Evaluating Saliva Sampling with Reverse Transcription Loop-mediated Isothermal Amplification to Improve Access to SARS-CoV-2 Diagnosis in Low-Resource Settings.

Standard diagnosis of SARS-CoV-2 by nasopharyngeal swab (NPS) and real-time reverse transcriptase-polymerase chain reaction (PCR) requires a sophisticated laboratory, skilled staff, and expensive reagents that are difficult to establish and maintain in isolated, low-resource settings. In the remote setting of tropical Sumba Island, eastern Indonesia, we evaluated alternative sampling with fresh saliva (FS) and testing with colorimetric loop-medicated isothermal amplification (LAMP). Between August 2020 and May 2021, we enrolled 159 patients with suspected SARS-CoV-2 infection, of whom 75 (47%) had a positive PCR on NPS (median cycle threshold [Ct] value: 27.6, interquartile range: 12.5-37.6). PCR on FS had a sensitivity of 72.5% (50/69, 95% confidence interval [CI]: 60.4-82.5) and a specificity of 85.7% (66/77, 95% CI: 75.9-92.6), and positive (PPV) and negative (NPV) predictive values of 82.0% (95% CI: 0.0-90.6) and 77.6% (95% CI: 67.3-86.0), respectively. LAMP on NPS had a sensitivity of 68.0% (51/75, 95% CI: 56.2-78.3) and a specificity of 70.8% (63/84, 95% CI: 58.9-81.0), with PPV 70.8% (95% CI: 58.9-81.0) and NPV 72.4% (95% CI: 61.8-81.5%). LAMP on FS had a sensitivity of 62.3% (43/69, 95% CI: 49.8-73.7%) and a specificity of 72.7% (56/77, 95% CI: 61.4-82.3%), with PPV 67.2% (95% CI: 54.3-78.4) and NPV 68.3% (95% CI: 57.1-78.1%). LAMP sensitivity was higher for NPS and FS specimens with high viral loads (87.1% and 75.0% for Ct value < 26, respectively). Dried saliva on filter paper was stable for 4 days at room temperature. LAMP on either NPS or FS could offer an accessible alternative for SARS-CoV-2 diagnosis in low-resource settings, with potential for optimizing sample collection and processing, and selection of gene targets.

Serum anti-Spike antibody titers before and after heterologous booster with mRNA-1273 SARS-CoV-2 vaccine following two doses of inactivated whole-virus CoronaVac vaccine

