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.

Posted in MMR, Resource

Transcriptomic profiling of different responder types in adults after a Priorix vaccination

Study design by Bartholomeus et al., Vaccine, 2020

Measles, mumps and rubella (MMR) used to be common childhood diseases until the advent of the MMR vaccine, which significantly reduced MMR cases worldwide. According to CDC, a single dose of the MMR vaccine is 93% effective against measles, 78% effective against mumps and 97% against rubella. However, in unprotected individuals, either due to vaccine non-responders or those with poor immunological memory over time, can still be at risk of MMR virus infection.

In this report, E Bartholomeus et al. attempts to study the transcriptomic signatures underlying differences in vaccine responses between individuals. 40 healthy individuals (between 20 and 30 years) whom have received at least one MMR vaccine dose earlier in life, were recruited. Whole blood was collected at days 0, 3 and 7 for whole blood transcriptomic analysis. Later time points of days 21, 150 and 365 was used to evaluate humoral responses to the vaccines (See schematic at top).

Subjects were categorised as: (a) High Ab: individuals with a relatively high Ab titer before vaccination that remained stable or further increased after vaccination (b) Low Ab: individuals with a relatively low Ab titer before vaccination that remained low after vaccination (c) Long response: individuals with a relatively low Ab titer before vaccination that increased after vaccination and stayed stable within the first year (d) Peak response: individuals with a relatively low Ab titer before vaccination that increased at day 21, and then decreased by day 150 and 365. Since 3 live-attenuated viruses are administered together, the analysis was stratified according to responses to each viruses as well.

Comparisons between categories yielded few differentially expressed genes (DEGs). Interestingly, the time comparisons per response group analysis showed that that individuals under the long response group had the most DEGs, as compared to the other categories, especially at day 7 post-vaccination. Pathway analyses reveal that most of these upregulated genes are related to the innate and humoral immune responses.

Posted in Malaria, Resource

Expression of genes associated with immunoproteasome processing of major histocompatibility complex peptides is indicative of protection with adjuvanted RTS,S malaria vaccine

Figure highlighting that subjects with increased expression of genes involved in formation of the immunoproteasome complex after RTS,S vaccination are associated with increased protection from malaria challenge. Specific genes involved highlighted in red. Source from MT Vahey, JID, 2010.

The RTS,S is a subunit recombinant vaccine expressed in yeast that represents the central repeat and C-terminal portion of the Plasmodium falciparum circumsporozoite protein (CSP) covalently linked with the S antigen of hepatitis B virus. The overall protective efficacy of RTS,S/ASO1B in malaria-naive adults is ∼50%.

The differences in the spectrum of the protective responses to adjuvanted RTS,S, and the ability to do an experimental challenge with the mosquito-borne falciparum malaria, offers an opportunity to decipher the host genomic mechanisms involved in vaccine efficacy against malaria.

In this study by MT Vahey et al., 2010, 39 vaccine recipients were assessed at study entry, on the day of the third vaccination, at 24 h, 72 h, and 2 weeks after vaccination, and on day 5 after challenge. Of 39 vaccine recipients, 13 were protected and 26 were not.

Most DEGs were detected at day 1 post vaccination, with the majority of these transcripts associated with proinflammatory responses. Most of these responses resolve at day 3 post-vaccination.

After day 5 of malaria challenge, prediction analysis of microarray (PAMR) identified 393 genes that are differentially expressed in subjects who are protected and unprotected. Most of these genes are related to apoptosis and cell cycle.

At 2 weeks after the third vaccination but before malaria challenge, 32 genes belonging to the proteasome degradation pathway separated protected vs unprotected subjects. Examples include PSME2 (a component of the 11S regulator), and PSMA4, PSMB6, and PSMB9 which forms the immunoproteasome involved in processesing of peptides for presentation to the MHC complex.

This study may highlight the critical role of T-cells as correlate of protection against malaria. Data deposited at Gene Expression Omnibus, with accession number GSE18323.

Posted in Adenovirus, Resource

Merck Ad5/HIV induces broad innate immune activation that predicts CD8+ T-cell responses but is attenuated by preexisting Ad5 immunity

Figure (top) showing the protein–protein interaction network that links between CD8+ T-cell response-associated genes and constituents of functional gene modules differentially regulated by MRKAd5/HIV. Study is done by Zak DE et al., PNAS, 2012.

The Step Study revealed that the MRKAd5/HIV had poor efficacy in protecting against HIV-1 infection, especially in subjects with pre-existing Ad5 immunity. However, no clear mechanisms have been identified to date why the seropositive subjects showed reduced efficacy. In this manuscript, Zak DE et al., describe the possible mechanisms of vaccine failure using a systems biology approach

35 healthy HIV-1-uninfected adults, 20–50 years old were recruited for the study. 10 subjects (3 seropositive and 7 seronegative subjects) PBMCs were isolated and transcript levels were analysed by microarray at 4–6, 24, 72, and 168 h post-vaccination.

