MI2025 – Poster Presenters

Ankyrons® are small, high-affinity binding reagents that are animal free recombinant proteins and are based on an ankyrin-repeat scaffold overcoming many of the limitations of traditional antibodies. At ProImmune, we are generating a growing catalog of Ankyron clones which currently covers 3,000+ targets across 100+ species including proteins from key infectious diseases such as Viruses (Dengue, Mpox, SARS Coronaviruses, etc.), Bacteria (Mycobacterium tuberculosis), Parasites (Malaria), Disease Vectors (Mosquitoes, Avian Species, Swine, etc.).

Ankyrons perform seamlessly across applications including immunofluorescence, immunohistochemistry, flow cytometry, and ELISA while offering critical advantages over antibodies. Unlike antibodies, the Ankyrons compact size enables superior cell and tissue penetration and precise imaging, while its Fc-free design eliminates background from Fc receptor binding. Discover how Ankyrons can advance your research with more reliable, flexible, and cost-effective alternatives to conventional antibodies.

Chlamydia trachomatis is the most common bacterial sexually transmitted infection globally, posing significant risks for reproductive health, including pelvic inflammatory disease, infertility, and ectopic pregnancy. As an obligate intracellular pathogen, C. trachomatis primarily targets vaginal epithelial cells, where it establishes persistent infections while evading immune surveillance. Type III interferons (IFN-λ) are key cytokines in mucosal immunity, offering localized protection with minimal systemic inflammation due to the restricted expression of their receptor complex (IFNLR1/IL-10R2) on epithelial tissues. This study investigates the interaction between type III IFNs and C. trachomatis infection in epithelial cells. Our study reveals that C. trachomatis interferes with the expression of signaling proteins, suggesting a potential immune evasion mechanism employed by C. trachomatis to dampen type III IFN signaling. Understanding this regulatory dynamic provides novel insight into host-pathogen interactions and highlights the potential of targeting the IFN-λ pathway to develop therapeutic interventions that enhance mucosal immunity while limiting inflammation during C. trachomatis infection.

Pulmonary aspergillosis encompasses several types of infections caused by ubiquitous fungi belonging to the genus Aspergillus. Immunosuppression, derived either from past disease, including asthma, cancer, and HIV infection, or cytotoxic therapies, including organ transplantation and high-dose corticosteroid administration, create a permissive environment for conidial growth and germination. This is associated with considerable mortality regardless of antifungal treatment. Interestingly, recent clinical observations suggest that pulmonary aspergillosis is the most common infection in patients with a history of tuberculosis treatment1,2. Many such cases are misdiagnosed as relapsed tuberculosis due to similarities in clinical and radiological presentation3. Although the permanent structural lung changes succeeding treatment of tuberculosis facilitate subsequent re-infection, effects on alveolar epithelial cells and macrophages, acting as the gatekeepers to pulmonary aspergillosis, as are yet to be recognised to a similar extent.

In this project, we sought to investigate changes in the stimulation of macrophages and alveolar epithelial cells in response to challenge by Aspergillus fumigatus following priming with BCG, a vaccine strain of Mycobacterium tuberculosis. We compared responses of immortalised bone marrow derived macrophages (iBMDMs), GM-CSF differentiated bone-marrow derived macrophages (BMDMs), and A549 cells. We found that BMDM and A549 cells are more responsive to aspergillus infection compared to iBMDMs. Contrary to published data, we observed that priming with BCG leads to reductions in caspase activity for both cell types at 24h and 48h post BCG priming compared to non-primed cells. Priming also leads to increased release of proinflammatory cytokines IL-1β, IL-6, and TNFα. Lastly, we observed that BMDM and A549 priming with BCG may promote aspergillus survival.

Enterovirus 71 (EV-A71) is the main cause severe hand, foot and mouth disease (HFMD) in the Asia-Pacific region. Although T cell–mediated immune responses potentially play a critical role in controlling severe disease, current knowledge regarding T cell immunity in patients with hand, foot, and mouth disease (HFMD) caused by EV-A71 remains unknown. These findings highlight for the first time the immunological significance of structural protein in particular VP2 in inducing the T cell response to EV-A71 infection, and underscore the need for further investigation into the immune response profiles underlying disease progression and correlate of protection in HFMD, which would inform the development of vaccines and therapeutic approaches.

