BNT162b2 mRNA Vaccine-Induced Immune Response in Oral Fluids and Serum.

OBJECTIVES: The COVID-19 vaccine is currently being administered worldwide to address the ongoing pandemic. Although these vaccines have proven effective in preventing severe disease, the level of immunity required to prevent respiratory mucosal infection remains less well understood. Therefore, it is desirable to develop a noninvasive screening strategy such as oral fluid to monitor secreted antibodies longitudinally as potential surrogates of mucosal immunity. METHODS: We evaluated the anti-spike protein antibodies in gingival crevicular fluid (GCF) and saliva and compared them to immune responses in the blood of 50 healthy health care workers following 2 doses of intramuscular Pfizer/BioNTech-BNT162b2 vaccine. RESULTS: The antibodies to SARS-CoV-2 spike and subdomain proteins (RBD, S1, S2, and NTD) were significantly higher in serum than oral fluids but showed a greater detection rate and higher median titres in GCF than saliva. For all tested SARS-CoV-2 antigens, IgG in GCF (as opposed to saliva) showed a more significant and stronger correlation with IgG in serum. Serum-neutralising antibodies (Nab) titres also displayed a significant and stronger correlation with anti-spike protein and their subdomains in GCF than saliva. Interestingly, the time post-second dose of vaccine and sex had a similar influence on IgG in serum and GCF. However, interferon (IFN)-γ-producing T-cell responses showed no association with SARS-Cov-2 IgG antibodies in serum, GCF, or saliva and neutralisation antibodies in serum. The correlation matrix of all measured parameters grouped serum and GCF IgG parameters separately from salivary IgG parameters indicating that GCF better represents the humoural response in serum than saliva. CONCLUSIONS: Within limitations, we propose that GCF could be a less invasive alternative to serum and more appropriate than saliva to detect antibody responses by current COVID-19 vaccines if the GCF collection procedure could be standardised. Further research is needed to investigate the suitability of GCF for community immune surveillance for vaccines.

RNase2 is a possible trigger of acute-on-chronic inflammation leading to mRNA vaccine-associated cardiac complication.

BACKGROUND: Post-mRNA vaccination-associated cardiac complication is a rare but life-threatening adverse event. Its risk has been well balanced by the benefit of vaccination-induced protection against severe COVID-19. As the rate of severe COVID-19 has consequently declined, future booster vaccination to sustain immunity, especially against infection with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, may encounter benefit-risk ratios that are less favorable than at the start of the COVID-19 vaccination campaign. Understanding the pathogenesis of rare but severe vaccine-associated adverse events to minimize its risk is thus urgent. METHODS: Here, we report a serendipitous finding of a case of cardiac complication following a third shot of COVID-19 mRNA vaccine. As this case was enrolled in a cohort study, pre-vaccination and pre-symptomatic blood samples were available for genomic and multiplex cytokine analyses. FINDINGS: These analyses revealed the presence of subclinical chronic inflammation, with an elevated expression of RNASE2 at pre-booster baseline as a possible trigger of an acute-on-chronic inflammation that resulted in the cardiac complication. RNASE2 encodes for the ribonuclease RNase2, which cleaves RNA at the 3' side of uridine, which may thus remove the only Toll-like receptor (TLR)-avoidance safety feature of current mRNA vaccines. CONCLUSIONS: These pre-booster and pre-symptomatic gene and cytokine expression data provide unique insights into the possible pathogenesis of vaccine-associated cardiac complication and suggest the incorporation of additional nucleoside modification for an added safety margin. FUNDING: This work was funded by the NMRC Open Fund-Large Collaborative Grant on Integrated Innovations on Infectious Diseases (OFLCG19May-0034).

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.

Durability of Vaccine-Induced and Natural Immunity Against COVID-19: A Narrative Review.

