Background Immunocompromised individuals were identified early in the pandemic as being at increased risk of severe COVID-19 and have demonstrated variable immune responses to SARS-CoV-2 vaccination. Although coordinated vaccination programmes are now well established, their long-term effects on sustained immunity in the present patient populations remain insufficiently understood. Methods The prospective SARS-CoV-2 mRNA vaccine trial COVAXID was conducted in a well-characterised, real-world cohort of 539 immunocompromised and healthy individuals across 21 subgroups, organised into six main categories. At the 36-month time point, 218 participants remained. Participants provided blood samples for assessment of binding antibody titres and pseudo-neutralisation activity against ancestral SARS-CoV-2 and 21 variants, including Omicron sub-lineages. T cell responses were evaluated in a defined subset of participants. Immunogenicity outcomes were analysed over a three-year period in relation to SARS-CoV-2 vaccination, SARS-CoV-2 infection, and immunoglobulin replacement therapy (IGRT). Findings Between years two and three, antibody titres and neutralisation capacity showed a consistent pattern of maintenance or increase across most study groups and subgroups. These increases were driven by cumulative exposure to vaccine booster doses, SARS-CoV-2 infection, and, in some cases, passive immunisation through IGRT. CD4+ and CD8+ T cell responses were detected across all study groups. Early immune responses were primarily vaccine-driven, whereas later immune profiles reflected substantial contributions from natural infection and anti-SARS-CoV-2 antibodies in IGRT products. Interpretation The findings support continued, tailored vaccination strategies for elderly and immunocompromised individuals. Integrating immune monitoring with infection history and adjunctive therapies may help refine booster policies, optimise protection, and strengthen future vaccination programmes for high-risk populations. Funding The present studies were supported by the European Research Council, Karolinska Institutet, Knut and Alice Wallenberg Foundation, Nordstjernan AB, Region Stockholm, and the Swedish Research Council.

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.

Older adults (> 65 years) residing in long-term care facilities (LTCFs) are at elevated risk of severe outcomes from respiratory infections. Infections often remain undetected or present atypically in this population, leading to underdiagnosis. Our study aimed to estimate the respiratory virus infection burden, independent of symptom presentation, among older adults in Swedish LTCFs in the post-pandemic period (2021–2024). We leveraged capillary blood samples and coupled national registry data from 1622 LTCF residents (median age = 87). A multiplex platform was used to quantify antigen-specific IgG and IgM responses to RSV (pre-/post-F, strain A-specific G-protein), influenza-A (H1N1 and H3N2 HA), influenza-B (HA) and SARS-CoV-2 (spike). Linear mixed-effects models were used to demonstrate the dynamics of antibody levels over time, adjusted for age, sex and comorbidities. RSV-specific antibody responses peaked in spring 2022 (p < 0.001), suggesting an impact of relaxed COVID-19-related restrictions on RSV exposure at LTCFs. RSV-specific antibodies subsequently declined over time until an increase during autumn 2023 (p < 0.001). Geographic variation in pre-F antibody levels suggested localised RSV outbreaks. The total estimated RSV burden at LTCFs was markedly higher than official reports of the Swedish Public Health Agency. Influenza antibody dynamics reflected seasonal trends and were strongly influenced by annual vaccination. A random forest classifier incorporating serological profiles with demographics, location and comorbidities significantly outperformed a model without serological data (AUC-ROC = 0.67 vs. 0.58), although discriminatory performance remained modest. Higher levels of RSV pre-F antibodies in autumn 2021 were associated with increased one-year mortality in logistic regression (OR = 1.43, p = 0.024). Exploratory survival analysis indicated a trend that elevated levels of RSV pre-F antibodies during low population immunity may confer a transiently elevated early hazard of death, although this did not reach statistical significance (HR = 4.50, p = 0.087). We observed substantial respiratory virus circulation among older adults in Swedish LTCFs and show that RSV burden is under-reported. The results highlight a need for further research into the role of RSV pre-F antibody levels in preventing severe outcomes, potentially via vaccination of LTCF residents. Our scalable serological surveillance system is a valuable approach to detect respiratory infections in LTCFs, independent of symptom presentation or healthcare-seeking behaviour.

