Posted in Resource

Systems vaccinology publications compiled

mRNA (Messenger ribonucleic acid): Disrupting the field of vaccinology | GSK

This year, I was very interested in systems vaccinology, and have placed a lot of my efforts summarising various systems vaccinology papers which I found interesting. However, the current layout of my blog didn’t allow presentation of all these publications in a format that users can quickly access to them. I have thus compiled them in my medium publication account, and in a systematic fashion in my medium website. Feel free to visit these small compilations of my blog entries from the hyperlink. Cheers!

Posted in Resource, VSV vectors

Systems Vaccinology Identifies an Early Innate Immune Signature as a Correlate of Antibody Responses to the Ebola Vaccine rVSV-ZEBOV

Immunologic parameters that are correlated with antibody responses to rVSV-EBOV. Source from Rechtien et al., Cell Reports, 2017.

Predicting and achieving vaccine efficacy remains a major challenge. Here, Rechtien et al used a systems vaccinology approach to disentangle the early innate immune responses elicited by the Ebola vaccine rVSV-Zaire Ebola virus (ZEBOV) to identify innate immune responses correlating with Ebola virus (EBOV)-glycoprotein (GP)-specific antibody induction. Of note, this replication-competent recombinant vaccine candidate is based on the vesicular stomatitis virus (rVSV)-based vaccine vector, which has been shown safe and immunogenic in a number of phase I trials.

The vaccine rVSV-ZEBOV induced a rapid and robust increase in cytokine levels, with a maximum peak at day 1, especially for CXCL10, MCP-1 and MIP-1β. Assessment of PBMCs revealed significant induction of co-stimulatory molecules, monocyte/DC activation and NK cell activation at day 1 post-vaccination. The expression of these molecules begin to decline at day 3.

Interestingly, CXCL10 plasma levels and frequency of activated NK cells at day 3 were found to be positively correlated with antibody responses. CD86+ expression in monocytes and mDCs at day 3 are negatively correlated with antibody responses (See figure on top).

The most number of upregulated genes were detected at day 1 post-vaccination. Critically, the early gene signature linked to CXCL10 pathway, including TIFA (TRAF-interacting protein with forkhead-associated domain) on day 1, SLC6A9 (solute carrier family 6 member 9) on day 3, NFKB1 and NFKB2 were most predictive of antibody responses.

Data is stored under NCBI GEO: GSE97590.

Posted in Dengue, Resource

Immunotranscriptomic profiling the acute and clearance phases of a human challenge dengue virus serotype 2 infection model

Differentially expressed genes at day 8 and 28 after rDEN2Δ30 infection. Source from Hanley JP et al., Nature Communications, 2021.

rDEN2Δ30 is a recombinant serotype 2 virus based on the American genotype 1974 Tonga DENV2 virus, which has been partially attenuated by deletion of 30 nucleotides in the 3′ untranslated region of the RNA genome (Δ30). rDEN2Δ30 infection is known to induce modest viremia in all flavivirus-naive subjects and a mild, transient non-pruritic rash in 80% of recipients.

rDEN2Δ30 infection could hence be a suitable model to evaluate molecular signatures responsible for asymptomatic or mild DENV-2 infection.

In this study by Hanley JP et al., RNA-seq was performed on whole blood collected from rDEN2Δ30-infected subjects at 0, 8, and 28 days post infection. rDEN2Δ30-induced reproducible but modest viremia and a mild rash as the only clinically significant finding in DENV-naive subjects.

Principal component analysis reveal minimal overlap between baseline (day 0) and peak viremia (day 8). The day 28 data (post viremia) partially overlapped with the baseline (day 0) and acute (day 8) timepoints. Pathways enriched in the type I and type II interferon and antiviral responses were upregulated at day 8, whereas pathways controlling translational initiation were downregulated. NF-κB, IL-17 signaling pathways, apoptosis, toll-like receptor signaling, response to viruses, ribosomes, and defense responses were also differentially regulated at day 28.

Myeloid cells including monocytes and activated dendritic cells were significantly increased during acute infection and returned to baseline. In contrast, regulatory T cells (Tregs) were significantly decreased during acute stage.

Gene ontology pathway analysis revealed that the viremia-tracking set of genes was enriched for both response to and regulation of type I and II interferon pathways, including JAK/STAT signaling. Genes encoding for proteins that directly inhibit viral genome replication and involved in protein ubiquitination and catabolism, especially ISG15 pathway, tracked with viremia. Day 28 revealed more varied pathways, including protein ubiquitination, cell migration, cytoskeletal reorganization, and angiogenesis.