Strong induction of innate immune responses were detected at the gene and protein level, peaking at 24 hours post-vaccination and resolving at 72 hours post-vaccination. In addition, protein levels of CXCL-10, ITAC, monocyte chemoattractant protein-1 (MCP-1), and MCP-2, as well as immunoregulatory IL-10 and IL-1Ra, were upregulated.

92% concordance between the in vivo and in vitro induction of IFN response genes was observed, indicating that these immune responses observed were modulated by viral infection. Genes not captured in vitro are related to cell lineages, possibly due to lack of cell trafficking in vitro.

When comparing subjects with Ad5 nAb titers ≤ 200 and >200, significant attenuation of the innate immune responses were seen in seropositive subjects compared to the seronegative subjects.

Two chemokines, MCP-1 and MCP-2, discriminated between the strong and moderate CD8-responders as compared to those subjects with poor or no CD8 responses.

Interestingly, the gene signatures that correlated with CD8 T-cell responses were seen at day 3, where members of the “Cytotoxicity,” “T cells,” and “Lymphoid lineage” modules were positively correlated (see above figure). This finding may support the notion that prolonged upregulation of the innate immune gene transcripts could influence CD8 responses.

Data is deposited in the Gene Expression Omnibus (GEO) database, GSE22822.

Posted in influenza, Resource

Innate gene signature distinguishes humoral versus cytotoxic responses to influenza vaccination

Hierarchical clustering showing how the changes in transcript levels at day 1 differ between patients. Two distinct clusters, C1 and C2 are seen. Source from Gonçalves et al., JCI, 2020.

Does the vaccine administration route affect host responses to vaccines? In this paper, Gonçalves et al investigates how vaccine administration via the intramuscular (i.m.), intradermal (i.d.) and transcutaneous (t.c.) routes affects innate and adaptive responses to the inactivated influenza vaccine.

60 healthy subjects, 18–45 years old recruited to compare the immunogenicity of the 2012–2013 seasonal trivalent inactivated influenza vaccine (TIV) administered by the t.c. (20 subjects), i.d. (20 subjects) and i.m. (20 subjects) routes.

The i.m. and i.d. route generated higher neutralising antibodies compared to the t.c. route. Higher vaccine-specific CD8+GRZ+ T cells was seen after t.c. and i.d. vaccination compared to i.m. However, vaccine-specific CD8+ T-cell responses were not significantly different between the conditions.

More differentially expressed genes were detected in i.m. and i.d., as compared to t.c.. Two distinct gene signature clusters (C1 and C2; see above figure) were observed, but the clusters did not segregate by vaccine administration route. The C1-C2 dichotomy is attributed to genes involved in multiple pathways, such as those for antigen-presentation, DC maturation, and IFN signaling, where subjects in the C1 cluster expressed higher levels of these transcripts (see above figure).

Instead, C1 and C2 clusters had significant differences in humoral and CD8+GRZ+ T cell responses. Of note, C1 individuals had significantly higher influenza-specific MN antibody titers, but lower frequency of TIV-specific CD8+GRZ+ T cell responders.

80 transcripts related to interferon signaling and antigen presentation pathways are correlated with neutralising antibody responses. On the other hand, 31 transcripts related to metabolic pathways were correlated with TIV-specific CD8+GRZ T cell responses.

The top positively correlated genes with antibody responses are CXCR2P1, C2, and CKS1B and the top negatively correlated genes are PRKAA1 and TMEM8B. The top genes involved in correlation with CD8+GRZ+ T cell response were MAP2K5, PVRL1, SARM1, and CXCR4.

Data deposited in ArrayExpress with the accession code E-MTAB-7741.

Posted in Meningococcal vaccines, Resource

Molecular signatures of antibody responses derived from a systems biological study of 5 human vaccines

Volcano plots comparing the differentially expressed genes after 3 days and 7 days post-vaccination with MCV4, MPSV4, YF-17D, TIV or LAIV vaccines. Source taken from Li et al., Nature Immunology, 2014.

Neisseria meningitidis is a leading cause of meningitis and septicemia with 1.2 million cases per year worldwide. Of note, bacterial polysaccharides do not trigger the same receptors that are involved in viral sensing. This brings an interesting question: Do carbohydrate vaccines induce the same or distinct molecular signatures as viral vaccines? To address this question, Shuzhao Li et al., 2014, compared the molecular signatures induced by meningococcal vaccines with yellow fever (YF-17D) and influenza vaccines (TIV and LAIV).

