Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as\n\n\na promising avenue for enhancing B cell responses to protein subunit vaccines. Here,\n\n\nwe evaluated B cell responses in rhesus macaques immunized with prefusion stabilized\n\n\nRespiratory Syncytial Virus (RSV) F glycoprotein trimer compared to\n\n\nnanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent\n\n\ndisplay skews antibody specificities and drives epitope-focusing of responding B cells.\n\n\nAntibody cloning and repertoire sequencing revealed that focusing was driven by\n\n\nexpansion of clonally distinct B cells through recruitment of diverse precursors. We\n\n\nidentified two antibody lineages that developed either ultrapotent neutralization or\n\n\npneumovirus cross-neutralization from precursor B cells with low initial affinity for the\n\n\nRSV-F immunogen. This suggests that increased avidity by multivalent display\n\n\nfacilitates the activation and recruitment of these cells. Diversification of the B cell\n\n\nresponse by multivalent nanoparticle immunogens has broad implications for vaccine\n\n\ndesign.

Objectives:Vaccine responses in haematopoietic stem cell transplant (alloHCT) recipients vary, with different degrees of B-cell reconstitution likely playing a key role. However, mechanistic understanding of the B-cell receptor (BCR) repertoire and its functional impact post-alloHCT remain limited. Methods:Within the scope of a mRNA SARS-CoV-2 vaccine phase IV clinical trial in alloHCT recipients (n = 77), we have measured antibody titers and avidity, and performed B-cell immunophenotyping and B-cell receptor repertoire sequencing in sub-populations. Results:AlloHCT patients receiving prime-boost mRNA vaccination within 12 months post-transplant exhibited lower vaccine-specific antibody levels and memory B-cell frequencies than vaccinated healthy controls. Responses were comparable to healthy controls in patients vaccinated later than 12 months post transplant. BCR repertoire sequencing showed reduced somatic hypermutation (SHM) levels in bulk IgG+ B cells from alloHCT patients. Although some alloHCT patients showed exceptional expansion of a few IgG clones of unknown specificity, their overall B-cell repertoires remained polyclonal. Vaccine-specific B-cell clonotypes detected in patients responding to vaccination showed similar proportional expansion and SHM as in controls. The level of immature CD24hiCD38hi transitional B cells pre-vaccination was negatively correlated to the vaccine response, and can be used as a predictor of antibody titres. Conclusion:Our data indicate that mRNA vaccination can stimulate expansion of vaccine-specific B cells to affinity mature in many alloHCT recipients, though restricted by the presence of immature B-cell populations.

Seasonal vaccination is the most important strategy for mitigating the large disease burden caused by influenza virus. Licensed inactivated influenza virus vaccines rely on slow, egg-based production, underscoring the need for faster, more effective platforms that elicit robust immune responses. Here, we compare the immunogenicity of an unmodified mRNA-lipid nanoparticle vaccine encoding influenza hemagglutinin (HA) from A/Michigan/45/2015 (H1N1)pdm09, A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Phuket/3073/2013 and B/Colorado/06/2017 with two approved, strain-matched, inactivated vaccines (Vaxigrip and Fluad) in non-human primates. The mRNA vaccine induced substantially stronger innate immune activation than Vaxigrip and Fluad, evidenced by rapid upregulation of genes involved in antiviral, antigen presentation and cell migration pathways, expansion of intermediate monocytes and increased secretion of pro-inflammatory cytokines. The mRNA vaccine elicited HA-specific antibodies against all four strains, with levels generally comparable to or exceeding those induced by Vaxigrip and Fluad, although this did not consistently translate to greater neutralizing capacity. Both the mRNA vaccine and Fluad generated higher frequencies of HA-specific memory B and T cell responses compared to Vaxigrip, with the mRNA vaccine inducing a particularly stronger response in the draining lymph nodes, potentially increasing antibody diversity and affinity. Altogether, these findings support the advancement of influenza mRNA vaccines as promising clinical candidates.

Nonhuman primates have a key role in the evaluation of novel therapeutics including vaccine and drug development. Monitoring biochemical and hematological parameters of macaques is critical to understand toxicity and safety, but general reference intervals following standardized guidelines remain to be determined. Here we compiled multiple internal datasets to define normal ranges of classical biochemical and hematological parameters in Indian and Chinese rhesus macaques as well as cynomolgus macaques. Furthermore, the combination of hematological data with phenotypic information of cells obtained by flow cytometry enabled analyses of specific immune cell subsets. We found that vaccination generally induced transient changes at 24 h in cell frequencies accompanied by fluctuation in selected liver enzymes and metabolites. However, most parameters remained within our identified reference intervals. These deviations did not lead to noticeable side effects. Fluctuation in selected biochemical and hematological parameters was accompanied with differentiation of CD14+CD16+ intermediate monocytes and upregulation of genes associated with interleukin-1 signaling. By contrast, two animals with noticeable side effects showed sustained deviations. This study provides insights into baseline and vaccine-induced biochemical and hematological profiles of healthy macaques, facilitating the interpretation of toxicity and safety assessments in preclinical trials of novel therapies.