Baseline transcript signatures can potentially predict whether the subjects would develop rash after rDEN2Δ30 infection. Higher baseline expression of myeloid nuclear differentiation antigen (MNDA), and cell surface associated cellular processes such as tetraspanin CD37, integral membrane 2B (ITM2B), and genes involved in autophagy (VMP1) was associated with protection from rash. These genes are mostly related to myeloid responses, membrane regulation, autophagy, K63 ubiquitination, and cell morphogenesis.

Transcriptomic signatures modulated by rDEN2Δ30 infection and severe dengue are distinct. Only one gene family, the guanine binding protein (GBP1/2) genes was differentially regulated in both severe dengue and during mild rDEN2Δ30 infection.

Data deposited im Gene Expression Omnibus under accession number GSE152255

Posted in HIV, Resource

Molecular Signatures of a TLR4 Agonist-Adjuvanted HIV-1 Vaccine Candidate in Humans

Molecular signatures associated with early (A) and late (B) humoral responses. Source from Anderson et al., 2018, Frontiers in Immunology

Immunisation with the stable trimeric recombinant HIV-1 envelope glycoprotein, CN54gp140, has been shown to induce potent humoral immune responses, especially when adjuvanted with TLR4 agonist adjuvants, such as monophosphoryl lipid A or GLA-AF (glucopyranosol lipid adjuvant-aqueous formulation). These adjuvants exert their adjuvanticity, at least in part, by activating the myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor domain-containing adapter inducing interferon-β (TRIF) pathways. However, the clinical efficacy to the CN54gp140 adjuvanted with GLA-AF is variable between individuals. Anderson et al characterised the host responses after vaccinating subjects with CN54gp140 adjuvanted with GLA-AF, and examined the gene signatures linked to vaccine immunogenicity. 

Healthy male (n = 8) or female (n = 6) volunteers aged between 18 and 45 and with no history of HIV-1 and HIV-2 infection received the vaccine, and whole blood was collected from these subjects at 6 hours, 1, 3 and 7 days after vaccination. 

Majority of total DEGs were observed within 24 h post vaccination compared to later time points at 3 days and 7 days post-immunisation. 

The DEGs reveal an enrichment of BTMs related to cell cycle regulation and signaling as well as those related to innate and adaptive immune responses.

NK cell-related enriched BTMs (M7.2, M61.0, and S1) were significantly repressed in the gene expression profiles from individuals with either late high serum IgA or IgG4 responders (See Figure on top). 

In particular, we identified a repression in BTM modules related to NK cells, especially at 3 and 7 days post-vaccination, for high serum IgM, IgA, and IgG4 antibody responders.

Flow cytometry was performed to determine that the changes were due to NK cell numbers or expression levels of proteins upon vaccination.

In the limited number of analyzed samples, frequency of CD3–CD56dim NK cell population in the blood of high antibody responder subjects was increased on 14 days post vaccination compared to the 0 h baseline. While more studies need to be done, the authors speculate that the repression of BTMs related to NK cells observed in the first 7 days post-vaccination reflects NK cells leaving the circulation early in the response. Given that NK cells are short lived, the enhanced frequency of NK cells for 14  days post vaccination is presumably attributed to secondary induction of NK cell differentiation processes in response to vaccination.

Posted in About me

Animal Coronavirus Diseases: Parallels with COVID-19 in Humans

Figure showing the comparisons between animal and human coronaviruses that are discussed in our review paper

Happy to share that we have just published a review paper on “Animal Coronavirus Diseases: Parallels with COVID-19 in Humans.” This review is written with Chao Nan Lin, whom I have worked very closely with since I was a PhD student. It is hence a great privilege to be able to collaborate with him on his topic of expertise: Animal coronaviruses. This manuscript describes the similarities and differences between animal and human coronaviruses with regards to genome organization and recombination, immunopathogenesis, transmission, viral shedding, diagnosis, treatment, and prevention. We hope that the synthesised knowledge can help us better in managing coronavirus epidemics and design of interventions that can reduce the spread of these viruses.

Posted in influenza, Resource

Systems Analysis of Immunity to Influenza Vaccination across Multiple Years and in Diverse Populations Reveals Shared Molecular Signatures

Figure showing that the number of DEGs related to innate immune responses are more highly expressed in the young compared to the elderly subjects after receiving the Influenza TIV. Source from Nakaya et al., 2015, Immunity.

Although vaccination is considered the most effective method for preventing influenza, it shows limited efficacy in the elderly. Here, Nakaya et al used a systems vaccinology approach to understand the mechanisms behind poor vaccine efficacy in the inactivated influenza vaccine (TIV) in the elderly.

212 individuals from the current study and 218 individuals from a previously published study (Franco et al., 2013) were included in analyses. 54 of these were elderly (>65 years old). As expected, antibody responses to the Influenza vaccine (TIV) decrease with age.

