Electron transport chain capacity expands yellow fever vaccine immunogenicity.

Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity.

Early peripheral blood MCEMP1 and HLA-DRA expression predicts COVID-19 prognosis.

BACKGROUND: Mass vaccination has dramatically reduced the incidence of severe COVID-19, with most cases now presenting as self-limiting upper respiratory tract infections. However, those with co-morbidities, the elderly and immunocompromised, as well as the unvaccinated, remain disproportionately vulnerable to severe COVID-19 and its sequelae. Furthermore, as the effectiveness of vaccination wanes with time, immune escape SARS-CoV-2 variants could emerge to cause severe COVID-19. Reliable prognostic biomarkers for severe disease could be used as early indicator of re-emergence of severe COVID-19 as well as for triaging of patients for antiviral therapy. METHODS: We performed a systematic review and re-analysis of 7 publicly available datasets, analysing a total of 140 severe and 181 mild COVID-19 patients, to determine the most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients. In addition, we included an independent cohort where blood transcriptomics of COVID-19 patients were prospectively and longitudinally monitored previously, to track the time in which these gene expression changes occur before nadir of respiratory function. Single cell RNA-sequencing of peripheral blood mononuclear cells from publicly available datasets was then used to determine the immune cell subsets involved. FINDINGS: The most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients were MCEMP1, HLA-DRA and ETS1 across the 7 transcriptomics datasets. Moreover, we found significantly heightened MCEMP1 and reduced HLA-DRA expression as early as four days before the nadir of respiratory function, and the differential expression of MCEMP1 and HLA-DRA occurred predominantly in CD14+ cells. The online platform which we developed is publicly available at https://kuanrongchan-covid19-severity-app-t7l38g.streamlitapp.com/, for users to query gene expression differences between severe and mild COVID-19 patients in these datasets. INTERPRETATION: Elevated MCEMP1 and reduced HLA-DRA gene expression in CD14+ cells during the early phase of disease are prognostic of severe COVID-19. FUNDING: K.R.C is funded by the National Medical Research Council (NMRC) of Singapore under the Open Fund Individual Research Grant (MOH-000610). E.E.O. is funded by the NMRC Senior Clinician-Scientist Award (MOH-000135-00). J.G.H.L. is funded by the NMRC under the Clinician-Scientist Award (NMRC/CSAINV/013/2016-01). S.K. is funded by the NMRC under the Transition Award. This study was sponsored in part by a generous gift from The Hour Glass.

A phase I/II randomized, double-blinded, placebo-controlled trial of a self-amplifying Covid-19 mRNA vaccine.

Coronavirus disease-19 (Covid-19) pandemic have demonstrated the importantance of vaccines in disease prevention. Self-amplifying mRNA vaccines could be another option for disease prevention if demonstrated to be safe and immunogenic. Phase 1 of this randomized, double-blinded, placebo-controlled trial (N = 42) assessed the safety, tolerability, and immunogenicity in healthy young and older adults of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N = 64) tested two-doses of ARCT-021 given 28 days apart. During phase 1, ARCT-021 was well tolerated up to one 7.5 µg dose and two 5.0 µg doses. Local solicited AEs, namely injection-site pain and tenderness were more common in ARCT-021vaccinated, while systemic solicited AEs, mainly fatigue, headache and myalgia were reported in 62.8% and 46.4% of ARCT-021 and placebo recipients, respectively. Seroconversion rate for anti-S IgG was 100% in all cohorts, except for the 1 µg one-dose in younger adults and the 7.5 µg one-dose in older adults. Anti-S IgG and neutralizing antibody titers showed a general increase with increasing dose, and overlapped with titers in Covid-19 convalescent patients. T-cell responses were also observed in response to stimulation with S-protein peptides. Taken collectively, ARCT-021 is immunogenic and has favorable safety profile for further development.

A Zika virus-specific IgM elicited in pregnancy exhibits ultrapotent neutralization.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

A protocol to assess cellular bioenergetics in flavivirus-infected cells.