Vaccines developed against SARS-CoV-2 have proven to be highly effective in preventing symptomatic infection. Similarly, prior infection with SARS-CoV-2 has been shown to provide substantial protection against reinfection. However, it has become apparent that the protection provided to an individual after either vaccination or infection wanes over time. Waning protection is driven by both waning immunity over time since vaccination or initial infection, and the evolution of new variants of SARS-CoV-2. Both antibody and T/B-cells levels have been investigated as potential correlates of protection post-vaccination or post-infection. The activity of antibodies and T/B-cells provide some potential insight into the underlying causes of waning protection. This review seeks to summarise what is currently known about the waning of protection provided by both vaccination and/or prior infection, as well as the current information on the respective antibody and T/B-cell responses.

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.

Immune gene expression analysis indicates the potential of a self-amplifying Covid-19 mRNA vaccine.

Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. An alternative form of the mRNA vaccine is the self-amplifying mRNA (sa-mRNA) vaccine, which encodes an alphavirus replicase that self-amplifies the full-length mRNA and SARS-CoV-2 spike (S) transgene. However, early-phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, ARCT-021, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual S-specific T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2 1 day post-vaccination. Moreover, the ARCT-021 signature correlated with day 7 YF17D live-attenuated vaccine transcriptomic responses. Altogether, our findings show that sa-mRNA vaccination induces innate immune responses that are associated with the development of adaptive immunity from other forms of vaccines, supporting further development of this vaccine platform for clinical application.

RNA Viruses, Pandemics and Anticipatory Preparedness.

RNA viruses are likely to cause future pandemics and therefore we must create and organize a deep knowledge of these viruses to prevent and manage this risk. Assuming prevention will fail, at least once, we must be prepared to manage a future pandemic using all resources available. We emphasize the importance of having safe vaccine candidates and safe broad-spectrum antivirals ready for rapid clinical translation. Additionally, we must have similar tools to be ready for outbreaks of RNA viruses among animals and plants. Finally, similar coordination should be accomplished for other pathogens with pandemic potential.

Chronic sequelae complicate convalescence from both dengue and acute viral respiratory illness.

Long Covid has raised awareness of the potentially disabling chronic sequelae that afflicts patients after acute viral infection. Similar syndromes of post-infectious sequelae have also been observed after other viral infections such as dengue, but their true prevalence and functional impact remain poorly defined. We prospectively enrolled 209 patients with acute dengue (n = 48; one with severe dengue) and other acute viral respiratory infections (ARI) (n = 161), and followed them up for chronic sequelae up to one year post-enrolment, prior to the onset of the Covid-19 pandemic. Baseline demographics and co-morbidities were balanced between both groups except for gender, with more males in the dengue cohort (63% vs 29%, p<0.001). Except for the first visit, data on symptoms were collected remotely using a purpose-built mobile phone application. Mental health outcomes were evaluated using the validated SF-12v2 Health Survey. Almost all patients (95.8% of dengue and 94.4% of ARI patients) experienced at least one symptom of fatigue, somnolence, headache, concentration impairment or memory impairment within the first week of enrolment. Amongst patients with at least 3-months of follow-up, 18.0% in the dengue cohort and 14.6% in the ARI cohort experienced persistent symptoms. The median month-3 SF-12v2 Mental Component Summary Score was lower in patients who remained symptomatic at 3 months and beyond, compared to those whose symptoms fully resolved (47.7 vs. 56.0, p<0.001), indicating that patients who self-reported persistence of symptoms also experienced functionally worse mental health. No statistically significant difference in age, gender distribution or hospitalisation status was observed between those with and without chronic sequelae. Our findings reveal an under-appreciated burden of post-infection chronic sequelae in dengue and ARI patients. They call for studies to define the pathophysiology of this condition, and determine the efficacy of both vaccines as well as antiviral drugs in preventing such sequelae.

TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating dengue virus infection.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

Monitoring human arboviral diseases through wastewater surveillance: Challenges, progress and future opportunities.

Arboviral diseases are caused by a group of viruses spread by the bite of infected arthropods. Amongst these, dengue, Zika, west nile fever and yellow fever cause the greatest economic and social impact. Arboviral epidemics have increased in frequency, magnitude and geographical extent over the past decades and are expected to continue increasing with climate change and expanding urbanisation. Arboviral prevalence is largely underestimated, as most infections are asymptomatic, nevertheless existing surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. Wastewater-based surveillance (WBS), which has been demonstrated to be useful for monitoring diseases with significant asymptomatic populations including COVID19 and polio, could be a useful complement to arboviral surveillance. We review the current state of knowledge and identify key factors that affect the feasibility of monitoring arboviral diseases by WBS to include viral shedding loads by infected persons, the persistence of shed arboviruses and the efficiency of their recovery from sewage. We provide a simple model on the volume of wastewater that needs to be processed for detection of arboviruses, in face of lower arboviral shedding rates. In all, this review serves to reflect on the key challenges that need to be addressed and overcome for successful implementation of arboviral WBS.