The two major classes of meningococcal vaccines tested are:

  1. The quadrivalent polysaccharide vaccine (MPSV4) containing polysaccharides from serogroups A, C, Y and W-135. Vaccine is given intramuscularly.
  2. The polysaccharide-protein conjugate vaccines, such as the quadrivalent conjugate vaccine (MCV4) that contains the same four polysaccharides conjugated to diphtheria toxoid. MCV4 given subcutaneously.

13 healthy subjects (age 18-45) received MPSV4 and 17 subjects received MCV4. Direct transcriptomic analysis on PBMCs was performed on days 0, 3 and 7 after vaccination.

The magnitude and duration of the polysaccharide-specific IgG response was greater with MPSV4 compared to a single dose of MCV4.

Peak levels of peripheral blood antibody secreting cells (ASCs) detected at day 7 post-vaccination.

Most DEGs for both vaccines were detected at day 7, with more DEGs in MCV4 vaccinees compared to MPSV4 vaccinees. Many of these DEGs were transcripts related to ASCs, which coincided with the increase in ASCs at day 7. XBP1 gene network was also enriched in MCV4 at day 7 post vaccination, similar to antibody signatures seen in subjects given the influenza inactivated vaccine. The signatures produced by MCV4 and MPSV4 were clearly distinct from the live-attenuated yellow fever and influenza vaccines.

Blood transcriptomic modules was used to decrypt immune responses produced by MCV and MPSV. Dendritic cell surface signature module, together with activation, complement and pro-inflammatory cytokines was correlated with anti-polysaccharide IgG in both MCV4 and MPSV4 vaccinees, suggesting the role of myeloid dendritic cells in antibody production. However, how these vaccines lead to myeloid dendritic cell activation still remains to be elucidated.

Data is deposited at Gene Expression Omnibus: GSE52245

Posted in Hepatitis B, Resource

Transcriptional profiles of adjuvanted hepatitis B vaccines display variable interindividual homogenity but a shared core signature

Clustergram showing similarities and differences in gene expression for different adjuvants. The day 1 and day 31 (day 1 after 2nd dose) for AS01 and AS03 adjuvants cluster together, whereas AS04 and Alum cluster together with the late responses (days 3-7). AS01 and AS03 induces greater number of DEGs compared to AS04 and Alum. Source from: Laurane et al., Science Translational Medicine, 2020

Adjuvants are an important component in inactivated vaccines, as they are known to trigger activation of innate immune responses that is essential for eliciting adaptive immune responses. In this study, Laurane et al., Science Translational Medicine, 2020 investigated how the transcriptional profile in humans is altered by 5 different types of adjuvants, namely:

  1. AS01B (MPL + QS-21)
  2. AS01E (half dose of MPL + QS-21)
  3. AS03 (a-tocopherol + squalene)
  4. AS04 (MPL+Alum)
  5. Alum

They also examined the inter-individual variations in vaccine responses to the different adjuvants. The antigen used is the Hepatitis B virus (HBV) surface antigen (HBs).

112 healthy subjects were given two intramuscular injections of HBs antigen (18 for AS01B, 23 for AS01E, 28 for AS03, 22 for AS04, 21 for Alum). Whole blood transcriptome profiled at 0, 3-6hrs, 1, 14 days after first dose and 0, 3-6hrs, 1, 3, 7 days after second dose. The second dose was given 30 days after first dose.

Most DEGs are detected at day 1 and day 31 post vaccination. Highest number of DEGs were seen in AS01B and AS01E , followed by AS03. Much lesser DEGs were seen with AS04 and Alum. The upregulated DEGs for AS01B, AS01E, and AS03 comprised of genes mostly related to innate immune genes and interferon responses. This is interesting because AS01 and AS03 components are different but yet, they induce transcriptional responses that are converging.

Interestingly, heterogeneity among individual responses for the AS03 recipients was higher compared to AS01 recipients.

Some of the day 1 genes related to innate response, interferon pathway and NK cells are correlated with humoral and cellular responses at day 44. However, this only accounted for <40% of the correlated genes. Genes from other pathways such as cell cycle and metabolism were also associated with the humoral and cellular responses at day 44.

Posted in influenza, Resource

Temporal Dynamics of Host Molecular Responses Differentiate Symptomatic and Asymptomatic Influenza A Infection

Figure describing the 8 distinct clusters of genes that are temporally modulated (0-108hrs) in the symptomatic (Sx) and the asymptomatic (Asx) volunteers after challenge with influenza virus. Source: Yongsheng H et al., PLOS Genetics, 2011.

