assay using droplet digital PCR (ddPCR), across different HIV-1 subtypes. STUDY DESIGN: The analytical sensitivity and specificity of an HIV-1 DNA ddPCR assay was determined using serial dilutions of a plasmid containing HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. RESULTS: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. CONCLUSIONS: This assay has applications for detecting occult HIV-1-infection in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in infants and adults, and in HIV-1 cure interventions.

SAMHD1 Inhibits LINE-1 Retrotransposition by Promoting Stress Granule Formation.

The SAM domain and HD domain containing protein 1 (SAMHD1) inhibits retroviruses, DNA viruses and long interspersed element 1 (LINE-1). Given that in dividing cells, SAMHD1 loses its antiviral function yet still potently restricts LINE-1, we propose that, instead of blocking viral DNA synthesis by virtue of its dNTP triphosphohydrolase activity, SAMHD1 may exploit a different mechanism to control LINE-1. Here, we report a new activity of SAMHD1 in promoting cellular stress granule assembly, which correlates with increased phosphorylation of eIF2α and diminished eIF4A/eIF4G interaction. This function of SAMHD1 enhances sequestration of LINE-1 RNP in stress granules and consequent blockade to LINE-1 retrotransposition. In support of this new mechanism of action, depletion of stress granule marker proteins G3BP1 or TIA1 abrogates stress granule formation and overcomes SAMHD1 inhibition of LINE-1. Together, these data reveal a new mechanism for SAMHD1 to control LINE-1 by activating cellular stress granule pathway.

The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA.

BACKGROUND: Highly active antiretroviral therapy (HAART) has transformed HIV-1 infection from a deadly disease to a manageable chronic illness, albeit does not provide a cure. The recently developed genome editing system called CRISPR/Cas9 offers a new tool to inactivate the integrated latent HIV-1 DNA and may serve as a new avenue toward cure. FINDINGS: We tested 10 sites in HIV-1 DNA that can be targeted by CRISPR/Cas9. The engineered CRISPR/Cas9 system was introduced into the JLat10.6 cells that are latently infected by HIV-1. The sequencing results showed that each target site in HIV-1 DNA was efficiently mutated by CRISPR/Cas9 with the target site in the second exon of Rev (called T10) exhibiting the highest degree of mutation. As a result, HIV-1 gene expression and virus production were significantly diminished with T10 causing a 20-fold reduction. CONCLUSIONS: The CRISPR/Cas9 complex efficiently mutates and deactivates HIV-1 proviral DNA in latently infected Jurkat cells. Our results also revealed a highly efficient Cas9 target site within the second exon of Rev that represents a promising target to be further explored in the CRISPR/Cas9-based cure strategy.

Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins.

Interferon-induced transmembrane (IFITM) proteins inhibit the entry of a large number of viruses. Not surprisingly, many viruses are refractory to this inhibition. In this study, we report that different strains of HIV and SIV are inhibited by human IFITM proteins to various degrees, with SIV of African green monkeys (SIV(AGM)) being mostly restricted by human IFITM2. Interestingly, SIV(AGM) is as much inhibited by human IFITM2 as by IFITM3 of its own host African green monkeys. Our data further demonstrate that the entry of SIV(AGM) is impaired by human IFITM2 and that this inhibition is overcome by the cholesterol-binding compound amphotericin B that also overcomes IFITM inhibition of influenza A viruses. These results suggest that IFITM proteins exploit similar mechanisms to inhibit the entry of both pH-independent primate lentiviruses and the pH-dependent influenza A viruses.

DDX17 promotes the production of infectious HIV-1 particles through modulating viral RNA packaging and translation frameshift.

RNA helicases are a large family of proteins that rearrange RNA structures and remodel ribonucleic protein complexes using energy derived from hydrolysis of nucleotide triphosphates. They have been shown to participate in every step of RNA metabolism. In the past decade, an increasing number of helicases were shown to promote or inhibit the replication of different viruses, including human immunodeficiency virus type 1. Among these helicases, the DEAD-box RNA helicase DDX17 was recently reported to modulate HIV-1 RNA stability and export. In this study, we further show that the helicase activity of DDX17 is required for the production of infectious HIV-1 particles. Over expression of the DDX17 mutant DQAD in HEK293 cells reduces the amount of packaged viral genomic RNA and diminishes HIV-1 Gag-Pol frameshift. Altogether, these data demonstrate that DDX17 promotes the production of HIV-1 infectious particles by modulating HIV-1 RNA metabolism.

The pre-S1 and antigenic loop infectivity determinants of the hepatitis B virus envelope proteins are functionally independent.

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides-estimated at three to four per virion-is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S- and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.