Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.

SARS-CoV-2 and its variants continue to infect hundreds of thousands every day despite the rollout of effective vaccines. Therefore, it is essential to understand the levels of protection that these vaccines provide in the face of emerging variants. Here, we report two demographically balanced cohorts of BNT162b2 vaccine recipients and COVID-19 patients, from which we evaluate neutralizing antibody titers against SARS-CoV-2 as well as the B.1.1.7 (alpha) and B.1.351 (beta) variants. We show that both B.1.1.7 and B.1.351 are less well neutralized by serum from vaccinated individuals, and that B.1.351, but not B.1.1.7, is less well neutralized by convalescent serum. We also find that the levels of variant-specific anti-spike antibodies are proportional to neutralizing activities. Together, our results demonstrate the escape of the emerging SARS-CoV-2 variants from neutralization by serum antibodies, which may lead to reduced protection from re-infection or increased risk of vaccine breakthrough.

Significance: RNA viruses usurp and reprogram host cells using short RNA genomes. RNA viruses encode the information required for their replication in both their primary sequences and higher-order structures formed when the RNA genome strand folds back on itself, but the extent of higher-order structure has remained unclear. We use a new high-throughput RNA structure probing technology to identify RNA regions with tertiary folds and discover that roughly one-third of the dengue virus RNA genome forms higher-order interactions, many in regions functionally important for replication. This work suggests that tertiary structure elements might be common in large RNAs, and that these regions might contain pockets targetable by small molecules in the design of antiviral therapeutics.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be important globally. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants has created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explored the effect of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies elicited in a cohort of 96 health care workers. We found robust neutralizing antibody responses among those with hybrid immunity; these hybrid immune responses neutralized all variants, including BA.2. Neutralizing titers were significantly improved for those with longer vaccine-infection intervals of up to 400 days compared with those with shorter intervals. These results indicate that anti–SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting, with greater potency seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.

Access to fast and reliable nucleic acid testing continues to play a key role in controlling the COVID-19 pandemic, especially in the context of increased vaccine break-through risks due to new variants. We report a rapid, low-cost (~ 2 USD), simple-to-use nucleic acid test kit for self-administered at-home testing without lab instrumentation. The entire sample-to-answer workflow takes < 60 min, including noninvasive sample collection, one-step RNA preparation, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in a thermos, and direct visual inspection of a colorimetric test result. To facilitate long-term storage without cold-chain, a fast one-pot lyophilization protocol was developed to preserve all required biochemical reagents of the colorimetric RT-LAMP test in a single microtube. Notably, the lyophilized RT-LAMP assay demonstrated reduced false positives as well as enhanced tolerance to a wider range of incubation temperatures compared to solution-based RT-LAMP reactions. We validated our RT-LAMP assay using simulated infected samples, and detected a panel of SARS-CoV-2 variants with successful detection of all variants that were available to us at the time. With a simple change of the primer set, our lyophilized RT-LAMP home test can be easily adapted as a low-cost surveillance platform for other pathogens and infectious diseases of global public health importance.