Autologous T-cell therapies against cancer face disadvantages in terms of production cost, logistics, and compromised T-cell fitness. The use of allogeneic T cells can partially bypass these limitations, but it brings new challenges involving host-versus-graft (HvG) and graft-versus-host (GvH) reactions. Our aim is to develop a strategy to bypass these two obstacles by leveraging on the substantial flexibility of mRNA electroporation technology in engineering allogeneic mRNA TCR-T cells.

Instead of reducing allogeneic T cell immunogenicity through irreversible genetic approaches, we propose to prevent HvG reactions by transiently suppressing the host's immune system with a finite treatment of immunosuppression (tacrolimus). At the same time, the functionality of these allogeneic T cells will be preserved through the conferment of transient tacrolimus resistance by introducing a modified version of calcineurin B into the T cells through mRNA electroporation. Moreover, to minimize the risk of GvH disease, we propose to utilize different cytokine cocktails to expand T cells to produce TCR-T cell products with inherently reduced GvH potential.

Outbred animal models of rapid and reproducible elicitation of potent, broadly neutralizing antibodies (bNAbs) against HIV-1 Envelope could advance vaccine research. We designed a simian-human immunodeficiency virus (SHIV.5MUT) bearing a V3-glycan epitope-targeting Envelope that elicited bNAbs within a year in 14/22 rhesus macaques (plasma neutralization breadth ≥50%; ID50 titers >1:1000), compared with 0/14 controls. As revealed by antibody/virus sequencing and polyclonal epitope mapping by cryo-EM, SHIV.5MUT first induced V1-directed antibodies that drove generation of shortened V1 loop escape variants, which then stimulated V3-glycan bNAb precursors. For mature bNAbs, Fab-Env cryo-EM structures revealed V3-recognition motifs resembling human bNAb counterparts. In summary, antibody-virus coevolution after infection with an epitope-targeting SHIV is a generalizable strategy for inducing bNAbs and can facilitate identification of prime and sequential boosting immunogens for HIV-1 vaccine development. Thus, my talk will focus on both the immunological and structural mechanisms of V3-glycan bNAb induction in rhesus macaques. Unpublished data.

Cerebral malaria (CM) is a severe complication caused by Plasmodium falciparum infection, leading to persistent neurological impairments in survivors. To understand the complex mechanisms and investigate advanced diagnostic and treatment strategies targeting human CM, we utilized Plasmodium coatneyi-infected rhesus macaques, a non-human primate model closely resembling P. falciparum infection in humans. Through differential gene expression analysis, our study demonstrated methylene blue’s efficacy in reversing the detrimental effects of infection on the brainstem. Furthermore, by comparing our brainstem dataset from P. coatneyi-infected Macaca mulatta with two additional transcriptomic datasets (P. coatneyi-infected M. mulatta blood and P. falciparum-infected human blood), we identified nine genes associated with CM severity. Most of these genes were expressed in neutrophils, indicating their potential as blood biomarkers for diagnosing P. falciparum-induced fatal CM. This research highlights the necessity for new CM treatments and reveals promising biomarkers that could improve diagnosis and prognosis in affected individuals.

Accurately identifying individuals at high risk for severe COVID-19 remains a critical challenge. Current risk stratification methods, which rely on clinical symptoms and broad factors such as age and comorbidities, often lack the precision needed for early intervention. Although genomic research has progressed significantly worldwide, efforts to identify population-specific genetic biomarkers, particularly in Malaysia, remain limited. This study analyzed genetic data from 409 recovered COVID-19 patients from Klang Valley, Malaysia, comprising 381 with history of non- severe and 28 with severe COVID-19. Ion AmpliSeq technology was employed to design a custom panel of 100 genes of interest, covering a 492.92 kb genomic region. Our analysis detected 326,199 variants in non-severe cases and 23,855 in severe cases, with subsequent filtering (SIFT score ≤ 0.05) identifying 525 and 179 unique variants, respectively. Statistical evaluation revealed five significant pathogenic variants associated with key biological processes, including immune regulation and host-pathogen interactions. In silico functional analyses further predicted their impact on protein structure and function. These findings underscore the role of genetic predisposition in severe COVID-19 and offer valuable insights for early diagnosis, personalized treatment, and the development of targeted therapies. This study provides a foundation for leveraging genetic insights to advance precision medicine in COVID-19 management, particularly for Malaysian and other underrepresented populations.

