Study design

This prospective cohort study comprised three study visits shown in Fig. 3. Each enrolled participant had up to 23 milliliters (ml) of venous blood collected at two time-points, baseline and at D28 (±3 days) following intramuscular influenza vaccination. During the first study visit, participants completed an interviewer-administered questionnaire by trained interviewers with data collected on demographic information, medical history, vaccination history, and health and physical activity habits (Rapid Assessment of Physical Activity—RAPA questionnaire)40.

Fig. 3: Outline of study visits, the DYNAMIC cohort study.

¶ anthropometric measurements comprised weight (kg), height (m), waist, and hip circumferences (cm). BMI (kg/m2), and waist: hip circumference ratios were calculated.

Ethical approval

The study was approved by the National Healthcare Group Institutional Review Board (DSRB 2016/00248). At the time of the study’s enrollment, all human subject studies with an intervention (such as the influenza vaccine for this study) were regulated by Singapore’s Health Sciences Authority (this study’s Clinical Trial Notification CTN1700053 and ClinicalTrials.gov NCT03399357).

Participants and recruitment

Adults aged 65 and older residing in the community in Singapore were prospectively enrolled after written informed consent at community sites and our institution’s research clinic from June to December 2017. In-person study visits concluded by February 2018. The community sites comprised a few sites that were part of the National Healthcare Group’s mid-year influenza vaccination program outreach for older adults, and other sites engaged in community initiatives for older adults (Supplementary Information).

Eligible older adults included those with well-controlled chronic conditions if they had not been hospitalized in the preceding 12 months. They should also have received their most recent annual seasonal influenza vaccine no more recently than 10 months prior. Immunocompromised adults were excluded. The detailed inclusion and exclusion criteria are shown in Box 1.

Box 1 Inclusion and exclusion Criteria for the DYNAMIC cohort study

Inclusion criteria

Age ≥65 years on the day of inclusion (not older than 100 years).

Able to give written informed consent.

Has NO immunosuppressive conditions and determined by medical evaluation, and clinical judgment to be generally healthy or have only stable medical conditions (no hospitalizations for worsening of disease in past 12 weeks prior to vaccination).

Note: Change in the dose of medication or therapy within the treatment category is allowed. Change to new therapy only allowed if not due to worsening disease.

Is eligible for seasonal influenza vaccine.

Able to attend and willing to comply with all scheduled visits and comply with all study procedures.

Exclusion criteria

A change to a new therapy category caused by worsening disease, or worsening chronic disease for any reason.

Symptoms suggestive of influenza, influenza-like illness, or respiratory illness.

Vaccination with any licensed or experimental influenza vaccine within the past 10 months.

Intent to receive any other investigational vaccine or agent during the course of the study.

Intent to receive other licensed vaccines during the course of the study (does not apply for a pandemic or post-exposure prophylaxis scenario).

Allergic to egg proteins (egg or egg products) and chicken proteins (serious allergy).

History of a life-threatening reaction to the vaccine used in the study, vaccine components (formaldehyde, cetylmethylammonium bromide (CTAB), polysorbate 80, gentamicin, or excipients such as potassium chloride) or to a vaccine containing any of the same substances.

Known or suspected immunodeficiency or receiving treatment with immunosuppressive therapy including cytotoxic agents (in past 6 months) e.g., transplant recipients on active immunosuppression, patients with cancer, HIV, or autoimmune disease.

Long-term systemic corticosteroid therapy (prednisolone ≥ 7.5 mg/day or equivalent for more than 2 consecutive weeks within the past 3 months).

Note: If systemic corticosteroids have been administered short term for the treatment of an acute illness, subjects will be excluded from the study until corticosteroid therapy has been discontinued for at least 30 days.

History of Guillain-Barré syndrome (GBS).

Serious chronic illness that, in the opinion of the investigator, is at a severity or stage which might interfere with study conduct or completion.a

Bariatric surgery, GI malabsorption disorders.

Recurrent Falls (≥2 falls in the past 12 months).

Osteoporosis with or without pathological fractures.

Current or recently completed high-dose vitamin D supplementation within the past 3 months (defined as daily cholecalciferol dose of 2000 IU or higher, weekly 50,000 IU or intramuscular calcitriol).

Receipt of any blood products, including immunoglobulin, within 6 months of study enrollment.

Donated blood within the last 58 days.

Current anticoagulant therapy or a history of bleeding diathesis (including thrombocytopenia with platelet count <50,000) that would contraindicate intramuscular (IM) injection (Note: antiplatelet drugs such as aspirin and clopidogrel are permitted).

Moderate or severe acute illness/infection (according to investigator judgment) on the day of vaccination, or febrile illness (temperature ≥ 37.5 °C). A prospective subject should not be included in the study until the condition has resolved or the febrile event has subsided.

Any medical condition or situation that would, in the opinion of the investigator, interfere with the evaluation of the study objectives.

aSerious chronic medical condition including: metastatic malignancy, a severe chronic obstructive pulmonary disease requiring supplemental oxygen, CKD stage 3 and above, end-stage renal disease with or without dialysis, clinically unstable cardiac disease, or any other disorder that, in the investigator’s opinion, should preclude the subject from participating in the study

Vaccine

All participants received the southern hemisphere 2017 IIV3 Influvac® containing an A/Hong Kong/4801/2014-H3N2-like virus, A/Michigan/45/2015-H1N1, and B/Brisbane/60/200841. The northern hemisphere IIV3 2017–18 composition was identical to the composition of the 2017 southern hemisphere vaccine42. Trivalent inactivated seasonal influenza vaccines were the standard clinically available vaccines in Singapore at the time of this study’s enrollment. The vaccine was maintained in a cold chain between +2 and +8 °C as recommended by the manufacturer before administration to participants via intramuscular injection in the deltoid muscle by a trained research nurse. Temperature monitoring was performed for vaccines administered at all study sites.