Adverse effects following anti-COVID-19 vaccination with mRNA-based BNT162b2 are alleviated by altering the route of administration and correlate with baseline enrichment of T and NK cell genes.

Ensuring high vaccination and even booster vaccination coverage is critical in preventing severe Coronavirus Disease 2019 (COVID-19). Among the various COVID-19 vaccines currently in use, the mRNA vaccines have shown remarkable effectiveness. However, systemic adverse events (AEs), such as postvaccination fatigue, are prevalent following mRNA vaccination, and the underpinnings of which are not understood. Herein, we found that higher baseline expression of genes related to T and NK cell exhaustion and suppression were positively correlated with the development of moderately severe fatigue after Pfizer-BioNTech BNT162b2 vaccination; increased expression of genes associated with T and NK cell exhaustion and suppression reacted to vaccination were associated with greater levels of innate immune activation at 1 day postvaccination. We further found, in a mouse model, that altering the route of vaccination from intramuscular (i.m.) to subcutaneous (s.c.) could lessen the pro-inflammatory response and correspondingly the extent of systemic AEs; the humoral immune response to BNT162b2 vaccination was not compromised. Instead, it is possible that the s.c. route could improve cytotoxic CD8 T-cell responses to BNT162b2 vaccination. Our findings thus provide a glimpse of the molecular basis of postvaccination fatigue from mRNA vaccination and suggest a readily translatable solution to minimize systemic AEs.

As Omicron Takes Hold and Other New Variants Arise, COVID-19 Testing Remains the Universally Agreed Tool to Effect Transition From Pandemic to Endemic State.

The COVID-19 pandemic has caused more than 448 million cases and 6 million deaths worldwide to date. Omicron is now the dominant SARS-CoV-2 variant, making up more than 90% of cases in countries reporting sequencing data. As the pandemic continues into its third year, continued testing is a strategic and necessary tool for transitioning to an endemic state of COVID-19. Here, we address three critical topics pertaining to the transition from pandemic to endemic: defining the endemic state for COVID-19, highlighting the role of SARS-CoV-2 testing as endemicity is approached, and recommending parameters for SARS-CoV-2 testing once endemicity is reached. We argue for an approach that capitalizes on the current public health momentum to increase capacity for PCR-based testing and whole genome sequencing to monitor emerging infectious diseases. Strategic development and utilization of testing, including viral panels in addition to vaccination, can keep SARS-CoV-2 in a manageable endemic state and build a framework of preparedness for the next pandemic.

A Dynamic Immune Response Shapes COVID-19 Progression.

The inflammatory response to SARS-coronavirus-2 (SARS-CoV-2) infection is thought to underpin COVID-19 pathogenesis. We conducted daily transcriptomic profiling of three COVID-19 cases and found that the early immune response in COVID-19 patients is highly dynamic. Patient throat swabs were tested daily for SARS-CoV-2, with the virus persisting for 3 to 4 weeks in all three patients. Cytokine analyses of whole blood revealed increased cytokine expression in the single most severe case. However, most inflammatory gene expression peaked after respiratory function nadir, except expression in the IL1 pathway. Parallel analyses of CD4 and CD8 expression suggested that the pro-inflammatory response may be intertwined with T cell activation that could exacerbate disease or prolong the infection. Collectively, these findings hint at the possibility that IL1 and related pro-inflammatory pathways may be prognostic and serve as therapeutic targets for COVID-19. This work may also guide future studies to illuminate COVID-19 pathogenesis and develop host-directed therapies.

Use of analgesics/antipyretics in the management of symptoms associated with COVID-19 vaccination.