CD4⁺ T cells are central orchestrators of immune responses in the context of infection and vaccinations, yet the molecular determinants of potent CD4⁺ T-cell responses remain largely unclear. We have established an assay to assess the TCR avidity of antigen-specific CD4+ and CD8+ T cells by analyzing the expression of the transcription factor IRF4, induced by SARS-CoV-2 infection and mRNA vaccinations. In this study, we aimed to define their transcriptional and T-cell receptor (TCR) features at single-cell resolution. To this end, we analyzed spike protein-specific CD4⁺ T cells in subjects at post-2nd dose of the BNT162b2 mRNA vaccine. PBMCs were cultured with SARS-CoV-2 spike protein for 24 hours, and specific CD4+ T cells were sorted as cells expressing activation-induced markers (AIM), followed by analysis with single-cell RNA and paired TCR sequencing. We found that high-avidity CD4⁺ T cells were clearly separated on the gene expression (GEX) UMAP space. High-avidity CD4⁺ T cells are exhibited stronger expression of TCR signaling and proliferation-related genes, with greater clonal expansion. The TCR repertoire analysis revealed minimal overlaps with low-avidity T cells, indicating that high-avidity T cells do not merely represent a highly activated status. Moreover, high-avidity CD4⁺ T cells were enriched with public clonotypes that exist in more than 2 individuals. Collectively, these findings highlight the unique biological features of high-avidity CD4⁺ T cells, and thus provide important insights into the conceptual framework for the generation of T cell–targeted vaccines.

The nasal cavity is the entry site for respiratory viruses. Understanding how the nasal cavity sustains memory CD8+ T cell is essential for improving respiratory virus management and vaccine development. Here, we sampled CD8+ T cells from the upper nasal turbinate and peripheral blood of healthy adults. We analysed their transcriptomic profile and antigen specificity for respiratory (SARS-CoV-2, Influenza) and non-respiratory (HCMV) viruses.
Transcriptomic analysis revealed that nasal CD8+ T cells failed to upregulate STAT1 following TCR stimulation, potentially enabling clonal expansion despite the antiproliferative effects of IFN signalling. They also exhibited a cytotoxic, Th1-like profile with tissue-residency markers but lacked TCF7 expression, suggesting limited self-renewal capacity. The CD8+ T cell analysis of antigen specificity demonstrates that local nasal exposure is indispensable for virus-specific CD8+ T cell detection. Only SARS-CoV-2 and influenza-specific but not HCMV-specific CD8+ T cells were detected in the nasal compartment. However, their persistence over time in the nasal cavity appears linked to repetitive viral exposure.
Our findings provide insights into the adaptations of nasal-resident CD8+ T cells and highlight challenges in eliciting durable nasal T cell immunity, with important implications for vaccine strategies against respiratory pathogens.

This presentation examines how maternal infections with SARS-CoV-2 and Enteroviruses may influence placental immune composition and function. Through the use of RT-qPCR and multicolour flow cytometry, we analysed maternal plasma and placental tissue to investigate viral presence and profile key immune subsets including T cells, NK cells, and B cells. The study aims to uncover how viral exposure during pregnancy shapes the immunological landscape of the placenta, with potential implications for maternal and neonatal health.

