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The ComFluCOV trial tested the safety and immunogenicity of COVID-19 and influenza vaccines co-administration. Binding and functional SARS-CoV2 anti-spike responses were measured using assays developed in response to the COVID-19 pandemic. The three assays used to measure the immunogenicity outcomes are reported here and their performance compared to inform future vaccine development. Adults aged over 18 were vaccinated with a COVID-19 and either an influenza vaccine or saline placebo. Serum sampled one month after vaccination was used to measure SARS-CoV2 anti-spike antibody concentrations using a commercial in-house enzyme-linked immunosorbent assay (ELISA), a commercial fast throughput electrochemiluminescence immunoassay (ECLIA) and a viral neutralisation assay (VNA). Geometric mean ratios were used to compare the response to COVID-19 with or without influenza vaccine with a threshold of 0.67 considered non-inferior. The relationship between the different assays was examined using Kendall rank correlations. The geometric mean ratios exceeded 0.67 using all assays for all COVID-19 and influenza vaccine combinations tested. Moderate rank correlations were found between the three assays. All three assays confirmed that vaccine co-administration did not significantly impact on immunogenicity of any of the vaccines tested. Trial registration: ISRCTN14391248, registered on 17/03/2021.

The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti-mumps virus antibody response after vaccination. Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

► A pneumococcal PlyD1 vaccine candidate was safe in adults. ► The PlyD1 vaccine candidate was immunogenic at 10, 25, and 50μg. ► PlyD1 vaccination induced toxin-neutralizing antibodies. Pneumococcal vaccines based on conserved protein antigens have the potential to offer expanded protection against Streptococcus pneumoniae. This study examined the safety and immunogenicity in adults of three doses of a pneumococcal single-antigen protein vaccine candidate formulated with aluminum hydroxide adjuvant and recombinantly derived, highly detoxified, genetically mutated pneumolysin protein (PlyD1). This phase I, randomized, placebo-controlled, observer-blinded, dose-escalating study enrolled adults (18–50 years). In a pilot safety study, participants received a single injection of 10μg PlyD1 and were observed for 24h. Following review of the pilot safety data, participants were randomized (2:1) to receive two injections of PlyD1 at one of three doses or placebo 30 days apart. Assignment of second injection and successive dose cohorts was made after blinded safety reviews after each injection at each dose level. Safety endpoints included rates of solicited injection site reactions, solicited systemic reactions, unsolicited adverse events (AEs), serious AEs (SAEs), and safety laboratory tests. Immunogenicity endpoints included geometric mean concentrations of anti-PlyD1 IgG as determined by ELISA and functional assessment in an in vitro toxin neutralization assay. The study included a total of 100 participants, including 10 in the pilot study and 90 in the randomized study. None of the participants in the pilot study had SAEs, allergic reactions, or other safety concerns. Ninety participants received two doses of or placebo (n=30) or active vaccine candidate at 10 (n=20), 25 (n=20), or 50μg (n=20). No vaccine-related SAE or discontinuation due to an AE occurred. Most solicited reactions were mild and transient. The most frequently reported solicited reactions were pain at the injection site and myalgia. Antigen-specific IgG levels and functional activity showed dose-related increases. When comparing the three dose levels, a plateau effect was observed at the 25μg dose. All dose levels were safe and immunogenic. Repeat vaccination significantly increased the level of anti-PlyD1 antibodies. Functional antibody activity was demonstrated in sera from vaccinated individuals (ClinicalTrails.gov no. NCT01444352).

