Direct genome sequencing of respiratory viruses from low viral load clinical specimens using target capture sequencing technology (preprint)

The use of metagenomic next-generation sequencing technology to obtain complete viral genome sequences directly from clinical samples with low viral load remains challenging--especially in the case of respiratory viruses--due to the low copy number of viral versus host genomes. To overcome this limitation, target capture sequencing for the enrichment of specific genomes has been developed and applied for direct genome sequencing of viruses. However, as the efficiency of enrichment varies depending on the probes, the type of clinical sample, etc., validation is essential before target capture sequencing can be applied to clinical diagnostics. Here we evaluated the utility of target capture sequencing with a comprehensive viral probe panel for clinical respiratory specimens collected from patients diagnosed with SARS-CoV-2 or influenza type A. We focused on clinical specimens containing low copy numbers of viral genomes. Target capture sequencing yielded approximately 180- and 2000-fold higher read counts of SARS-CoV-2 and influenza A virus, respectively, than metagenomic sequencing when the RNA extracted from specimens contained 59.3 copies/L of SARS-CoV-2 or 544 copies/L of influenza A virus, respectively. In addition, the target capture sequencing identified sequence reads in all SARS-CoV-2- or influenza type A-positive specimens with <26 RNA copies/L, some of which also yielded >70% of the full-length genomes of SARS-CoV-2 or influenza A virus. Furthermore, the target capture sequencing using comprehensive probes identified co-infections with viruses other than SARS-CoV-2, suggesting that this approach will not only detect a wide range of viruses, but also contribute to epidemiological studies.

Two doses of mRNA vaccine elicit cross-neutralizing memory B-cells against SARS-CoV-2 Omicron variant (preprint)

SARS-CoV-2 Beta and Omicron variants have multiple mutations in the receptor-binding domain (RBD) allowing antibody evasion. Despite the resistance to circulating antibodies in those who received two doses of mRNA vaccine, the third dose prominently recalls cross-neutralizing antibodies with expanded breadth to these variants. Herein, we longitudinally profiled the cellular composition of persistent memory B-cell subsets and their antibody reactivity against these variants following the second vaccine dose. The vaccination elicited a memory B-cell subset with resting phenotype that dominated the other subsets at 4.9 months. Notably, most of the resting memory subset retained the ability to bind the Beta variant, and the memory-derived antibodies cross-neutralized the Beta and Omicron variants at frequencies of 59% and 29%, respectively. The preservation of cross-neutralizing antibody repertoires in the durable memory B-cell subset likely contributes to the prominent recall of cross-neutralizing antibodies following the third dose of the vaccine. One Sentence Summary Fully vaccinated individuals preserve cross-neutralizing memory B-cells against the SARS-CoV-2 Omicron variant.

Viral and Immunological Characteristics of COVID-19 Vaccine Breakthrough Infections (preprint)

Since little is known about viral and host characteristics of breakthrough infections after COVID-19 vaccination, a nationwide investigation of breakthrough cases was initiated in Japan. 130 cases (90%+ received mRNA vaccines) were reported with respiratory specimens in 117 cases and sera in 68 cases. A subset of cases shed infectious virus regardless of symptom presence or viral lineages. Viral lineages for breakthrough infections matched both temporally and spatially with the circulating lineages in Japan with no novel mutations in spike receptor binding domain that may have escaped from vaccine-induced immunity were found. Anti-spike/neutralizing antibodies of breakthrough infections in the acute phase owing to vaccine-induced immunity were significantly higher than those from unvaccinated convalescent individuals but were comparable to vaccinated uninfected individuals, and followed by boosting in the convalescent phase. Symptomatic cases had low anti-spike/neutralizing antibodies in the acute phase with robust boosting in the convalescent phase, suggesting the presence of serological correlate for symptom development in COVID-19 vaccine breakthrough infections.