TCR-T cell therapy holds great promise for solid tumour treatment, but its feasibility relies critically on identifying antigen-specific T cell receptors (TCRs) capable of recognizing cancer cells. This typically involves characterising tumour-infiltrating lymphocytes (TILs); however, the majority of TILs are bystander cells, making the identification of antigen-specific tumour-reactive T cells a laborious and costly "needle in a haystack" problem. Hence, we aim to establish and validate an antigen-agnostic, gene-signature based approach to rapidly identify antigen-specific T cells based solely on their transcriptional profiles. We stimulated peripheral blood mononuclear cells (PBMCs) in vitro with peptide pools derived from SARS-CoV-2 (Spike), Influenza A (IAV; HA, NA, MP1, MP2, NP), and EBV (EBNA1, LMP1, LMP2A, BZLF1) and sorted total activated T cells, containing both TCR- and bystander- activated populations, for single-cell RNA (scRNA-seq) and T cell receptor sequencing (TCR-seq). In parallel, PBMCs were stained with the corresponding MHC Class I multimers to identify known CoV-2/IAV/EBV- specific TCR clonotypes. By integrating both datasets, we classified peptide-activated T cells, of known specificity defined by multimers, as TCR-activated when stimulated by matching peptide pools, and as bystander-activated when stimulated by non-matching peptide pools. We subsequently applied a TCR activation–associated gene signature derived from a public dataset to these two populations and observed an increase in gene signature module scores in TCR-activated T cells, but not in bystander or unstimulated T cells, validating the signature’s ability to robustly distinguish TCR-activated T cells from bystanders. Using this validated signature, we selected putative antigen-specific T cells directly from the scRNA-seq data of peptide-activated T cells. Future studies will functionally validate these candidates and extend this approach to rapidly identify tumor-reactive TILs in solid tumors.

Obesity is a significant risk factor for worsening SARS-CoV-2 infection, leading to complications such as malnutrition, systemic inflammation, and myocardial dysfunction. However, the precise mechanisms by which obesity increase susceptibility and severity of SARS-CoV-2 remain poorly understood. Based on these preliminary findings, we hypothesize that ACE2 expression is increased in differentiated adipocytes, which may enhance SARS-CoV-2 infection in these cells.

We propose to pursue three specific aims to identify novel host factors that facilitate viral infection in adipocytes: (1) To measure the expression levels of ACE2 receptors at various stages of adipocyte maturation. (2) To identify important Gene related to SARS-COV-2 entry into Adipocyte (3) To Validate identified host factors through targeted knockout and overexpression studies. To this end, we will utilize a bidirectional study of genome-wide CRISPR overexpression library system and CRISPR knockout library system in combination with ACE2 overexpression to mimic the ACE2 expression levels observed in obese individuals. We will then employ SARS- CoV-2 spike pseudovirus (Vpp) infection in adipocytes. This approach enables systematic identification and validation of host factors essential for viral entry. Our findings may offer key insights into the molecular dynamics of SARS-CoV-2 infection within adipocytes and may establish a novel therapeutic strategy targeting virus-host interactions. These insights will be important for developing novel therapeutic strategies against SARS-CoV-2 infection.

Many coronaviruses, including members of the alphacoronavirus and betacoronavirus genera, utilize the host angiotensin converting enzyme 2 (ACE2) as the entry receptor for viral infection. Due to the structural similarities between many animal ACE2 with the human homolog, there is potential for zoonotic spillover events. As such, prophylactic or therapeutic treatment options against ACE2-utilizing coronaviruses are required. To address this urgent need, ACE2 decoy receptors were generated and optimized to broadly neutralize both alpha- and betacoronaviruses that exploit the ACE2 pathway for viral entry and infection. Through in silico methods to identify key interaction residues and mutations for favorable binding energetics, a library of ACE2 decoys was generated and synthesized for testing. Our results revealed that the key ACE2 interaction residues differed across betacoronaviruses, as well as between beta- and alphacoronaviruses. The hits that were identified demonstrated breadth in binding to all tested SARS-CoV-2 variants, as well as other human and animal sarbecovirus, merbecovirus, and setracovirus subgenera. Notably, our hits showed at least 7-fold improvements in binding KD and neutralization IC50 against recent variants of SARS-CoV-2, as well as up to 100-fold improvements against animal sarbecoviruses. Overall, the improved ACE2 decoy molecules offer a promising avenue for broad-spectrum coronavirus neutralization as well as provide valuable insights into the ACE2-virus interaction dynamics. Our findings also highlight the adaptability and efficacy of combining computational and high-throughput screening approaches to identify future-proof therapeutics in response to emerging and diverse viral threats.

