Immunologic Changes during Pandemic (H1N1) 2009, China

We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-γ, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later.

Flow cytometric results showing development of peripheral blood lymphocyte subsets during the disease course were divided into 3 groups on the basis of time of illness onset until date of blood sample collection: 1-3, 4-6, or 7-10 days. These groups were used because the longest time from onset of illness to hospitalization was 3 days for all patients, and the peak temperature for patients was observed 3 days after illness onset.
Mean counts and percentages of all T-and B-lymphocyte subsets increased after 3 days of illness compared with results obtained during the fi rst 3 days of illness ( Figure 1). However, the increase in CD8 and B cells was not signifi cant. Another study showed a decrease in CD4, CD8, and B cells ≤2 days of symptom onset in patients with pandemic (H1N1) 2009 than in healthy persons (4). Our results show impaired adaptive immune responses and a gradual increase during recovery in mildly affected patients.  We measured serum cytokine concentrations and hemagglutination inhibition (HAI) antibody titers in patients during hospitalization and the follow-up period ( Figure  2). We observed an increase in interleulin-6 (IL-6) levels 1 day after illness onset, which were 6.0-fold higher than the baseline level, and a 2.3-fold increase in interferon-γ (IFN-γ) levels. These levels decreased to baseline levels 5 days after illness onset, although the IL-6 level 5 days after illness onset was higher than levels 15 and 37 days after illness onset. The maximum IL-10 level 1 day after symptom onset was 3.2-fold higher than the baseline level. This level decreased to a value lower than the baseline level within 4 days, and then gradually increased to the baseline level 37 days after illness onset. Serum IL-6, IFN-γ, and IL-10 levels were not related to patient temperature 1 day after symptom onset, peak temperature during the disease, or period of fever. These levels showed minor differences that were not related to cough or sore throat in patients.
Only 1 patient had an HAI antibody titer ≥10 (titer 20) 1 day after illness onset. The HAI geometric mean titer increased 5 days after symptom onset compared with that 1 day after symptom onset and continued to increase until it reached a peak level of 137.9 at 37 days after symptom onset (25.5-fold increase). Peak HAI antibody titers ≥40 and ≥4-fold increases were observed in 27 (96.4%) patients.

Conclusions
Bermejo-Martin et al. reported increased serum levels of IL-6, IFN-γ, and tumor necrosis factor-α in patients with pandemic (H1N1) 2009 during the fi rst 5 days after symptom onset; no difference in levels of these 3 cytokines was observed in patients with mild disease and controls (5). However, similar to another report (4), we detected increases of IL-6 and IFN-γ levels in patients with mild disease during the fi rst 3 days after symptom onset. These different patterns may be caused by different intervals from time of symptom onset to date of sample collection (5 days vs. 3 days) because IL-6 and IFN-γ levels in our study quickly decreased to baseline levels ≤7 days after symptom onset. These results suggest that serum IL-6 and IFN-γ  , and IL-6 are medians (pg/mL). Serum HAI antibody titers were transformed by using the natural logarithm and are shown as means. Baseline cytokine concentrations on the y-axis are values for healthy persons. *p<0.05 when IL-6 or HAI antibody levels were compared with those at day 5; †p<0.05 when IL-10 level was compared with those at baseline; ‡p<0.05 when IL-10 level was compared with those at days 5 or 15; §p<0.05 when value was compared with that at any other time point; ¶p<0.05 when value was compared with those at days 5, 15, or 49. levels may be increased in patients with pandemic (H1N1) 2009 within the fi rst 3 days after symptom onset, followed by a decrease to baseline levels ≤5 days after symptom onset in patients with mild disease or a continuous increase in severely affected patients. IL-6 and IFN-γ are associated with antiviral immune responses during infl uenza infection (6)(7)(8). However, continuous, excessive release of IL-6 three days after illness onset likely contributed to serious pulmonary infl ammation and tissue injury, as has been documented for severe acute respiratory syndrome and 1918 pandemic infl uenza, but this release could be tempered by production of IL-10 (6,7,(9)(10)(11).
The proportion of persons 18-60 years of age with a ≥4-fold increase in HAI titer who received 1 dose (15 μg) of monovalent pandemic (H1N1) 2009 nonadjuvant vaccine was 96.2%, and the proportion with an increased HAI titer ≥40 was 97.1%, results similar to those of a recent study (12). However, the geometric mean titer in healthy vaccinated persons was 237.8, a 34.5-fold increase over the prevaccination titer, which was greater than that for patients naturally infected with pandemic (H1N1) 2009 virus (12). This fi nding may have resulted from impaired adaptive immune responses against pandemic (H1N1) 2009 virus in the initial phase, which included decreased numbers of CD4 and B lymphocytes and an increase in T regulatory cells (4).
In conclusion, our data indicated changes in cellular profi les during pandemic (H1N1) 2009 virus infection; showed that transient production of IL-6, IFN-γ, and IL-10 are main effectors of the early innate immune response against pandemic (H1N1) 2009 virus; and indicated that adaptive immune responses are impaired in the initial phase after infection. These factors may help clarify the pathogenesis of pandemic (H1N1) 2009 virus and provide new approaches in overcoming severe infections.