Aberrant cellular bioenergetics has detrimental consequences in host cells. For instance, pathogenic Zika virus strains can suppress mitochondria respiration and glycolytic functions, disrupting cellular bioenergetics that leads to apoptosis. Herein, we describe methods for flavivirus propagation, titering and infection, cell preparation, and procedures for mitochondrial and glycolytic stress tests. The protocol enables assessment of cellular respiration and glycolytic flux in flavivirus-infected cells. For complete details on the use and execution of this protocol, please refer to Yau et al. (2021).

A mouse model of lethal respiratory dysfunction for SARS-CoV-2 infection.

The global spread of SARS-CoV-2 has made millions ill with COVID-19 and even more from the economic fallout of this pandemic. Our quest to test new therapeutics and vaccines require small animal models that replicate disease phenotypes seen in COVID-19 cases. Rodent models of SARS-CoV-2 infection thus far have shown mild to moderate pulmonary disease; mortality, if any, has been associated with prominent signs of central nervous system (CNS) infection and dysfunction. Here we describe the isolation of SARS-CoV-2 variants with propensity for either pulmonary or CNS infection. Using a wild-type SARS-CoV-2 isolated from a COVID-19 patient, we first found that infection was lethal in transgenic mice expressing the human angiotensin I-converting enzyme 2 (hACE2). Fortuitously, full genome sequencing of SARS-CoV-2 from the brain and lung of these animals showed genetic differences. Likewise, SARS-CoV-2 isolates from brains and lungs of these also showed differences in plaque morphology. Inoculation of these brain and lung SARS-CoV-2 isolates into new batch of hACE2 mice intra-nasally resulted in lethal CNS and pulmonary infection, respectively. Collectively, our study suggests that genetic variants of SARS-CoV-2 could be used to replicate specific features of COVID-19 for the testing of potential vaccines or therapeutics.

Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset.

BACKGROUND: RNA vaccines against coronavirus disease 2019 (COVID-19) have demonstrated ∼95% efficacy in phase III clinical trials. Although complete vaccination consisted of 2 doses, the onset of protection for both licensed RNA vaccines was observed as early as 12 days after a single dose. The adaptive immune response that coincides with this onset of protection could represent the necessary elements of immunity against COVID-19. METHODS: Serological and T cell analysis was performed in a cohort of 20 healthcare workers after receiving the first dose of the Pfizer/BioNTech BNT162b2 vaccine. The primary endpoint was the adaptive immune responses detectable at days 7 and 10 after dosing. FINDINGS: Spike-specific T cells and binding antibodies were detectable 10 days after the first dose of the vaccine, in contrast to receptor-blocking and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) neutralizing antibodies, which were mostly undetectable at this early time point. CONCLUSIONS: Our findings suggest that early T cell and binding antibody responses, rather than either receptor-blocking or virus neutralizing activity, induced early protection against COVID-19. FUNDING: The study was funded by a generous donation from The Hour Glass to support COVID-19 research.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

Temporal dynamics of the host molecular responses underlying severe COVID-19 progression and disease resolution.

BACKGROUND: The coronavirus disease-19 (COVID-19) pandemic has cost lives and economic hardships globally. Various studies have found a number of different factors, such as hyperinflammation and exhausted/suppressed T cell responses to the etiological SARS coronavirus-2 (SARS-CoV-2), being associated with severe COVID-19. However, sieving the causative from associative factors of respiratory dysfunction has remained rudimentary. METHODS: We postulated that the host responses causative of respiratory dysfunction would track most closely with disease progression and resolution and thus be differentiated from other factors that are statistically associated with but not causative of severe COVID-19. To track the temporal dynamics of the host responses involved, we examined the changes in gene expression in whole blood of 6 severe and 4 non-severe COVID-19 patients across 15 different timepoints spanning the nadir of respiratory function. FINDINGS: We found that neutrophil activation but not type I interferon signaling transcripts tracked most closely with disease progression and resolution. Moreover, transcripts encoding for protein phosphorylation, particularly the serine-threonine kinases, many of which have known T cell proliferation and activation functions, were increased after and may thus contribute to the upswing of respiratory function. Notably, these associative genes were targeted by dexamethasone, but not methylprednisolone, which is consistent with efficacy outcomes in clinical trials. INTERPRETATION: Our findings suggest neutrophil activation as a critical factor of respiratory dysfunction in COVID-19. Drugs that target this pathway could be potentially repurposed for the treatment of severe COVID-19. FUNDING: This study was sponsored in part by a generous gift from The Hour Glass. EEO and JGL are funded by the National Medical Research Council of Singapore, through the Clinician Scientist Awards awarded by the National Research Foundation of Singapore.