Cross-reactivity is a phenomenon in which T cells recognise and respond to new antigens that are structurally similar, but not identical to the original target antigen they were originally primed against. In the context of SARS-CoV-2, the presence of pre-existing, cross-reactive T cells has been widely reported in individuals with no prior exposure to the virus, highlighting ubiquitous pre-existing T cell memory attributed to previous exposure to common cold coronaviruses. The protection afforded by cross-reactivity against new, emerging antigens remains a topic of contentious debate, and is influenced by variables such as T cell avidity – defined as the overall sensitivity to its cognate antigen. High avidity T cells are highly responsive to antigens and therefore are theorised to exhibit higher functionality for enhanced viral protection. In contrast, low avidity T cells do not undergo robust activation, and hence could possess lacklustre effector capabilities that impedes optimal responses by occupying the immunological synapse. Whether pre-existing, cross-reactive T cells could be reactivated by SARS-CoV-2 and the immunological outcomes following reactivation remains obscure.

Here, we profiled the dynamics of cross-reactive CD4+ T cell responses in a longitudinal cohort following SARS-CoV-2 mRNA vaccination by analysing HCoV-229E-specific CD4 T cells. Peripheral blood mononuclear cells (PBMCs) collected at baseline, 2 weeks post-primary dose, 2 weeks post-second dose, and 6 months post-second dose were stimulated with whole spike proteins of SARS-CoV-2 (CoV2) and HCoV-229E (229E). Pre-existing CoV-2-reactive T cells were observed before vaccination in most individuals. mRNA vaccination induced robust expansion of CoV2-specific and 229E-specific CD4 T cells. Surprisingly, frequencies of high avidity 229e-specific, but not low avidity 229e-specific, CD4 T cells expanded after two doses of vaccination. High avidity CoV-2- and 229E-specific T cells shared similar features and were dominated by Th1 and Tfh phenotypes. By contrast, low-avidity T cells showed enrichment for inflammatory profiles. All in all, this study provides novel insights on the protective role of cross-reactive T cells.

Seasonal influenza vaccines require annual updates due to antigenic drift and waning immunity, highlighting the need for a universal flu vaccine targeting conserved viral epitopes. The extracellular domain of M2 (M2e) is a leading universal vaccine antigen candidate, but its weak immunogenicity hinders clinical development.

To enhance M2e immunogenicity, we developed a dendritic cell (DC)-targeting vaccine by fusing M2e to the heavy chains of a Clec9A-specific monoclonal antibody (Clec9A-M2e). We show that a prime-boost regimen with only 2µg of the Clec9A-M2e construct (adjuvanted with poly I:C) induced very high systemic M2e-specific IgG titres that afforded full protection against lethal H1N1/PR8 challenge in young adult mice, with minimal body weight loss up to 6 months post-boost. We also employed an antibody-dependent cell cytotoxicity (ADCC) reporter assay to demonstrate the functionality of immune sera collected. The Clec9A-M2e prime-boost immunization also generated significant M2e-specific T cell responses both systemically (spleen) and locally (lungs).

In aged mice, Clec9A-M2e induced significant but lower M2e-specific antibody, T cell responses, and ADCC activity, correlating with only 50% protection and moderate lung viral reduction upon challenge. In young mice with pre-existing flu immunity, a single Clec9A-M2e dose boosted M2e-specific antibody titres to levels comparable to prime-boosted flu-naïve mice. Aged mice with pre-existing immunity required two doses of Clec9A-M2e to achieve similar immune responses.

Taken together, these results support that the Clec9A-targeting strategy represents a promising vaccine delivery platform able to overcome the weak immunogenicity of M2e and induce strong immune responses upon respiratory immunization.