Sanger sequencing remains widely used in various experimental contexts, often in combination with flow cytometry for indexing specific cell populations. However, existing software lacks the capability to automate quality control (QC) of raw Sanger sequencing data and integrate it with flow cytometry information on a large scale. Here, we introduce scifer, an R package now available in the latest release of Bioconductor (3.20) showcasing its effectiveness in seamlessly integrating these types of data as demonstrated by analyses of B cell and T cell receptor sequences. Scifer preprocesses raw data from index sorts and immune receptor Sanger sequencing. It identifies high-quality sequences based on selected parameters, such as length, Phred scores, and heavy-chain complementarity-determining region 3 (HCDR3) quality. As a result, the quality of germline assignments is significantly increased and spurious variable gene mutations are reduced. Scifer is automated and can process thousands of sequences in less than an hour. Its output provides quality control reports, FASTA files, summarized tables, and electropherograms for manual inspection. In summary, scifer is a user-friendly software that speeds up the analysis of immune receptor repertoire sequences, offering wide applicability.

mRNA vaccines are likely to become widely used for the prevention of infectious diseases in the future. Nevertheless, a notable gap exists in mechanistic data, particularly concerning the potential effects of sequential mRNA immunization or preexisting immunity on the early innate immune response triggered by vaccination. In this study, healthy adults, with or without documented prior SARS-CoV-2 infection, were vaccinated with the BNT162b2/Comirnaty mRNA vaccine. Prior infection conferred significantly stronger induction of proinflammatory and type I IFN-related gene signatures, serum cytokines, and monocyte expansion after the prime vaccination. The response to the second vaccination further increased the magnitude of the early innate response in both study groups. The third vaccination did not further increase vaccineinduced inflammation. In vitro stimulation of PBMCs with TLR ligands showed no difference in cytokine responses between groups, or before or after prime vaccination, indicating absence of a trained immunity effect. We observed that levels of preexisting antigen-specific CD4 T cells, antibody, and memory B cells correlated with elements of the early innate response to the first vaccination. Our data thereby indicate that preexisting memory formed by infection may augment the innate immune activation induced by mRNA vaccines.

The immune responses to Novavax’s licensed NVX-CoV2373 nanoparticle Spike protein vaccine against SARS-CoV-2 remain incompletely understood. Here, we show in rhesus macaques that immunization with Matrix-MTM adjuvanted vaccines predominantly elicits immune events in local tissues with little spillover to the periphery. A third dose of an updated vaccine based on the Gamma (P.1) variant 7 months after two immunizations with licensed NVX-CoV2373 resulted in significant enhancement of anti-spike antibody titers and antibody breadth including neutralization of forward drift Omicron variants. The third immunization expanded the Spike-specific memory B cell pool, induced significant somatic hypermutation, and increased serum antibody avidity, indicating considerable affinity maturation. Seven months after immunization, vaccinated animals controlled infection by either WA-1 or P.1 strain, mediated by rapid anamnestic antibody and T cell responses in the lungs. In conclusion, a third immunization with an adjuvanted, low-dose recombinant protein vaccine significantly improved the quality of B cell responses, enhanced antibody breadth, and provided durable protection against SARS-CoV-2 challenge.

The mRNA vaccines have proven to be very effective in preventing severe disease and death from SARS-CoV-2 in the general population. However, in patients with chronic kidney disease (CKD) in dialysis or with kidney transplants (KT) the vaccine responses vary, with severe breakthrough infections as a consequence. In this intervention study we investigated the magnitude and quality of the responses to mRNA vaccination administered prior to kidney replacement therapy (KRT). Twenty patients with CKD G4/5 and nine healthy controls were followed for 12 months after receiving two doses of BNT162b2 four weeks apart and a booster dose after 3-6 months. Induction of anti-Spike and anti-RBD IgG in plasma followed the same kinetics in CKD patients and controls, with a trend towards higher titers in controls. In accordance, there was no differences in the establishment of Spike-specific memory B-cells between groups. In contrast, the CKD patients showed lower levels of anti-Spike IgG in saliva and Spike-specific CD8+ T-cells in blood, possibly influencing the capacity of viral clearance which can contribute to an elevated risk of severe breakthrough infections. In conclusion, we found that CKD patients, despite having a reduced mucosal and cytotoxic immunity to BNT162b2, demonstrated a serological response in plasma similar to healthy controls. This suggests that immunization prior to RRT is efficient and motivated. (EudraCT-nr 2021-000988-68).