Laboratory methods

Blood samples were stored at +2 to +8 °C following collection and delivered to a central laboratory at Singapore Immunology Network (SIgN) for processing. Peripheral blood mononuclear cells (PBMCs) and serum were stored at −80 °C and only thawed at the time of assays. Assays on PBMCs assessing markers of cell-mediated immunity will be reported separately.

25-hydroxyvitamin D (25-(OH) D) assay

25-Hydroxyvitamin D2 and 25-hydroxyvitamin D3 were measured on baseline sera for participants who completed all study visits at the Mayo Clinic Immunochemical Core Laboratory by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (ThermoFisher Scientific, Franklin, Massachusetts 02038 and Applied Biosystems-MDS Sciex, Foster City, CA 94404). Intra-assay C.V.’s for 25-(OH) D2 were 4.4%, 3.3%, and 4.2% at 14, 41, and 124 ng/mL, respectively. Interassay C.V.’s were 6.1%, 6.2%, and 4.7% at 15, 43, and 128 ng/mL, respectively. Intra-assay C.V.’s for 25-(OH) D3 were 3.8%, 2.4%, and 4.7% at 25, 54, and 140 ng/mL, respectively. Interassay C.V.’s were 6.4%, 6.8%, and 5.0% at 24, 52, and 140 ng/mL respectively. As 25-(OH) D2 were universally low, the total 25-(OH) D was used for analyses.

Haemagglutination-inhibition (HAI) assays

HAI assays were performed on the baseline and D28 sera at the WHO Influenza Collaborating Centre for Reference and Research on Influenza in Australia, using standard protocols with egg-grown influenza viruses contained in trivalent influenza inactivated vaccine the subjects had received, namely A/Hongkong/4801/2014-H3N2 like virus, A/Singapore/GP1908/2015-H1N1 (antigenically identical to the A/Michigan/45/2015-H1N1 contained in the vaccine) and B/Brisbane/60/200843. The data used for the analyses for B/Brisbane/60/2008 were from assays performed with the ether-split virus. Turkey red blood cells were used for all assays. Additional dilutions were performed as needed for high HAI titers.

Statistical analysis

We estimated the sample size based on the study objective for 25-(OH) D status on HAI response. In our null hypothesis, the proportion of vitamin-D-deficient and non-deficient subjects with HAI seroconversion would be the same. We obtained a conservative estimate of 30% to be vitamin D deficient for our study population based on existing literature, including studies investigating Vitamin D status/deficiency in Singapore20,26,44,45,46, and assumed 20% of vitamin-D-deficient patients and 40% of non-deficient patients will seroconvert to any of the influenza vaccine strains. To detect this difference in proportion with a Pearson’s Chi-square test, we set the level of significance at 5% and a power of 0.8 to derive a total sample size of 237 required for the study (Supplementary Table 2).

The HAI titers appear in results as A/HK/H3N2 (abbreviated for A/Hong Kong/4801/2014-H3N2 like virus), A/MI/H1N1 (A/Michigan/45/2015-H1N1), and B-split (B/Brisbane/60/2008). We assessed HAI response based on (1) the proportion who seroconverted post-vaccination for each vaccine strain, as defined by at least a 4-fold rise in HAI titer at D28 compared to baseline with a titer of at least 1:40, and (2) D28/baseline log2 HAI fold rise. We explored the proportion of participants seroprotected (titer of ≥1:40) at D28 compared to baseline, but due to high baseline seroprotection rates for two of the three vaccine strains, the proportion seroprotected at D28 is a less meaningful outcome for our study objectives.

Clinically relevant variables were discussed among clinicians and based on a literature review before identifying possible factors of antibody fold rise. These included variables of interest in the aims of this study, such as metabolic co-morbidities (diabetes mellitus, hypertension, hyperlipidemia), chronic pulmonary disease, baseline 25-(OH) D, anthropometric measures of obesity (waist circumference, hip circumference, and body-mass index), demographic variables (age, gender, ethnicity), physical activity, baseline HAI titer, and prior influenza vaccine history.

Descriptive statistics were performed, and differences in patient characteristics between seroconversion status for each strain’s HAI responses were assessed based on Mann–Whitney U-test for continuous variables and Chi-square test (or Fisher’s exact test when appropriate) for categorical variables. Due to the nature of a two-fold serial dilution performed to measure antibody titer, the resulting antibody fold rise can be expressed in the form of an exponential expression. As such, the exponent in the expression for each HAI fold rise was used as outcome measurements by calculating log2 [HAI fold rise] to restrict the range and ensure the normality of data47. Multiple linear regression was then performed on each of these outcome measurements to adjust for potential confounding due to all other included variables. Similarly, the exponent in the exponential expression for each baseline HAI titer was also used as the independent variable. Test of the trend was conducted for the ordinal variables where appropriate, by treating it as a categorical variable represented as ordered integers in the regression model. Modifying effects of prior vaccination with different influenza vaccines on HAI responses were also investigated with regression models, which included interaction terms between the vaccine type and baseline HAI titer. All analyses were performed in R3.6.2, and statistical significance was deemed when a p < 0.05 (two-sided) was achieved48.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.