COVID-19 vaccines are effective and important to control the ongoing pandemic, but vaccine reactogenicity may contribute to poor uptake. Analgesics or antipyretic medications are often used to alleviate vaccine side effects, but their effect on immunogenicity remains uncertain. Few studies have assessed the effect of analgesics/antipyretics on vaccine immunogenicity and reactogenicity. Some studies revealed changes in certain immune response parameters post-vaccination when analgesics/antipyretics were used either prophylactically or therapeutically. Still, there is no evidence that these changes impact vaccine efficacy. Specific data on the impact of analgesic/antipyretic medications on immunogenicity of COVID-19 vaccines are limited. However, available data from clinical trials of licensed vaccines, along with recommendations from public health bodies around the world, should provide reassurance to both healthcare professionals and vaccine recipients that short-term use of analgesics/antipyretics at non-prescription doses is unlikely to affect vaccine-induced immunity.

Differential immunogenicity of homologous versus heterologous boost in Ad26.COV2.S vaccine recipients.

BACKGROUND: Protection offered by coronavirus disease 2019 (COVID-19) vaccines wanes over time, requiring an evaluation of different boosting strategies to revert such a trend and enhance the quantity and quality of Spike-specific humoral and cellular immune responses. These immunological parameters in homologous or heterologous vaccination boosts have thus far been studied for mRNA and ChAdOx1 nCoV-19 vaccines, but knowledge on individuals who received a single dose of Ad26.COV2.S is lacking. METHODS: We studied Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals (n = 55) who were either primed with Ad26.COV2.S only (n = 13) or were boosted with a homologous (Ad26.COV2.S, n = 28) or heterologous (BNT162b2, n = 14) second dose. We compared our findings with the results found in individuals vaccinated with a single (n = 16) or double (n = 44) dose of BNT162b2. FINDINGS: We observed that a strategy of heterologous vaccination enhanced the quantity and breadth of both Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals. In contrast, the impact of the homologous boost was quantitatively minimal in Ad26.COV2.S-vaccinated individuals, and Spike-specific antibodies and T cells were narrowly focused to the S1 region. CONCLUSIONS: Despite the small sample size of the study and the lack of well-defined correlates of protection against COVID-19, the immunological features detected support the utilization of a heterologous vaccine boost in individuals who received Ad26.COV2.S vaccination. FUNDING: This study is partially supported by the Singapore Ministry of Health's National Medical Research Council under its COVID-19 Research Fund (COVID19RF3-0060, COVID19RF-001, and COVID19RF-008), The Medical College St. Bartholomew's Hospital Trustees - Pump Priming Fund for SMD COVID-19 Research.

Immune Immunomodulation in Coronavirus Disease 2019 (COVID-19): Strategic Considerations for Personalized Therapeutic Intervention.

We are learning that the host response to severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) infection is complex and highly dynamic. Effective initial host defense in the lung is associated with mild symptoms and disease resolution. Viral evasion of the immune response can lead to refractory alveolar damage, ineffective lung repair mechanisms, and systemic inflammation with associated organ dysfunction. The immune response in these patients is highly variable and can include moderate to severe systemic inflammation and/or marked systemic immune suppression. There is unlikely to be a "one size fits all" approach to immunomodulation in patients with coronavirus disease 2019 (COVID-19). We believe that a personalized, immunophenotype-driven approach to immunomodulation that may include anticytokine therapy in carefully selected patients and immunostimulatory therapies in others is the shortest path to success in the study and treatment of patients with critical illness due to COVID-19.

Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

Hypoxia and viral infectious diseases.

Oxygen-sensing mechanisms allow cells to adapt and respond to changes in cellular oxygen tension, including hypoxic conditions. Hypoxia-inducible factor (HIF) is a central mediator in this fundamental adaptive response, and has critical functions in normal and disease physiology. Viruses have been shown to manipulate HIFs during their life cycle to facilitate replication and invasion. Conversely, HIFs are also implicated in the development of the host immune system and response to viral infections. Here, we highlight the recent revelations of host-pathogen interactions that involve the hypoxic response pathway and the role of HIF in emerging viral infectious diseases, as well as discussing potential antiviral therapeutic strategies targeting the HIF signaling axis.