Vaccines have been shown to cause differential expression of genes and increase antibody titers against antigens. Influenza vaccines may have an effect on unexplained disorders such as Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). Immunological changes have been identified following immunization with trivalent influenza vaccine (TIV). The objective of this pilot study was to examine the consequences of TIV on cytokine and cytotoxic genes in CFS/ME. Peripheral blood mononuclear cells were preferentially isolated from whole blood of 7 CFS/ME patients and 8 controls. Following total RNA extraction and synthesis of cDNA, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression levels of mRNAs for cytotoxic genes (perforin (PRF1), granzyme A (GZMA), granzyme B (GZMB) and cytokine genes. GZMB was significantly increased overall in the CFS/ME patients compared to the controls. GZMA was significantly increased 28 days after vaccination while PRF1 was reduced prevaccination but increased 14 days post-vaccination in the CFS/ME patients. There were no sig nificant changes in cytokine genes pre or post vaccination. Administration of TIV may increase the expression of lytic genes in CFS/ME and this may contribute to the increase in cytotoxic activity we observed in these patients post vaccination .
The effectiveness of seasonal vaccines such as the influenza vaccine is related to the ability to establish antigenicity involving the type of vaccine generated and the strain of influenza vaccine present in the periphery [
Administration of vaccines in patients with immunecompromised disorders is controversial, as there is a tendency for the vaccine to either exacerbate the symptoms or further suppress the already compromised immune system, thus causing detriment to health [
We have previously illustrated that administration of TIV may alter some immune indices in CFS/ME patients including cytotoxic activity, FOXP3 and the secretion of certain cytokines [
Ethical approval was obtained from the Bond University Human Research Ethics Committee (R0852A). The study included CFS/ME participants (n = 8; age = 48 years) and non-fatigued controls (n = 7; age = 38 years) [
Venous blood samples (40 mL) drawn from all participants were collected into EDTA tubes and analysed within three hours of collection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-hypaque (GE Healthcare, Uppsala, Sweden). Samples were collected prior to vaccination and 14 and 28 days post vaccination.
Total RNA was extracted from PBMCs using the miRNeasy isolation kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Concentration and purity of RNA was determined using the NanoDrop 3300 (Thermo Scientific, Waltham, MA). Following RNA extraction and quantification, synthesis of RNA to cDNA was accomplished with the SuperScriptTM III First-Strand synthesis SuperMix for reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Synthesized cDNA were diluted 1:20 and stored at −20˚C prior to RT-qPCR.
RT-qPCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The final reaction volume (10 µL) included 1 × iQ SYBR-Green Supermix (Bio-Rad, Hercules, CA), 200 nM of each primer and 4 µL of diluted cDNA. The RT-qPCR conditions were 95˚C for 3 minutes, followed by 45 cycles of 95˚C for 10 s, 62˚C for 30 s and generation of melt curves at 65˚C to 95˚C for the detection of secondary amplicons [
Determination of the most stable mRNA gene for normalization purposes was performed on the bio-rad CFX manager analysis software which allowed the comparison of M-values for each of the housekeeping genes. GAPDH, PGK1, 18S and ACTIN-β were the housekeeping genes tested for stability. M-values greater than 1 were rejected while an M-value less than 1 for a particular gene was considered the most stable.
The data generated from the PCR were evaluated using the CFX Manager v1.6 (Bio-Rad, Hercules, CA). Interassay variations were normalized using a calibrator sample. Normalized expression (ΔΔCq) for the genes of interest was normalized to a calibrator control and reference gene GAPDH. All values for expression were log2 transformed. The results were analysed using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Results were statistically significant where P ≤ 0.05. Non-parametric Mann-Whitney test was used in analysing all data.
As the stability of a reference gene is paramount for accurate outcomes in gene expression studies, a number of reference genes were inspected to establish the best possible gene for this study. GAPDH was found to be the most stable of the other housekeeping genes PGK1, 18S and ACTIN-β. GAPDH was determined to be the most stable reference gene as its average expression was less than one (
The expression levels of 6 genes were examined in the CFS/ME patients and non-fatigued controls. These included mRNA levels for lytic genes, PRF1, GZMA and GZMB, and cytokine genes IFN-G, TGF-B and TNF-A. In comparison to the non-fatigued controls, the CFS/ME patients, lytic proteins were significantly altered before and after vaccination. PRF1 was the only gene that was significantly decreased in the CFS/ME patients prior to vaccination (
days after vaccination in the CFS/ME group (Figures 2 and 3). An overall increase in GZMB was noticed in the pooled CFS/ME patients sample in comparison to the non-fatigued controls (
The results from this pilot investigation suggest that TIV may enhance the expression of lytic genes in patients with immune-compromised disorders such as CFS/ME. Prior to vaccination there was a trend towards reduced mean fold in PRF1 expression in the CFS/ME patients compared to the non-fatigued controls. Both granzyme genes were similar in expression in the two groups. However, PRF1 and GZMA were significantly increased 14 days and 28 days after vaccination, respectively. There were no observable changes in cytokine expression prior or post vaccination.
Previous reports have provided evidence in support of molecular changes following vaccination. In most of these studies, an upregulation in immune related genes has been observed in healthy individuals after vaccination. The pattern of response and the effects on molecular processes are time dependent and most often extreme expression changes have been observed in the first 24 hours following vaccination [
PRF1, GZMA and GZMB are translated in to perforin and granzyme proteins which are predominantly found
in cytotoxic lymphocytes. These proteins are used to induce cellular apoptosis of viral infected cells [14,15]. During cytotoxic activity against viral infection, perforin forms pores in the cell membrane via a calcium dependent pathway enabling the release and delivery of granzymes into the target cell where they induce apoptosis [16-18].
In the present study influenza vaccine enhanced the expression of PRF1 and this may be consistent with the finding that cytotoxic activity increased significantly 14 days after vaccination in the CFS/ME patients compared to the non-fatigued controls [
The exact cause for the increase in GZMA after vaccination is currently unknown however, GZMA is known to promote pro-inflammatory cytokine release, hence the increase in GZMA may have induced significant increases in pro-inflammatory cytokines including TNF-α [
At the molecular level vaccination against influenza may have a significant influence on the expression of immune related genes at different time points. These effects occur only in the short run and do not persist overtime. Therefore at the initial stages of vaccine introduction in CFS/ME, these effects may be beneficial as they may cause a significant improvement in mechanisms such as cytotoxic activity that are observed to be decreased in CFS/ME. Hence, vaccination may be protective in the short run causing an improvement in immune function. Thus, it can be postulated that the increase in cytotoxic activity in the CFS/ME patients occurred as a consequence of influenza vaccination, leading to an increase in PRF1 and a subsequent increase in cytotoxic activity.
The limitations with this study pertain to the sample size, as a larger sample size may substantiate the results. As this was a pilot study, further investigations are now required using a large sample size and a broad spectrum of genes. This study has demonstrated salient changes in cytotoxic-related genes that may correlate with the functional status of NK cells in CFS/ME patients following vaccination. Moreover, whether these effects are detrimental to CFS/ME immune health is yet to be proven as changes in lytic genes did not persist for the duration of the study.