SLE patients have reduced humoral immune responses following influenza vaccination
African American patients comprised 44% of this cohort and over 90% of participants were female (). As shown in , we looked at log10 transformed ratios of post-vaccination to pre-vaccination measurements. Using the INA approach we classified patients as either high or low responders based upon their overall anti-influenza response. Although controls showed a greater increase in the total amount of native antibody after vaccination compared to all SLE patients (p = 0.035), neither the high responding patients (Bmax, , p = 0.254) nor the low responding patients (Bmax, , p = 0.081) alone showed a significantly smaller increase in total native antibody after vaccination compared to controls. Both the high (p = 0.008) and low (p < 0.001) responding patients had a significantly smaller increase in apparent affinity after vaccination compared to controls (Ka, ). While a non-significant difference in hemagglutination inhibition was noted between SLE patients and controls (p=0.17), it should be noted that few of these individuals (patients or controls) had substantial increases in HAI titers after vaccination. Furthermore, low responding patients showed a significantly smaller increase in HAI after vaccination than either high responding patients (HAI, , p < 0.001) or healthy controls (HAI, , p < 0.001). Using the INA approach to look at differences of pre- and post-vaccination, controls showed significantly improved humoral influenza responses after vaccination than patients (p < 0.001), and this effect is driven by the poor overall antibody responses of low responding patients ().
Demographics of this influenza vaccination SLE patient cohort.
SLE patients with poor antibody responses have reduced humoral immunity compared to healthy controls following influenza vaccination
Based on the INA approach used to classify patients as either high or low responders, we can see that the high responder group of patients generated an anti-influenza response which was much closer to the behavior of the controls than that of the low responding patients. For each measure of responsiveness (Bmax, Ka, HAI and overall response), the low responder group had impaired responses compared to both the high responder and control groups, particularly when evaluating HAI differences and overall differences. In the following experiments we use this classification of high and low responders to further examine correlates of poor SLE vaccine response.
African-American SLE patients are more likely to have strong influenza vaccination responses
African-American patients were more likely to have strong influenza vaccination responses than European American patients (p=0.03, ). Specifically, African-American patients were 3 times more likely to be high responders than European Americans (95% CI: 1.07, 9.94). This racial association with vaccination response was not due to age since the ages between the two groups did not significantly differ (42±12.4 vs. 46±15.8, respectively). This discrepancy may be due to the impact of HLA haplotypes on vaccine responsiveness, although this requires further investigation.
Patients ranged in age from 20 to 89 years, with 42% between 29 and 45. The relationship between age and the response to vaccination was examined since older individuals may demonstrate decreased influenza antibody levels (38
). Although we had only a small number of elderly individuals, we found no relationship between age and the vaccine response. Due to the predilection of SLE for females, the small number of male patients did not allow evaluation of gender differences.
Poor influenza vaccination responses are associated with hematologic manifestations and more SLE ACR classification criteria
We next analyzed age at diagnosis, number of cumulative ACR criteria, and select ACR classification criteria to determine whether these factors correlated with the subsequent response (). Low responders had more ACR criteria (median=6) than high responders (median=5, p=0.05). Indeed, 23 of the 36 (64%) low responders met six or more of the ACR SLE criteria compared to 15 of 36 (42%) high responders.
We then analyzed the types of ACR criteria found in both groups. Twenty-five out of 36 low responders (69.4%, ) exhibited at least one hematological criterion, compared to 12 out of 36 high responders (33.3%, p=0.009). All of the individuals with hemolytic anemia were low responders (, p=0.01). The low responders also had a higher prevalence of thrombocytopenia (22.2% vs. 13.9%), lymphopenia (38.9% vs. 27.8%), and leucopenia (36.1% vs. 22.2%), although these were not statistically significant (). While not statistically significant, nine of the thirty-six (25%) high responders exhibited discoid rash compared to three of thirty-six (12.5%) low responders (p=0.1). No differences were seen in the prevalence of renal criteria or arthritis.
Patients with low influenza vaccination responses have more hematologic SLE classification criteria and more steroid use
Poor influenza humoral immune responses are more common in lupus patients using steroids
While some report that medication has no effect on anti-influenza antibodies (11
), others have shown decreases in antigen-specific responses in patients taking azathioprine or corticosteroids equivalent to ≥10 mg prednisone daily (19
). Twenty-four of 36 (67%) patients who were low responders took prednisone at a level equivalent to ≥10 mg/day compared to 17 of 36 patients (47%) who were high responders (p=0.04, ). Although the sample sizes are small, higher doses of corticosteroids (≥20 mg daily) did not appear to further lessen the magnitude of the influenza immune response. No other immunomodulatory medication was associated with a low response.
Of course, the number of individuals taking these medications is relatively small. Therefore, we divided patients into those on “low immunosuppressive therapies” (no medications, hydroxychloroquine only, or hydroxychloroquine in combination with the equivalent of 7.5 mg of prednisone daily) versus those on “high immunosuppressive therapies” (methotrexate, mycophenolate mofetil, azathiprine, cyclophosphamide, greater than 7.5mg of prednisone daily, or a combination of these). Of the thirty-nine individuals on “high immunosuppressive therapies”, twenty-one were high responders (54%). Of the thirty-three individuals on “low immunosuppressive therapies”, eighteen were high responders (55%). Thus, while steroid use is associated with a lower antibody response to influenza vaccination, other immunosuppressive medications do not account for the decreased responses in some patients.