Blood Transcriptomic Modules related to the induction of interferons and activation of dendritic cells were enriched on days 1 and 3 after TIV vaccination, whereas modules related to T cells at these time points were negatively associated with the antibody response. On day 7, there was a robust enrichment of antibody secreting cells (ASC) and cell cycle-related modules. The enriched modules in young and elderly were similar, particularly those related to the interferon response and activation of dendritic cells on day 1. However, the magnitude of expression of interferon-related genes was significantly higher in young individuals (See top figure).

Combining all datasets, several B-cell- and T-cell-related modules at pre-vaccination was positively correlated with an increased antibody response to vaccination. In contrast, modules related to monocytes were negatively correlated with antibody responses, supporting the concept that inflammatory responses at baseline might be detrimental to the induction of vaccine-induced antibody responses.

Consistent with the neutralizing antibody responses, B-cell and plasmablast modules (BTM S3) showed reduced expression in the elderly compared to the young on day 7. However, Natural killer (NK) cell and monocyte modules were enriched in the elderly at day 3 and day 7 after vaccination.

To examine if the transcriptional changes were due to changes in these specific cell types, flow cytometry was performed. Proportions of total NK cells in elderly subjects were higher than those of young subjects at baseline and all time points studied (days 0–14). The NK-cell activation markers were also more prominent in the elderly. Similarly, increased quantities of monocytes were seen in the elderly, with higher CCR5 expression at all time-points tested.

Differential expression of miRNA is also evident between the elderly and young, which suggests that miRNA could be important regulators of the immune response to influenza vaccination.

Data is deposited in GEO as GSE74817.

Posted in Resource, VSV vectors

Human Transcriptomic Response to the VSV-Vectored Ebola Vaccine

Principal Component Analysis plot showing the transcriptomic differences between days 0, 1, 2 and 3 after rVSV∆G-ZEBOV-GP vaccination. Source from F Santoro et al., 2021

rVSV∆G-ZEBOV-GP is a recombinant vaccine based on the Vesicular Stomatitis Virus (VSV), where the original VSV glycoprotein encoding gene was deleted and replaced with the surface glycoprotein (GP) encoding gene from the Ebolavirus Zaire strain (ZEBOV).

The vaccine was shown to be safe, although occasionally associated with transient reactogenicity. However, the host response to the vaccine has not been thoroughly investigated.

In this manuscript by F Santoro et al., 2021, the blood transcriptomic response to high dose vaccination (107 and 5 × 107 pfu) with rVSV∆G- ZEBOV-GP was analysed in 51 volunteers. Whole blood data was taken from day 0, 1, 2, 3, 7, 14, 21 and 28.

Vaccination resulted in greatest host transcriptomic changes at day 1, which lasts till day 3 (see top figure). Notably, the massive transcriptomic changes on days 1-2 corresponds to the timing of occurrence of mild to moderate reactogenicity events (chills, fever, headache, fatigue or myalgia) in 50 out of 51 vaccinees

Viral load differences did not affect host responses to vaccine, except for the MZB1 gene, coding for Marginal Zone B And B1 Cell Specific Protein.

Most blood transcriptomic module correlations with anti-ZEBOV GP IgGs were detected at day 14 post-vaccination. As expected, B cell activation and BCR signaling modules were observed to correlate with vaccine immunogenicity. Other modules that were significantly correlated at day 14 involve pathways such as calcium signalling, cell adhesion and activating transcription factor networks, which are possibly related to signal transduction.

Transcriptomic data are available in the Zenodo database.

Posted in BNT162b2 mRNA vaccine, Resource

Systems vaccinology of the BNT162b2 mRNA vaccine in humans

Interferon responses were most evident after 1 day post BNT162b2 vaccination, especially after the second vaccination. Source from Arunachalam et al., Nature, 2021

The Pfizer-BioNTech vaccine, BNT162b2 has ~95% efficacy, but little is known about the host immune responses involved. In this study by Arunachalam et al., the authors examined the host immune responses to this mRNA vaccine.

Immune responses were examined in 56 volunteers, each receiving two doses of the vaccine. Neutralizing antibodies were increased after primary vaccination, which boosted significantly after the second vaccination. Spike-specific CD4 and CD8 responses were more evident only after the second vaccination. No significant correlation were seen between T-cell, neutralizing antibodies and age were noted.

Phosphorylation of STAT1 and STAT3 in B-cells, T-cells, monocytes and pDCs were seen after 1 day post-vaccination, and especially after second vaccination. This observation was correlated with an induction of IFN-gamma and CXCL-10 protein expression at 1-2 days post-vaccination. Similarly, the induction of these cytokines was greater after second vaccination.

Consistent with the results from protein analysis, the transcriptional responses was greater after the second vaccination compared to the first vaccination (See top figure). Most of these responses were related to monocyte and inflammatory modules.