A Non-structural 1 Protein G53D Substitution Attenuates a Clinically Tested Live Dengue Vaccine.

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain.

Persistent Dengue Infection in an Immunosuppressed Patient Reveals the Roles of Humoral and Cellular Immune Responses in Virus Clearance.

Detailed understanding of the roles of humoral and cellular immune responses in sterilizing dengue virus (DENV) infection in humans is required to inform effective vaccine development. We report an unusual case of persistent DENV infection in a lymphopenic renal transplant recipient who was therapeutically immunosuppressed to prevent organ rejection. Following resolution of symptomatic dengue, this patient remained positive for DENV3 RNA in the blood for 4 months and viruric up to 9 months post-infection despite demonstrable levels of serum neutralizing antibodies throughout this period. Full resolution of DENV infection instead coincided with recovery of CD8+ T cell counts during reversal from lymphopenia. Taken collectively, our observations suggest a role for cellular immunity in sterilizing DENV infection in humans. Any dengue vaccine should thus be able to induce both humoral and cellular immunity that respectively prevent symptomatic infection and enable effective viral clearance.

Antibody-Dependent Dengue Virus Entry Modulates Cell Intrinsic Responses for Enhanced Infection.

Dengue is caused by infection with any one of four dengue viruses (DENV); the risk of severe disease appears to be enhanced by the cross-reactive or subneutralizing levels of antibody from a prior DENV infection. These antibodies opsonize DENV entry through the activating Fc gamma receptors (FcγR), instead of infection through canonical receptor-mediated endocytosis, to result in higher levels of DENV replication. However, whether the enhanced replication is solely due to more efficient FcγR-mediated DENV entry or is also through FcγR-mediated alteration of the host transcriptome response to favor DENV infection remains unclear. Indeed, more efficient viral entry through activation of the FcγR can result in an increased viral antigenic load within target cells and confound direct comparisons of the host transcriptome response under antibody-dependent and antibody-independent conditions. Herein, we show that, despite controlling for the viral antigenic load in primary monocytes, the antibody-dependent and non-antibody-dependent routes of DENV entry induce transcriptome responses that are remarkably different. Notably, antibody-dependent DENV entry upregulated DENV host dependency factors associated with RNA splicing, mitochondrial respiratory chain complexes, and vesicle trafficking. Additionally, supporting findings from other studies, antibody-dependent DENV entry impeded the downregulation of ribosomal genes caused by canonical receptor-mediated endocytosis to increase viral translation. Collectively, our findings support the notion that antibody-dependent DENV entry alters host responses that support the viral life cycle and that host responses to DENV need to be defined in the context of its entry pathway.IMPORTANCE Dengue virus is the most prevalent mosquito-borne viral infection globally, resulting in variable manifestations ranging from asymptomatic viremia to life-threatening shock and multiorgan failure. Previous studies have indicated that the risk of severe dengue in humans can be increased by a specific range of preexisting anti-dengue virus antibody titers, a phenomenon termed antibody-dependent enhancement. There is hence a need to understand how antibodies augment dengue virus infection compared to the alternative canonical receptor-mediated viral entry route. Herein, we show that, besides facilitating viral uptake, antibody-mediated entry increases the expression of early host dependency factors to promote viral infection; these factors include RNA splicing, mitochondrial respiratory chain complexes, vesicle trafficking, and ribosomal genes. These findings will enhance our understanding of how differences in entry pathways can affect host responses and offer opportunities to design therapeutics that can specifically inhibit antibody-dependent enhancement of dengue virus infection.

Metabolic perturbations and cellular stress underpin susceptibility to symptomatic live-attenuated yellow fever infection.

Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored1-4, the pathophysiology that differentiates symptomatic from asymptomatic infection remains undefined. To understand the molecular underpinnings of symptomatic infection, the blood transcriptomic and metabolomic profiles of individuals were examined before and after inoculation with the live yellow fever viral vaccine (YF17D). It was found that individuals with adaptive endoplasmic reticulum (ER) stress and reduced tricarboxylic acid cycle activity at baseline showed increased susceptibility to symptomatic outcome. YF17D infection in these individuals induced maladaptive ER stress, triggering downstream proinflammatory responses that correlated with symptomatic outcome. The findings of the present study thus suggest that the ER stress response and immunometabolism underpin symptomatic yellow fever and possibly even other flaviviral infections. Modulating either ER stress or metabolism could be exploited for prophylaxis against symptomatic flaviviral infection outcome.

A systematic approach to the development of a safe live attenuated Zika vaccine.

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.

Dengue virus compartmentalization during antibody-enhanced infection.

Secondary infection with a heterologous dengue virus (DENV) serotype increases the risk of severe dengue, through a process termed antibody-dependent enhancement (ADE). During ADE, DENV is opsonized with non- or sub-neutralizing antibody levels that augment entry into monocytes and dendritic cells through Fc-gamma receptors (FcγRs). We previously reported that co-ligation of leukocyte immunoglobulin-like receptor-B1 (LILRB1) by antibody-opsonized DENV led to recruitment of SH2 domain-containing phosphatase-1 (SHP-1) to dephosphorylate spleen tyrosine kinase (Syk) and reduce interferon stimulated gene induction. Here, we show that LILRB1 also signals through SHP-1 to attenuate the otherwise rapid acidification for lysosomal enzyme activation following FcγR-mediated uptake of DENV. Reduced or slower trafficking of antibody-opsonized DENV to lytic phagolysosomal compartments, demonstrates how co-ligation of LILRB1 also permits DENV to overcome a cell-autonomous immune response, enhancing intracellular survival of DENV. Our findings provide insights on how antiviral drugs that modify phagosome acidification should be used for viruses such as DENV.

Cross-reactive antibodies enhance live attenuated virus infection for increased immunogenicity.

Vaccination has achieved remarkable successes in the control of childhood viral diseases. To control emerging infections, however, vaccines will need to be delivered to older individuals who, unlike infants, probably have had prior infection or vaccination with related viruses and thus have cross-reactive antibodies against the vaccines. Whether and how these cross-reactive antibodies impact live attenuated vaccination efficacy is unclear. Using an open-label randomized trial design, we show that subjects with a specific range of cross-reactive antibody titres from a prior inactivated Japanese encephalitis vaccination enhanced yellow fever (YF) immunogenicity upon YF vaccination. Enhancing titres of cross-reactive antibodies prolonged YF vaccine viraemia, provoked greater pro-inflammatory responses, and induced adhesion molecules intrinsic to the activating Fc-receptor signalling pathway, namely immune semaphorins, facilitating immune cell interactions and trafficking. Our findings clinically demonstrate antibody-enhanced infection and suggest that vaccine efficacy could be improved by exploiting cross-reactive antibodies.

Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in ß-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

First experimental in vivo model of enhanced dengue disease severity through maternally acquired heterotypic dengue antibodies.

Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations. Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.

Dengue virus growth, purification, and fluorescent labeling.

The early events of the dengue virus life cycle involve virus binding, internalization, trafficking, and fusion. Fluorescently labeled viruses can be used to visualize these early processes. As dengue virus has 180 identical copies of the envelope protein attached to the membrane surface and is surrounded by a lipid membrane, amine-reactive (Alexa Fluor) or lipophilic (DiD) dyes can be used for virus labeling. These dyes are highly photostable and are ideal for studies involving cellular uptake and endosomal transport. To improve virus labeling efficiency and minimize the nonspecific labeling of nonviral proteins, virus concentration and purification precede fluorescent labeling of dengue viruses. Besides using these viruses for single-particle tracking, DiD-labeled viruses can also be used to distinguish serotype-specific from cross-neutralizing antibodies. Here the details of virus concentration, purification, virus labeling, applications, and hints of troubleshooting are described.