No differences were detected in baseline autoantibody specificities of patients with poor vaccination responses
To help determine what predicts a low response to vaccination in SLE patients, autoantibody levels were measured at the time of enrollment. Nearly all patients were ANA positive () and high and low responders did not significantly differ in regards to ANA titer (median 1:1080 and 1:953, respectively; data not shown). No significant differences in the dsDNA antibody titer were observed between the responders (median 1:90 for each; data not shown).
While a trend toward higher cardiolipin and lower nRNP antibody frequencies was seen in the low responders, these differences were not significant, which may relate to the low number of individuals positive for these antibodies (). No differences were seen between the high and low responders in the proportion of individuals positive for any of the other autoantibody specificities at baseline. There was also no difference in the average number of autoantibodies in high and low responders (average 2.6 and 2.4, respectively).
Low responders have increases in autoantibody levels and new specificities after vaccination
We next examined the impact of vaccination upon autoantibody specificities and levels. shows the number of individuals who had new onset, a two-fold or greater increase, or a two-fold or greater decrease in the specified antibody. These data show patterns of changes in certain autoantibodies, including ANA, anti-La, and anti-cardiolipin among the high responders, and ANA, anti-Ro, and anti-RiboP among the low responders. Nineteen (26%) of the patients had a change in their ANA titer at two weeks post vaccination: two with new onset, eight with increased titer, and nine with decreased titer. Low responders were more likely to have increased ANA titer than high responders (14% versus 8%, respectively), with 5 of the thirty-six low responders having an increase in ANA titer post vaccination. Eight of the high responders (22%) had decreased ANA titers compared to only 1 (3%) of the low responders. Indeed, among high responders, post-vaccination decreases in ANA were twice as common as increased or new ANA expression. Among low responders, decreases in ANA titers were one-sixth as common as increased or new ANA expression (p=0.05, ).
Patients with low responses to influenza vaccination have increases in autoantibody levels and new specificities after vaccination
The small number of patients exhibiting changes in antibodies to La, Ro, cardiolipin, and RiboP do not allow a definitive statistical evaluation of differences between high and low responders. However, both groups had new onset of autoantibodies directed against La and cardiolipin (). Three low responders and one high responder had new onset/increased antibodies to the La antigen. Both the high responder group and the low responder group had two individuals with new onset/increased antibodies to cardiolipin.
Low responders to influenza vaccination are more likely to have a disease flare
We next examined disease activity following vaccination. Five individuals (6.9%), four of which were low responders, had an increase in SLEDAI of three or more points at 6 weeks post vaccination. Eight individuals (11%), four high and four low responders, had a three point SLEDAI change at 12 weeks post vaccination. Overall, no increased frequency nor severity of flares were seen in the time period immediately after vaccination (6 weeks) compared to a time period further from vaccination (12 weeks). However, this method does not capture some indications of disease flare such as medication changes or hospitalizations.
To further refine our method of determining flares, the SELENA-SLEDAI flare index was used (24
). Using this index, fourteen patients (19.4%) flared at six weeks post vaccination including 10 (13.8%) with mild/moderate and 4 (5.6%) with severe flares (). At twelve weeks there were nineteen (26.4%) flares including 16 (22.2%) mild/moderate and 3 (4.2%) severe flares (). Of the patients flaring at 6 weeks, 10 (71.4%) were low responders and 4 (28.6%) were high responders (, p = 0.01). By twelve weeks after vaccination, no significant difference was observed in the flare rate between high and low responders, 53% and 47% respectively (). Using data collected from previous studies where patients did not receive influenza vaccination, we observed that 1 of 8 patients (12.5% vs 19.4% in our study) had a mild/moderate flare when observed over a six to eight week time frame. Of 41 patients observed over a nine to eighteen week timeframe without vaccination, 8 (19.5% vs 26.4% in our study) had mild/moderate flares. The differences between flare rates over similar time periods in vaccinated versus non-vaccinated SLE patients were not statistically significant.
SLE disease flares are more frequent in low responders at 6 weeks, but not 12 weeks, after influenza vaccination
Serum Interferon alpha activity associates with disease flare following vaccination
We next asked if there were characteristics that would predict which patients would flare following vaccination. To this end we compared groups of patients that flared at six weeks post vaccination (n = 14) to a group of matched patients that did not experience a flare. We found that age at diagnosis, number of SLE ACR criteria, and baseline disease activity did not correlate with a flare. Select SLE ACR criteria were more prevalent in the individuals experiencing a flare and these included renal disease (43% vs. 29%), central nervous system involvement (21% vs. 0), and hematological disorder (50% vs. 29%). In contrast, fewer individuals with serositis (29% vs. 50%) or oral ulcers (57% vs. 71%) were in the flare group. Unfortunately, the small numbers of patients in these groups do not allow a definitive statistical evaluation of the impact of these criteria upon flare.
Since increased IFNα induced gene expression correlates with higher disease activity in SLE patients (37
), we examined serum IFNα activity. We found that patients experiencing a flare six weeks post vaccination had higher baseline serum IFNα activity than patients that did not have a flare (mean activity 19.3 vs. 2.7, p = 0.04, ). While patients that experienced a flare had a decrease in IFNα activity at two weeks post vaccination (mean activity of 10.95), this activity was still higher than seen in individuals that did not experience a flare (). This difference in serum IFNα activity was not observed in the individuals that had a flare at twelve weeks post vaccination (mean activity 2.9 vs. 5.3, p = 0.2, ). Of note, we found no significant correlation between IFNα activity level and the overall magnitude of the influenza-specific response.
Patients that flare at six weeks post vaccination had higher baseline IFN alpha serum activity