The COVID-19 pandemic has sparked significant interest in understanding the immune responses of individuals who have experienced both SARS-CoV-2 vaccination and infection. While extensive research has explored both acute and long-term immune responses, less is known about their impact on allergic responses and immune dysregulation. This study investigates allergy-related immune markers in vaccinated COVID-19 convalescent individuals, specifically total IgE, specific anti-SARS-CoV-2 IgE (sIgE), and pro-inflammatory cytokines (IL-17A, IL-8, IL-2, IL-4, and IL-6) using ELISA, while mast cells were analysed using flow cytometry. Total IgE and sIgE levels were significantly correlated with age where younger individuals showed significantly higher levels of both total IgE and sIgE compared to older individuals. History of allergy was significantly linked to increased total IgE but showed no association with sIgE. Mast cell levels did not significantly vary by gender, age, allergy status, or infection severity. Cytokine analysis revealed a significant increase in IL-4 levels following SARS-CoV-2 peptide stimulation in peripheral blood mononuclear cells (PBMCs) from vaccinated COVID-19 recovered individuals. This effect was more pronounced in those with a history of severe infection, suggesting a Th2-skewed shift upon re-exposure, potentially impacting post-COVID-19 immune regulation and hypersensitivity. Other cytokines (IL-17A, IL-8, IL-2 and IL-6) showed no significant differences, though some trended upward or downward. Vaccinated COVID-19 recovered individuals, particularly those with a history of severe infection, exhibit altered immune profiles characterized by elevated IL-4 levels, suggesting a potential Th2-skewed immune response. These immune alterations, alongside age and allergy history effects on IgE levels, highlight the need for further research to refine treatment strategies and improve clinical management for individuals at risk of immune dysregulation.

Malaria is a life-threatening disease that continues to pose a major global health challenge, impacting millions of individuals each year and is caused by parasites of the Plasmodium species. Of the five known human-infecting species, Plasmodium falciparum remains the most extensively studied due to its high prevalence and association with severe clinical outcomes. . P. falciparum is able to invade mouse red blood cells (mRBCs) but fails to complete its maturation cycle within this non-native erythrocytic environment. We aimed to gain insights into the species barrier between human and mouse erythrocytes in order to identify crucial cellular pathways for drug discovery by using mRBCs infected with P. falciparum. To achieve this, we conducted microarray hybridisation followed by comprehensive transcriptomic analyses, including both differential gene expression and weighted gene co-expression network analyses. The functions of the key differentially-expressed genes highlight pathways potentially responsible for impaired ring stage development observed in mRBCs, and serves as a stepping stone to develop new drugs against P. falciparum.

Autoimmune demyelinating diseases (AIDD) of the nervous system, including multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD), are characterized by immune-mediated destruction of the myelin sheath, leading to impaired neural transmission and a range of neurological symptoms such as visual disturbances, motor dysfunction, and cognitive decline. AIDD is widely recognized to be driven by genetic susceptibility and environmental factors, particularly infectious agents. Notable pathogens include bacteria such as Mycoplasma pneumoniae, Chlamydia pneumoniae, and Staphylococcus aureus, as well as viruses including herpesviruses and human endogenous retroviruses. These infections may disrupt immune tolerance and induce long-term changes in immune surveillance, contributing to autoimmunity through mechanisms such as chronic immune activation and molecular mimicry. Immune dysregulation following infections may extend beyond lymphocyte responses to affect other immune compartments, including the less well-explored but important innate lymphoid cells (ILCs). Our study showed an increased percentage of ILC1 in active MS and ILC2 during remission using flow cytometric analysis, indicating dynamic, disease-phase-specific shifts in ILC subsets. Functional assays showed that ILCs from MS patients produced higher levels of IFNγ and IL-17A upon stimulation, suggesting functional reprogramming in the disease environment. These findings provide insights into the innate immune contributions to AIDD pathogenesis, potentially revealing shared and distinct immunological pathways linked to post-infection immune dysregulation.