Background Sandfly-borne phleboviruses are present in North Africa where they can infect humans in regions where Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin is present affecting both humans and dogs. We investigated the capacity of dogs to be used as sentinels for sandfly-borne phleboviruses as previously shown for leishmaniasis. Findings A total of 312 sera were collected from guard dogs in two different bioclimatic regions (governorates of Kairouan and Bizerte) of Tunisia where zoonotic visceral leishmaniasis has been reported. These sera were tested for the presence of neutralising antibodies against 3 phleboviruses: Toscana virus, Punique virus and Sicilian virus. In the governorate of Kairouan, seroprevalence rates of 7.5%, 43.5%, and 38.1% were observed for Toscana, Punique and Sicilian virus, respectively. A high proportion of sera from the governorate of Bizerte were hemolyzed and showed high cytotoxicity for the cells and subsequently precluded detailed interpretation of this batch. However, validated results for 27 sera were in agreement with data observed in the governorate of Kairouan. Conclusions Toscana virus is present in the governorate of Kairouan but at a lower rate compared to Punique and Sicilian viruses. These three sandfly-borne phleboviruses can infect dogs. Direct detection and isolation of the viruses are now to be attempted in animals as well as in humans. Our findings showed that guard dogs are good sentinels for virus transmitted by sandflies and strongly suggested that the high seroprevalence rates observed in dogs merit further attention.

Avian influenza continues to circulate and remains a global health threat not least because of the associated high mortality. In this study antibody persistence, booster vaccine response and cross-clade immune response between two influenza A(H5N1) vaccines were compared. Participants aged over 18-years who had previously been immunized with a clade 1, A/Vietnam vaccine were re-immunized at 6-months with 7.5 μg of the homologous strain or at 22-months with a clade 2, alum-adjuvanted, A/Indonesia vaccine. Blood sampled at 6, 15 and 22-months after the primary course was used to assess antibody persistence. Antibody concentrations 6-months after primary immunisation with either A/Vietnam vaccine 30 μg alum-adjuvanted vaccine or 7.5 μg dose vaccine were lower than 21-days after the primary course and waned further with time. Re-immunization with the clade 2, 30 μg alum-adjuvanted vaccine confirmed cross-clade reactogenicity. Antibody cross-reactivity between A(H5N1) clades suggests that in principle a prime-boost vaccination strategy may provide both early protection at the start of a pandemic and improved antibody responses to specific vaccination once available. ClinicalTrials.gov NCT00415129.

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

The ability of viruses to mutate and evade the human immune system and neutralizing antibodies remains an obstacle to antiviral and vaccine development. Many neutralizing antibodies, including some approved for emergency use authorization (EUA), reduced or lost activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we introduce a geometric deep learning algorithm that efficiently enhances antibody affinity to achieve broader and more potent neutralizing activity against such variants. We demonstrate the utility of our approach on a human antibody P36-5D2, which is effective against SARS-CoV-2 Alpha, Beta, and Gamma but not Delta. We show that our geometric neural network model optimizes this antibody’s complementarity-determining region (CDR) sequences to improve its binding affinity against multiple SARS-CoV-2 variants. Through iterative optimization of the CDR regions and experimental measurements, we enable expanded antibody breadth and improved potency by ∼10- to 600-fold against SARS-CoV-2 variants, including Delta. We have also demonstrated that our approach can identify CDR changes that alleviate the impact of two Omicron mutations on the epitope. These results highlight the power of our deep learning approach in antibody optimization and its potential application to engineering other protein molecules. Our optimized antibodies can potentially be developed into antibody drug candidates for current and emerging SARS-CoV-2 variants.

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC 50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2. A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. To address this, Shi and colleagues present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV2-Nluc), and show that it has potential for large-scale vaccine evaluation and neutralizing antibody testing.

Vaccine-induced neutralizing antibodies (nAbs) are key biomarkers considered to be associated with vaccine efficacy. In United States government-sponsored phase 3 efficacy trials of COVID-19 vaccines, nAbs are measured by two different validated pseudovirus-based SARS-CoV-2 neutralization assays, with each trial using one of the two assays. Here we describe and compare the nAb titers obtained in the two assays. We observe that one assay consistently yielded higher nAb titers than the other when both assays were performed on the World Health Organization’s anti-SARS-CoV-2 immunoglobulin International Standard, COVID-19 convalescent sera, and mRNA-1273 vaccinee sera. To overcome the challenge this difference in readout poses in comparing/combining data from the two assays, we evaluate three calibration approaches and show that readouts from the two assays can be calibrated to a common scale. These results may aid decision-making based on data from these assays for the evaluation and licensure of new or adapted COVID-19 vaccines.

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.