BackgroundThe inactivated whole-virus vaccine CoronaVac (SinoVac) is the COVID-19 vaccine most administered worldwide. However, data on its immunogenicity and reactogenicity to heterologous boosting with mRNA vaccines are lacking. MethodsIn a cohort of hospital staff in Jakarta, Indonesia, who received two-dose CoronaVac six months prior (median 190 days, IQR165-232), we measured anti-Spike IgG titers on paired serum samples taken before and 28 days after a 100g mRNA-1273 (Moderna) booster. We performed correlations and multivariable ordinal regressions. FindingsAmong 304 participants, the median age was 31 years (range 21-59), 235 (77.3%) were women, 197 (64.8%) had one or more previous SARS-CoV-2 infections (including 155 [51.0%] who had a post-CoronaVac breakthrough infection. Pre-boost IgG titers correlated negatively with the time since the latest documented "virus exposure" (either by the second CoronaVac or SARS-CoV-2-infection whichever most recent). Previous SARS-CoV-2 infection and a longer time interval between second vaccine and mRNA-1273 boost were associated with a higher pre-boost IgG titer. Post-booster, the median IgG titer increased 9.3-fold, from 250 (IQR32-1389) to 2313 (IQR1226-4324) binding antibody units (BAU/mL) (p<0.001). All participants, including seven whose pre-boost IgG was below assay detection limits, became seropositive and all reached a substantial post-boost titer ([≥]364 BAU/mL). Post-boost IgG was not associated with pre-boost titer or previous SARS-CoV-2 infection. Booster reactogenicity was acceptable, with 7.9% of participants experiencing short-lived impairment of activities of daily living (ADL). InterpretationA heterologous, high-dose mRNA-1273 booster after two-dose CoronaVac was highly immunogenic and safe, including in those most in need of improved immunity. FundingWellcome Trust, UK Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe inactivated whole-virus vaccine CoronaVac (SinoVac) is the COVID-19 vaccine most administered worldwide, at around 2 billion doses in 54 countries. Concerns that CoronaVac has lower immunogenicity than virus vector or mRNA vaccines, with pronounced decreases of neutralising antibody titres within a few months, and reduced effectiveness in the older population, highlight the urgent need for immunogenic, safe and well-tolerated booster schedules, especially with Omicron rapidly emerging. We used the terms "SARS-CoV-2", "COVID-19", "vaccine", "booster" to search PubMed and medRxiv up to Dec 22th, 2021, with no language or date restrictions, to identify clinical trials and real-world studies reporting on the immune responses and reactogenicity to a "third booster" of currently approved COVID-19 vaccines. Previous research reported that neutralising antibody responses elicited by all currently approved vaccines (mRNA, adenovirus-vectored, inactivated, and protein subunit) declined to varying degrees after 6-8 months after full-schedule vaccination. Several clinical trials have evaluated heterologous ("mix and match") vaccination schedules, demonstrating robust immune responses in adults. After two-dose CoronaVac, BNT162b2 (Pfizer-BioNTech) boost was significantly more immunogenic than a homologous booster against wild-type and Variants of Concern (VOCs) Beta, Gamma and Delta, and AZD1222 boost increased spike RBD-specific IgG 9-10-fold, with high neutralizing activity against the wild type and VOCs. Compared to previous SARS-CoV-2 variants, current vaccine boosters appeared to neutralise Delta to a slightly lesser degree, and Omicron to a substantially lesser degree, although preliminary data from Moderna found that the authorised dose (50g) of the mRNA-1273 boost increased antibodies 37-fold and the high-dose (100g) boost 83-fold. Added value of this studyTo our knowledge, this study is the first to provide critical real-world evidence that heterologous boosting with high-dose mRNA-1273 vaccine after CoronaVac is highly immunogenic, safe and well-tolerated in adults. After a primary course of two-dose CoronaVac, we found that a high-dose (100g) mRNA-1273 booster was immunogenic for all participants in a highly exposed cohort of hospital staff in Jakarta, Indonesia, in the context of Delta predominance, particularly for those with the lowest pre-boost antibody levels. All participants became seropositive and all reached a substantial post-boost titer ([≥]364 BAU/mL), up to a median 9.3-fold increase. Booster reactogenicity was acceptable, with 7.9% of participants experiencing short-lived impairment of activities of daily living Implications of all the available evidenceThe study findings contribute to informing policy makers on flexible options in deploying COVID-19 vaccines in mix-and-match schedules, with particular relevance for countries that are largely dependent on inactivated vaccines. Further trials are warranted that assess clinical endpoints of optimized doses of mRNA-1273 booster, and variant-specific or multivalent vaccines in response to decreased protection against emerging SARS-CoV-2 VOCs.

Recognition of CpG oligodeoxynucleotides by human Toll-like receptor 9 and subsequent cytokine induction.

Toll-like receptor 9 (TLR9) recognizes a synthetic ligand, oligodeoxynucleotide (ODN) containing cytosine-phosphate-guanine (CpG). Activation of TLR9 by CpG ODN induces a signal transduction cascade that plays a pivotal role in first-line immune defense in the human body. The three-dimensional structure of TLR9 has not yet been reported, and the ligand-binding mechanism of TLR9 is still poorly understood; therefore, the mechanism of human TLR9 (hTLR9) ligand binding needs to be elucidated. In this study, we constructed several hTLR9 mutants, including truncated mutants and single mutants in the predicted CpG ODN-binding site. We used these mutants to analyze the role of potential important regions of hTLR9 in receptor signaling induced by phosphorothioate (PTO)-modified CpG ODN and CpG ODNs only consist entirely of a phosphodiester (PD) backbone, CpG ODN2006x3-PD that we developed. We found truncated mutants of hTLR9 lost the signaling activity, indicating that both the C- and N-termini of the extracellular domain (ECD) are necessary for the function of hTLR9. We identified residues, His505, Gln510, His530, and Tyr554, in the C-terminal of hTLR9-ECD that are essential for hTLR9 activation. These residues might form positive charged clusters with which negatively charged CpG ODN could interact. Furthermore, we observed ODN-PD induced interleukin-6 (IL-6) through TLR9 in a CpG-sequence-dependent manner in human peripheral blood mononuclear cells and B cells, whereas ODN-PTO induced IL-6 in a CpG-sequence-independent manner. These finding are relevant for the mechanism of hTLR9 activation by CpG ODNs.