A deep understanding of the molecular underpinnings underlying severe viral disease outcome in humans is critical for the development of drugs and therapeutics. Controlled human infection studies, in which volunteers are intentionally infected with a pathogen, can advance our understanding of disease pathogenesis as the incubation time, time-course of disease progression, symptomatic rates and immune responses can be closely monitored. In this manuscript published by Yongsheng H et al., PLOS Genetics, 2011, the authors investigated how the transcripts are differentially modulated in the symptomatic and asymptomatic subjects after challenge with the live influenza (H3N2/Wisconsin) strain

17 healthy adults were inoculated with live influenza (H3N2/Wisconsin) strain at 3 different doses (1∶10, 1∶100, 1∶1000, 1∶10000). 9 subjects were symptomatic whereas 8 were asymptomatic. Changes in host peripheral blood gene expression measured at -12, 0, 12, 21, 29, 36, 45, 53, 60, 69, 77, 84, 93, 101 and 108 hrs. 

Increasing doses of virus does not correlate with increased symptomatic outcome. This finding is congruent with our previous findings showing vaccine viremia does not influence symptomatic rates (Chan et al., JCI insight, 2017). In contrast, gene signature patterns were strongly associated with disease severity.

Using EDGE with false discovery rate (FDR) significance level (q-value)<0.01, 5,076 genes were temporally changed, comparing between symptomatic and asymptomatic phenotypes. Self-organizing maps (SOM) identified eight distinct classes with differential expression dynamics (See figure above).

Cluster 3 reveal genes that are uniquely increased in symptomatic subjects. These include PRR genes such as Toll-like receptor 7 (TLR7), the RNA helicases (RIG-I), and interferon induced with helicase C domain 1 (IFIH1). In addition, 11 genes from the TLR signaling pathway, including MyD88, TRAF6, and STAT1. NOD1, RIPK2, NOD2, NLPR3, and CASP5 and CASP1 and IL1b were increased in symptomatic subjects but not in the asymptomatic subjects.

Cluster 6 genes identified genes that are uniquely increased in the asymptomatic subjects. Enriched pathways were enriched are related to cellular response to oxidative stress. These include superoxide dismutase (SOD1) and serine/threonine kinase 25 (STK25 or SOK1), which have been linked to anti-oxidant/stress response and reduced concentration of ROS. This cluster also contain genes related to ribosomal synthesis, suggesting that protein biosynthesis could be protective against severe disease.

Both raw and normalized gene expression data are available at GEO (GSE30550).

Posted in Resource, shingles vaccine

Metabolic Phenotypes Of Response to Vaccination in Humans

Metabolic responses to Zostavax vaccines over time (days 1, 3, 7). Source: Li et al., Cell, 2018

Zostavax is a licensed live-attenuated vaccine for prevention of HZ (shingles) in individuals aged 50 and above. HZ is caused by varicella zoster virus (VZV) reactivation, and Zostavax has been previously shown to confer ~50% reduction in HZ. Interestingly, the efficacy against HZ was 63.9% in subjects who were 60–69 years old but only 37.6% in subjects older than 70 years. In this article published in Cell, Li et al investigates why the efficacy of Zostavax could be different in the young and old subjects, using a multi-omics approach. Main findings are summarised below:

77 participants enrolled. 33 were young adults between the ages of 25 and 40 years and 44 were older subjects between the ages of 60 and 79 years.

PBMCs collected at baseline prior to vaccination and at days 1, 3, 7, 14, 28, 90, and 180 post-vaccination

Weak CD8+ T-cell responses seen in all subjects, but CD4 T-cell responses detected in the majority of subjects. Blood Tfh-like cells (CD4+CXCR5+CXCR3+ICOS+ T cells), known to be important in providing B-cell help, was also increased in both the young and elderly adults after vaccination.

The younger subjects had a greater increase of VZV-specific IgG antibody after vaccination compared to the elderly subjects.

Transcriptomics show increased interferon-stimulated genes at days 1 and 3, but the induction of these innate immune genes were of a smaller magnitude than the Yellow Fever live-attenuated vaccine. This is then followed by increased immunoglobulin transcripts at day 7. Of note, the increase in immunoglobulin genes coincided with the increase in antigen-specific plasmablast cells.

Comparing between young and elderly subjects, most differences were seen at baseline, including increased expression of genes in gene modules related to inflammation, cytosolic DNA sensing, and NK cells.

Among the different cell population phenotypes measured, day 7 IFN-γ+ T cells is the most significant predictor of IgG response. High activity in inositol phosphate metabolism is also associated with reduced T cell and B cell responses.

Sterol regulatory binding protein 1 and its targets is most predictive of Tfh response, IgG response, and the associations between genes and metabolite networks, including chemokine signaling (CCL23, CCL19, and CCR3), TNF/MAPK signaling (TNFSF11, VACM1, and DUSP6), complement genes (C1QA and C1QB), killer cell immunoglobulin-like receptors (KIR3DL3, KIR2DL3, and KIR2DS5), and lipid metabolic enzymes (ACSM2A, ACSM2B, and DGAT2).

Data is available at GEO: GSE79396