Recent advances in de novo protein design have greatly outpaced standard protein biochemistry workflows, making experimental validation a bottleneck. Here, we describe workflows to address the scale, speed and reproducibility of common in vitro protein testing methods, enabling at least an order of magnitude increase in throughput while reducing wetlab time. Semi-Automated Protein Production (SAPP) is a rapid, modular, scalable and cost-effective protocol, enabling up to milligram-scale protein production and standardized characterization – including yield, dispersity, and oligomeric state – of hundreds of designs per day, at the cost-equivalent of a few DNA oligos per construct. End-to-end protocol execution takes 48 hours, with ~6 hours spent benchside using standard laboratory equipment. We showcase the platform by rapidly screening redesigned fluorescent proteins, as well as identifying de novo binders that potently neutralize respiratory syncytial virus. We also developed a barcoding and demultiplexing protocol (DMX) to further reduce gene synthesis cost 5-fold by leveraging oligo pools as input DNA for the generation of thousands of sequence-verified arrayed clones. These protocols which combine optimized molecular biology, automated analysis, and optional open-source robotics should be widely adoptable, accelerating protein design. De novo protein design outpaces experimental validation. Here, authors present SAPP and DMX — scalable, semi-automated workflows enabling cloning, purification and characterization of hundreds to thousands of designed proteins per day at low cost.

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.

Despite waning of virus-neutralizing antibodies, protection against severe SARS-CoV-2 in the majority of immune individuals remains high, but the underlying immune mechanisms are incompletely understood. Here, rhesus macaques with pre-existing immunity from Novavax WA-1 and/or P.1 vaccines and WA-1 or P.1 infection are immunized with a bivalent WA-1/Omicron BA.5 Novavax vaccine ten months after the last exposure. The boost vaccination primarily increases the frequency of cross-reactive spike (S)-specific antibodies and B cells instead of inducing de novo BA.5-specific responses. Reinfection with heterologous Omicron XBB.1.5 six months after the boost vaccination results in low levels of virus replication in the respiratory tract compared with virus-naïve results from other studies. Whereas systemic S-specific immunity remains largely unchanged in all animals, the animals with complete protection from infection exhibit a stronger influx of S-specific IgG, monocytes, B cells and T cells into the bronchioalveolar space combined with expansion of CD69 + CD103 + lung tissue-resident, S-specific CD8 T cells compared to actively infected animals. Our results underscore the importance of localized respiratory immune responses in mediating protection from Omicron reinfection and provide guidance for future vaccine development.

mRNA vaccines represent a new era with several novel constructs underway. We compared the responses of high doses and multiple repetitive immunizations of a nucleoside-modified mRNA construct to a sequence-codon-optimized unmodified mRNA construct encoding the identical model antigen (HIV-1 gag). Rhesus macaques were immunized five times at 2-week intervals, with a final boost 20 weeks later. At 24 h post-vaccination, both unmodified (160 μg) and modified (400 μg and 800 μg) mRNA constructs elicited clear but transient increase of plasmacytoid dendritic cells, intermediate CD14+ CD16+ monocytes, and neutrophils along with secretion of type I interferon (IFN)-related and inflammatory cytokines. Unmodified mRNA induced higher interleukin-7 (IL-7) and IFN-α levels, whereas modified mRNA induced higher IL-6 levels. Transcriptomic profiling showed significant upregulation of genes related to type I IFN signaling, antigen presentation, and innate immune activation induced by both mRNA constructs. The high-dose modified mRNA induced a higher number of differentially expressed genes at prime, which further increased after the fifth immunization. These differences in innate immune activation nonetheless led to similar levels and kinetics of gag-specific antibody and T cell responses. These findings offer insights into the immunogenic and reactogenic potential of different mRNA vaccine modalities, guiding future vaccine and therapy development.

Seasonal influenza continues to be a global health problem. Current existing vaccines and antivirals against influenza have limited effectiveness, and typically do not stay ahead of the viral evolutionary curve. Broad-spectrum antiviral agents that are effective therapeutically and prophylactically are much needed. We have created a promising new broad-spectrum anti-influenza agent using molecular engineering of a lectin from bananas, H84T, which is well-tolerated and protective in small animal models. However, the potency and effect of H84T on human immune cells and influenza-specific immune responses are undetermined. We found that H84T efficiently inhibited influenza A virus (IAV) replication in primary human dendritic cells (DCs) isolated from blood and tonsil, preserved DC viability and allowed acquisition and presentation of viral antigen. Excitingly, H84T-treated DCs subsequently initiated effective expansion of IAV-specific CD8 T cells. Furthermore, H84T preserved the capacity of IAV-exposed DCs to present a second non-IAV antigen and induce robust antigen-specific CD8 T cell expansion. Our data support H84T as a potent antiviral in humans as it not only effectively inhibits IAV infection, but also preserves induction of robust pathogen-specific adaptive immune responses against diverse antigens, which likely is clinically beneficial.

