Highlights d Adenovirus transduction of human ACE2 enables SARS-CoV-2 infection of BALB/c mice d High levels of viral RNA and infectious SARS-CoV-2 accumulate in lungs d Mice transduced with human ACE2 develop viral pneumonia after SARS-CoV-2 infection d Neutralizing mAbs protect from SARS-CoV-2-induced lung infection and inflammation

Summary Influenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, leading to a transient wave of circulating antibody-secreting plasmablasts 1 – 3 . This recall response contributes to “original antigenic sin,” the selective boosting of antibody specificities from prior exposures to influenza virus antigens 4 . It remains unclear whether such vaccination can also induce germinal centre (GC) reactions in the draining lymph node (LN) where diversification and maturation of recruited B cells can occur 5 . Here we used ultrasound-guided fine needle aspiration to serially sample the draining LNs and investigate the dynamics and specificity of GC B cell responses after influenza vaccination in humans. We show that influenza vaccine-binding GC B cells can be detected as early as 1 week after vaccination. In 3 out of 8 participants, we detected vaccine-binding GC B cells up to 9 weeks after vaccination. Between 12% and 88% of the responding GC B cell clones overlapped with those detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation (SHM) and encoded broadly cross-reactive monoclonal antibodies (mAbs). In contrast, vaccine-induced B cell clones detected only in the GC compartment exhibited significantly lower SHM frequencies and predominantly encoded strain-specific mAbs, suggesting a naïve B cell origin. Electron microscopy-based epitope mapping revealed that some of these strain-specific mAbs recognized epitopes that were not targeted by the early plasmablast response. Our results indicate that influenza virus vaccination of humans can elicit a GC reaction to which B cell clones targeting novel epitopes are more likely to be recruited, thereby broadening the spectrum of vaccine-induced protective antibodies against this rapidly mutating pathogen.

Germinal centres (GC) are lymphoid structures where B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs) [1][2][3][4][5] . SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans [6][7][8] . The fate of responding GC B cells as well as the functional consequences of such persistence have not been elucidated. We detected SARS-CoV-2 spike (S)-specific MBCs in 42 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine six months earlier. S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies (mAbs), we tracked the evolution of 1540 S-specific B cell clones. We show that early blood S-specific plasmablasts -on averageexhibited the lowest SHM frequencies. In comparison, SHM frequencies of S-specific GC B cells increased by 3.5-fold within six months after vaccination. S-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-S antibody avidity in blood and affinity as well as neutralization capacity of BMPC-derived mAbs. This study documents how the striking persistence of SARS-CoV-2 vaccination-induced GC reaction in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus. B cell response to mRNA vaccinationWe have previously shown that vaccination of humans with The Pfizer-BioNTech SARS-CoV-2 mRNA vaccine, BNT162b2 induces a robust but transient circulating plasmablast (PB) response and a persistent germinal centre (GC) reaction in the draining lymph nodes 6 . Whether these persistent GC responses lead to the generation of affinity-matured memory B cells (MBCs) and long-lived bone marrow-resident plasma cells (BMPCs) remains unclear. To address this question, we analyzed long-term B cell responses in the participants enrolled in our previously described observational study of 43 healthy participants (13 with a history of SARS-CoV-2 infection) who received two doses of BNT162b2 (Extended Data Tables 1) 6,7 . Long-term blood samples (n=42) and fine needle aspirates (FNAs) of the draining axillary lymph nodes (n=15) were collected 29 weeks post-vaccination (Fig. 1a). Bone marrow aspirates were collected 29 (n=11) and 40 weeks (n=2) post-vaccination, with the latter time point used only for B cell receptor (BCR) repertoire profiling (Fig. 1a). None of the participants who contributed FNA or bone marrow specimens had SARS-CoV-2 infection history. GC B cells were detected in FNAs from all 15 participants (Fig. 1b, c, left panels, Extended Data Fig. 1a, Extended Data Table 2). All 14 participants with FNAs collected prior to week 29 generated S-binding GC B cell responses of varying magnitudes (Fig 1b, c, r...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

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The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs). It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS-CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naïve B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.