Rare adverse effects have been associated with Covid-19 mRNA vaccination. Although there are some reports of post-vaccination death, the relationship between vaccination and fatality remains elusive. Here, we present pre- and post-mortem analyses of a deceased case following Covid-19 mRNA vaccination. In-depth pathological analyses revealed systemic microthrombosis and massive heart ischemia. An imbalance in the vWF-ADAMTS13 axis and a cytokine storm likely contributed to the dysregulation of coagulation system. A cytokine storm developed within 24 hours after the booster shot, evidenced by the remarkably high serum levels of IFN-g, MIG, IP-10, IL-1Ra, and IL-6. scRNA-seq of immune cells revealed that pericardial fluid obtained at autopsy contained a large fraction of macrophages. These macrophages expressed various pro-inflammatory cytokines, including IL1B, TNF, IL6, and CXCL8, and could produce these cytokines in vitro. Culture with both patient serum and recombinant S protein further activated the patient’s macrophages in an FcgR dependent manner, suggesting the involvement of the immune complex including the vaccine antigen in their activation. Collectively, our data suggest that mRNA booster vaccination triggered a macrophage-driven cytokine storm, which was followed by fatal acute heart failure due to microthrombosis in this current case.

Dengue virus (DENV) poses a significant disease burden globally with an estimated 390 million infections annually. The envelope (E) protein of DENV is glycosylated at two highly conserved asparagine (N) sites (N67 and N153). While the functions of these glycans have been extensively studied in vitro, their biological roles have largely been overlooked in vivo.

A DENV2 mutant lacking N153-linked glycans (N153Q mutant) was engineered and found to be mildly impaired in vitro but drastically attenuated in a symptomatic mouse model of severe dengue, as evidenced by accelerated viral clearance. B cell depletion and knockout (KO) in mouse model restored N153Q parental virulence, suggesting the involvement of B cells in N153Q mutant attenuation. Homologous passive transfer of purified IgM from infected B-cell proficient mice into B cell-KO mice cleared N153Q mutant from blood circulation. In vitro neutralization assay using mouse sera showed that WT and N153Q viruses were not significantly neutralized. These data suggest that N153Q mutant attenuation was due to non-neutralizing IgM-mediated viral clearance mechanism. Interestingly, heterologous passive transfer of purified IgM from WT-infected mice into N153Q-infected B cell-KO mice did not clear the mutant, suggesting that infection with WT DENV and N153Q mutant induced distinct antibody repertoires.

Furthermore, using plasma samples from convalescent dengue patients and monoclonal antibodies, in vitro neutralization assays showed that N153Q DENV1-4 and N154Q ZIKV mutants were more susceptible than WT to IgG-mediated neutralization. Glycoproteomics combined with molecular dynamics (MD) simulations revealed that the presence of the glycan and its composition on E protein heavily influenced IgG binding.
These findings represent a novel immune evasion strategy for orthoflaviviruses and have important implications for the development of antibodies, live attenuated vaccine and antiviral.

Dengue is an acute mosquito-borne viral disease that poses major global public health concerns, caused by four related but genetically distinct orthoflaviviruses – dengue virus-1 to 4 (DENV-1 to -4). Epidemiological observations have consistently shown that, unlike the first DENV infection that produces type-specific immunity, the second infection elicits broad protection against subsequent infections. However, the B cell receptor (BCR) development following the second compared to the first infection remains unclear. Herein, we leveraged two related live-attenuated orthoflaviviral vaccines, yellow fever (YF17D) and chimeric Japanese encephalitis-yellow fever (JE/YF17D), to simulate sequential orthoflaviviral infections in healthy human volunteers. Participants were first primed with JE/YF17D and then inoculated with YF17D 28 days later; neutralizing antibody titers to both JEV and YF17D were detected at 28 days after YF17D inoculation. FACS sorting, single-cell 3’ reverse transcription, BCR target enrichment, and full-length deep sequencing were carried out on participants’ PBMCs. Even though JEV and YFV share ~70% of the membrane and envelope protein sequence, we found de novo generation of cross-reactive BCR clones after YF17D inoculation. The CDR3 hypervariable region sequences were shared among dominating clones after each dose, but with distinct V(D)J recombination and/or light-heavy chain pairing events, suggesting the insignificance of affinity maturation in sequential vaccination. Skewed V gene usage, preferential V-J pairing, as well as convergent evolution across subjects were also revealed. Various paired, full-length BCR sequences were reconstructed to compare against published orthoflaviviral monoclonal antibody databases. Previously reported Zika virus (ZIKV)-neutralizing antibodies were identified despite clean serological baselines, suggesting strong interspecies protection potential from sequential vaccination. Together, through controlled human infection with live attenuated vaccines, we demonstrated the detailed composition of BCRs with prospective broad-spectrum activity against orthoflaviviruses, which may also serve as a prototype study for human-pathogen interaction research and novel vaccination regime development.

