Overall, we isolated 5 neutralising mAbs against NL63, 4 against the RBD and 1 against the S2 stalk. The S2 mAb had unusually high neutralising potency comparable to RBD mAbs, and targeted a region that is extended in α-CoVs, potentially revealing a site of vulnerability absent in β-CoVs.

Interestingly, while NL63 was able to escape these neutralising mAbs in vitro, these escape mutations were not found in over 207 sequences spanning 1983-2023, matching previous data indicating that NL63 does not undergo adaptive evolution unlike HCoV OC43 and 229E (elifesciences.org/articles/64509).

The escape mutations for the non-RBD neutralising mAb (NLH02) were located within the membrane proximal HR2 region of the S2 subunit (within the stalk of spike), suggesting a neutralisation mechanism that interferes with viral fusion as described previously for S2 mAbs against SARS-CoV-2.

To further map where these mAbs bind to on spike, we generated escape variants to each of the neutralising mAbs. Escape mutations for the RBD neutralising mAbs were concentrated on the three discontinuous receptor binding motif loops of RBD.

4 of the 5 neutralising mAbs were specific for the receptor binding domain (RBD: Domain S1B) and could inhibit the RBD from binding to its receptor ACE2

We isolated 9 monoclonal antibodies against the entry protein (spike) of NL63, 5 of which had neutralising activity against the prototypic Amsterdam-1 isolate and other contemporary NL63 isolates that we cultured from PCR+ nasal swabs. Binding and neutralising IC50 values summarised below:

Link to paper: academic.oup.com/jimmunol/adv...

5/ Settling the #LeftvsRight debate: Ipsilateral prime-boost could be an optimal strategy for rapidly maximising humoral immunity during early outbreak phases, with particular relevance for vaccine strategies requiring multidose schedules.

4/ In our heterologous WT/BA.1 prime-boost model, we observed differential recognition patterns in cross-reactive GC B cells depending on vaccination site. Same-arm boosting also provided a transient advantage in breadth against the BA.1 spike variant.

3/ Alternate side vaccination established independent GCs at distinct lymph nodes. Interestingly, by day 28, antibody levels between both vaccination approaches became comparable in magnitude, durability, and neutralisation capacity.

2/ We found that same side vaccination accelerated antibody production, with significantly higher titers at day 19, coupled with sustained GCs in the primary draining lymph nodes.

5/ Settling the #LeftvsRight debate: Ipsilateral prime-boost could be an optimal strategy for rapidly maximising humoral immunity during early outbreak phases, with particular relevance for vaccine strategies requiring multidose schedules.

4/ In our heterologous WT/BA.1 prime-boost model, we observed differential recognition patterns in cross-reactive GC B cells depending on vaccination site. Same-arm boosting also provided a transient advantage in breadth against the BA.1 spike variant.

3/ Alternate side vaccination established independent GCs at distinct lymph nodes. Interestingly, by day 28, antibody levels between both vaccination approaches became comparable in magnitude, durability, and neutralisation capacity.

2/ We found that same side vaccination accelerated antibody production, with significantly higher titers at day 19, coupled with sustained GCs in the primary draining lymph nodes.