Single-cell transcriptomics reveal that the interferon signatures were broadly induced across cell types, much of which are driven by monocytes and dendritic cells. NK cell activation was only apparent at 1 day post second vaccination, which then disappears on the second day.

Comparing transcriptomics responses with other vaccines, the mRNA vaccine responses behave most similarly to the adjuvanted vaccines and live-viral vectors at day 1 post-vaccination. However, the authors did not detect any B-cell signatures in any of their time-points tested, despite seeing a significant increase in plasmablasts. Instead, the pathways related to interferon and inflammation were most correlated with neutralizing antibody and CD8 T-cell responses.

Posted in influenza, Resource

High-Resolution Temporal Response Patterns to Influenza Vaccine Reveal a Distinct Human Plasma Cell Gene Signature

Antibody titers, cell subset numbers, and gene transcript expression in different subjects across the different subjects. Source from Henn AD. et al., Scientific Reports, 2013

Influenza vaccines produce highly variable B cell responses among individuals, making it difficult to predict who will achieve protective antibody titers after vaccination.

In this paper by Henn AD et al., 2013, daily sampling of serum, peripheral blood mononuclear cells (PBMC), B cells and plasma cells from 14 human subjects was performed over 11 days post-influenza inactivated vaccine administration. Peripheral blood was drawn during the week prior to vaccination (pre-V), immediately before vaccination (day 0), daily for days 1–10 and on day 21 post-vaccination.

Most differentially expressed genes were detected at days 5-6 post vaccination, and this corresponded with the heightened IgM, IgG, plasmablast and activated ASC responses in most subjects. Many of these transcripts were validated to be B cell differentiation genes.

742 genes were differentially regulated temporally, and the majority of these genes were significantly correlated with CD27hiCD38hiCD138− plasmablasts. These genes are termed as the plasma cell gene signature (PCgs).

Ten of the top 30 categories of functionally related genes in the PCgs involved ER function and protein production. These findings are consistent with involvement of the PCgs in program-level upregulation of antibody production machinery and the unfolded protein response seen during plasma cell development.

Of interest, the other genes involved in the PCgs were expressed by the myeloid/DC lineages, many of which peaked at day 1. This is consistent with the notion that the magnitude of the innate immune response is also associated with antibody responses from influenza vaccination.

Posted in influenza, Resource

Systems biology of immunity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood

Figure showing that the differentially expressed genes (DEGs) were significantly higher in adults compared to children under 2 years of age. Overall, the adjuvanted inactivated influenza vaccine generated more DEGs than the unadjuvanted counterpart. Source from Nakaya et al., PNAS, 2016.

The trivalent inactivated influenza vaccine (TIV) is poorly immunogenic and has low effectiveness under 2 years of age. In this study by Nakaya et al., 2016 , the investigators studied the innate, adaptive and molecular responses to the seasonal TIV and MF59-adjuvanted TIV (ATIV) in 90 children from 14- to 24-months of age.

At day 28 post-boost, HAI geometric mean titers were higher in ATIV vaccinees compared with TIV. However, the magnitude of the plasmablast response was much lower in children than in adults. TIV and ATIV induced a similar magnitude of IgM- and IgG-secreting plasmablast cells in children.

MF59-adjuvanted TIV vaccine induced a higher expansion of multicytokine-producing vaccine-specific CD4+ T cells, mostly producing TNF-α and IL-2.

ATIV induced more alterations in gene expression at days 1, 3, and 7 post-boost compared with TIV. However, the numbers of DEGs were much smaller than in an adult cohort. There was high heterogeneity in the individual host responses, which could have accounted for the fewer DEGs detected.

Gene set enrichment analysis (GSEA) on individual subject’s responses revealed that at day 1 post vaccination, the positively enriched modules are M75 “antiviral interferon signature,” S5 “dendritic cell signature,” M16 “Toll-like receptor (TLR) and inflammatory signaling.” Among the negatively enriched modules, several modules related to T-cell function, NK cells, and cell cycle were found, including M7.1 “T cell activation,” M7.2 “enriched in NK cells,” and M4.0 “cell cycle and transcription”

GSEA was also applied to rank genes based on correlation with HAI titers. These modules include M75 “antiviral interferon signature,” M165 “enriched in activated dendritic cells,” and several others. These modules were positively correlated with HAI response at days 1, 7, and 28 following the booster shot.

The kinetics of enrichment of two blood transcriptomic modules associated with antibody-secreting cells (M156.1 and S3) show that enrichment is higher on days 7 and 28 for ATIV. The M156.1 module “plasma cell, immunoglobulins” was only significant at day 28 postboost, suggesting that unlike in adults, the expansion of antibody secreting cells may occur after day 7.

Overall, these findings highlight the differences in host immune responses between children and adults.