The non-structural protein 1 (NS1) of dengue virus (DENV) is a highly conserved 46-55 kDa protein which contains two N-glycosylation sites at positions 130 and 207 (N130 and N207). Secreted hexameric NS1 (sNS1) has been proposed to play an important role in dengue pathogenesis. However, the role of the two N-glycans in NS1’s biological functions has not been carefully examined. In this study, we generated stable DENV mutants that lack glycan structures at either N sites of NS1 and subsequently determined the in vitro and in vivo fitness in cell lines and symptomatic animal model, respectively. We show that the lack of glycan at either N sites of NS1 did not impair the structure, conformation, and lipid cargo of the hexameric sNS1 as well as the replication and growth of the virus in both mosquito and mammalian cell lines. However, while the N130 de-glycosylated mutant displayed a parental in vivo fitness in IFNAR-/- mice, the N207 de-glycosylated mutant was significantly attenuated as evidenced by higher survival rate and faster clearance of the virus in circulation, compared to the WT virus. Further experiment revealed that infection with N207 de-glycosylated mutant induces a lower initial inflammatory host response. Our results suggested that the glycans at position N207 but not N130 on NS1, play an important role in DENV in vivo fitness and virulence, by modulating the initial inflammatory response.

With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, dengue ranks among the top ten threats worldwide. It is caused by dengue virus, a flavivirus with four distinct serotypes. Dengue places a large economic burden on endemic countries and has the potential for severe disease manifestationin the form of Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). With no approved antivirals to treat infected patients, vaccines have been recognised to be the foundation to reduce DENV burden. However, dengue vaccine development has proven to be a challenge as an imbalanced immune response towards one serotype over the other could lead to antibody dependent enhancement (ADE) and eventual severe dengue. There are two live-attenuated dengue vaccines (Dengvaxia, Sanofi and QDENGA, Takeda Pharmaceuticals) that have been approved for human use, but only in specific age groups and have varied efficacies against the four DENV serotypes.

Our group utilises the Clec9A-targeting system to develop a subunit DENV vaccine, which involves a rat anti-mouse Clec9A antibody with a DENV2 envelope domain III (EDIII) genetically fused to the C-terminus of the heavy chain. Clec9A is a receptor that is specifically expressed in dendritic cells of the cDC1 subtype. cDC1s are highly efficient in processing antigens and cross-presentation on MHC I and MHC II, making Clec9A a promising DC surface receptor for antigen delivery. In addition, antibodies generated against EDIII are strongly neutralising and predominantly serotype-specific, minimising the risk of ADE. Optimisation of the immunisation regimen Balb/C mice immunised with a two-dose regimen of the Clec9A-EDIII construct (priming, followed by boosting one month after prime) have shown sustained anti-EDIII IgG titres and neutralising antibody titres up to 9 months post-boost. Clec9A-EDIII immunisation also generated EDIII-specific spleen Tfh cell response and poly-functional CD4+ T cells secreting IFN-γ, IL-2, and TNF-α at 1 week post boost. More work will be done to characterise the B cell responses following immunisation as well as exploring an alternative construct combining EDIII with a CD8+ DENV epitope to elicit both humoral and cellular protection.