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.

Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has a remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signaling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development.

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients.

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.

COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly impacted public health and the economy worldwide. Most of the currently licensed COVID-19 vaccines act by inhibiting the receptor-binding function of the SARS-CoV-2 spike protein. The constant emergence of SARS-CoV-2 variants resulting from mutations in the receptor-binding domain (RBD) leads to vaccine immune evasion and underscores the importance of broadly acting COVID-19 vaccines. Inactivated whole virus vaccines can elicit broader immune responses to multiple epitopes of several antigens and help overcome such immune evasions. We prepared a psoralen-inactivated SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) and evaluated its immunogenicity and efficacy in nonhuman primates (NHPs) when administered with the Advax-CpG adjuvant. We also evaluated the SARS-CoV-2 PsIV as a booster shot in animals vaccinated with a DNA vaccine that can express the full-length spike protein. The Advax-CpG-adjuvanted SARS-CoV-2 PsIV elicited a dose-dependent neutralizing antibody response in the NHPs, as measured using a serum microneutralization assay against the SARS-CoV-2 Washington strain and the Delta variant. The animals vaccinated with the DNA vaccine followed by a boosting dose of the SARS-CoV-2 PsIV exhibited the highest neutralizing antibody responses and were able to quickly clear infection after an intranasal challenge with the SARS-CoV-2 Delta variant. Overall, the data show that the Advax-CpG-adjuvanted SARS-CoV-2 PsIV, either by itself or as a booster shot following nucleic acid (NA) vaccines, has the potential to protect against emerging variants.

Background Immunocompromised patients with primary and secondary immunodeficiencies have shown impaired responses to SARS-CoV-2 mRNA vaccines, necessitating recommendations for additional booster doses. However, longitudinal data reflecting the real-world impact of such recommendations remains limited. Methods This study represents a two-year follow-up of the COVAXID clinical trial, where 364 of the original 539 subjects consented to participate. 355 individuals provided blood samples for evaluation of binding antibody (Ab) titers and pseudo-neutralisation capacity against both the ancestral SARS-CoV-2 strain and prevalent Omicron variants. T cell responses were assessed in a subset of these individuals. A multivariate analysis determined the correlation between Ab responses and the number of vaccine doses received, documented infection events, immunoglobulin replacement therapy (IGRT), and specific immunosuppressive drugs. The original COVAXID clinical trial was registered in EudraCT (2021-000175-37) and clinicaltrials.gov (NCT04780659). Findings Several of the patient groups that responded poorly to the initial primary vaccine schedule and early booster doses presented with stronger immunogenicity-related responses including binding Ab titres and pseudo-neutralisation at the 18- and 24-month sampling time point. Responses correlated positively with the number of vaccine doses and infection. The vaccine response was blunted by an immunosuppressive state due to the underlying specific disease and/or to specific immunosuppressive treatment. Interpretation The study results highlight the importance of continuous SARS-CoV-2 vaccine booster doses in building up and sustaining Ab responses in specific immunocompromised patient populations. Funding The present studies were supported by the European Research Council, Karolinska Institutet, Knut and Alice Wallenberg Foundation, Nordstjernan AB, Region Stockholm, and the Swedish Research Council.

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.

T cells are critical in mediating the early control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection. However, it remains unknown whether memory T cells can effectively cross-recognize new SARS-CoV-2 variants with a broad array of mutations, such as the emergent hypermutated BA.2.86 variant. Here, we report in two separate cohorts, including healthy controls and individuals with chronic lymphocytic leukemia, that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection or vaccination demonstrate resilient immune recognition of BA.2.86. In both cohorts, we found largely preserved SARS-CoV-2 spike-specific CD4+ and CD8+ T cell magnitudes against mutated spike epitopes of BA.2.86. Functional analysis confirmed that both cytokine expression and proliferative capacity of SARS-CoV-2 spike-specific T cells to BA.2.86-mutated spike epitopes are similarly sustained. In summary, our findings indicate that memory CD4+ and CD8+ T cells continue to provide cell-mediated immune recognition to highly mutated emerging variants such as BA.2.86.