Licensed rabies virus vaccines based on whole inactivated virus are effective in humans. However, there is a lack of detailed investigations of the elicited immune response, and whether responses can be improved using novel vaccine platforms. Here we show that two doses of a lipid nanoparticle-formulated unmodified mRNA vaccine encoding the rabies virus glycoprotein (RABV-G) induces higher levels of RABV-G specific plasmablasts and T cells in blood, and plasma cells in the bone marrow compared to two doses of Rabipur in non-human primates. The mRNA vaccine also generates higher RABV-G binding and neutralizing antibody titers than Rabipur, while the degree of somatic hypermutation and clonal diversity of the response are similar for the two vaccines. The higher overall antibody titers induced by the mRNA vaccine translates into improved cross-neutralization of related lyssavirus strains, suggesting that this platform has potential for the development of a broadly protective vaccine against these viruses.

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Effective humoral immune responses require well-orchestrated B and T follicular helper (Tfh) cell interactions. Whether these interactions are impaired and associated with COVID-19 disease severity is unclear. Here, longitudinal blood samples across COVID-19 disease severity are analysed. We find that during acute infection SARS-CoV-2-specific circulating Tfh (cTfh) cells expand with disease severity. SARS-CoV-2-specific cTfh cell frequencies correlate with plasmablast frequencies and SARS-CoV-2 antibody titers, avidity and neutralization. Furthermore, cTfh cells but not other memory CD4 T cells, from severe patients better induce plasmablast differentiation and antibody production compared to cTfh cells from mild patients. However, virus-specific cTfh cell development is delayed in patients that display or later develop severe disease compared to those with mild disease, which correlates with delayed induction of high-avidity neutralizing antibodies. Our study suggests that impaired generation of functional virus-specific cTfh cells delays high-quality antibody production at an early stage, potentially enabling progression to severe disease.

Targeting CD40 by agonistic antibodies used as vaccine adjuvants or for cancer immunotherapy is a strategy to stimulate immune responses. The majority of studied agonistic anti-human CD40 antibodies require crosslinking of their Fc region to inhibitory FcγRIIb to induce immune stimulation although this has been associated with toxicity in previous studies. Here we introduce an agonistic anti-human CD40 monoclonal IgG1 antibody (MAB273) unique in its specificity to the CD40L binding site of CD40 but devoid of Fcγ-receptor binding. We demonstrate rapid binding of MAB273 to B cells and dendritic cells resulting in activation in vitro on human cells and in vivo in rhesus macaques. Dissemination of fluorescently labeled MAB273 after subcutaneous administration was found predominantly at the site of injection and specific draining lymph nodes. Phenotypic cell differentiation and upregulation of genes associated with immune activation were found in the targeted tissues. Antigen-specific T cell responses were enhanced by MAB273 when given in a prime-boost regimen and for boosting low preexisting responses. MAB273 may therefore be a promising immunostimulatory adjuvant that warrants future testing for therapeutic and prophylactic vaccination strategies.

A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-μg doses of CVnCoV, CureVac’s severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth.

Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4(+) and CD8(+) T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4(+) T cells and robust expansions of oligoclonal effector-memory CD45RA(+) CD8(+) T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes.

Abstract Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer. Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021–000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1 .

AbstractObjectivesLoss of vaccine‐induced antibodies (Abs) after chemotherapy against paediatric acute lymphoblastic leukaemia (ALL) is common and often necessitates re‐immunisation after cessation of treatment. Even so, some ALL survivors fail to mount or to maintain protective Abs. Germinal centres (GCs) are clusters of proliferating B cells in follicles of secondary lymphoid tissues (SLTs) formed during adaptive immune responses and the origins of long‐lived memory B and plasma cells that are the source of Abs. Furthermore, productive GC reactions depend on T follicular helper (TFH) cells. To understand why chemotherapy induces deficits in Ab responses, we examined how SLTs were affected by chemotherapy.MethodsRhesus macaques were infused with either three cycles of the anthracycline doxorubicin or saline, followed by immunisation with a de novo and booster antigen. Spleen and lymph nodes were removed, and memory B, bulk T and TFH cells were examined.ResultsDespite adequate GC morphology, a diminished memory and IgG+ B‐cell population along with diminished total and booster vaccine‐specific IgG‐producing memory B cells were noted in the spleens of macaques with past doxorubicin exposure compared to the saline‐treated controls (P < 0.05). Intact bulk T and TFH cells were found in the SLTs of treated macaques, which displayed higher CD40L upregulation capacity by their splenic CXCR5+ helper T cells (P < 0.01). In contrast to the spleen, the immune cell populations studied were comparable between the lymph nodes of both saline‐ and doxorubicin‐treated macaques.ConclusionOur findings suggest that the splenic memory B‐cell subset, compared to its lymph node counterpart, is more severely altered by anthracycline treatment.

Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP) in non-human primates. While both intradermal (ID) and intramuscular (IM) administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.