Recent global reports of pregnant women, especially in the third trimester, being disproportionately affected by 2009 A/H1N11–6 are consistent with reports from past influenza pandemics and support the decade-long public health recommendation to routinely immunize pregnant women with trivalent inactivated influenza vaccine to protect both women and their infants.7 Despite these recommendations, vaccination rates, although recently improved,8,9 remain suboptimal and there have been surprisingly few reports of vaccine immunogenicity among pregnant women.10–15 We report immunologic results from our influenza vaccine cohort study, which enrolled pregnant and postpartum women who had received influenza vaccine as part of their routine standard of care.
MATERIALS AND METHODS
This study was part of the Mount Sinai Viral Immunity in Pregnancy project, which was funded by a National Institutes of Health–National Institute of Allergy and Infectious Diseases contract (Immune Responses to Virus Infections During Pregnancy; Contact No. HHSN266200500028C). The project had two different cohort studies whose overarching aim was to characterize the immunologic adaptations that occur as pregnancy progresses. The vaccination cohort study enrolled antepartum and postpartum women to assess factors influencing the immunologic responses to trivalent inactivated influenza vaccine and to evaluate the spread of influenza-like illness among household members. The study design involved a baseline visit with blood draw, a postvaccination visit with blood draw, and monthly contact visits until the end of flu season (April each year).
The study and all modifications were approved by the Mount Sinai School of Medicine institutional review board (MSSM # 05-0054) and study recruitment started in the 2006–2007 influenza vaccine season and continued through consecutive influenza vaccine seasons. Consistent with Advisory Committee on Immunization Practices recommendations, during year 4, we modified our protocol to include women receiving either or both of the two different recommended influenza vaccinations, the monovalent inactivated vaccine against the circulating pandemic 2009 H1N1 as well as the standard seasonal 2009–2010 trivalent inactivated influenza vaccine.8,16 Patients receiving care at either one of two on-campus practice sites (the faculty practice or the resident-teaching practice) who were receiving trivalent inactivated influenza vaccine for clinical indications were eligible for study participation. There were no exclusions based on maternal comorbid medical conditions. Patients were enrolled throughout pregnancy and at two times postdelivery (either within 72 hours of delivery while an inpatient and again at approximately 6 weeks postpartum). Serum samples were obtained prevaccination or the day of vaccination and again at 4–8 weeks postvaccination. The specimen biorepository was linked to comprehensive maternal data (age, weight, comorbid medical conditions, concomitant medications or vaccinations, obstetric history, allergies, asthma and atopy, depression and stress assessments, influenza vaccination history, alcohol and drug use, and smoking and second-hand smoke exposures), pregnancy outcome data, and information about the spread of influenza-like illness among pregnant women and their household members.
Immunologic responses to influenza A were assessed by standard hemagglutination inhibition methods. Hemagglutination inhibition titer was determined by the ability of serially diluted receptor-destroying enzyme-treated serum to inhibit hemagglutination of chicken (H1 strains) or turkey (H3 strains) red blood cells in round-bottomed 96-well plates. Viruses used were either pseudotyped (6:2 recombinants) to match vaccine strains (Wisconsin/67/2005, Brisbane 10/2007, Brisbane 59/2007) or wild-type vaccine strains (New Caledonia/20/99, Solomon Islands/03/2006). Appropriate responses were assessed for both H1N1 and H3N2 strains of each year; additionally, response to California 04/2009 was assessed for patients who received the vaccine for the circulating pandemic H1N1 in 2009–2010.
Adequacy of serologic responses (seroconversion and seroprotection) were assessed using the criteria adopted by regulatory agencies to support influenza vaccine licensure.17 Seroconversion rates were defined as the proportion of patients with a fourfold or greater increase in reciprocal hemagglutination inhibition antibody titer at the postvaccination visit compared with prevaccination or a reciprocal hemagglutination inhibition titer of 40 or greater from a starting value less than 10. Seroconversion rates has been accepted as a surrogate for clinical vaccine efficacy because studies have demonstrated a strong correlation between a fourfold rise in serum hemagglutination inhibition antibody titer and disease protection. By convention, seroprotection rates were defined as the proportion of participants with hemagglutination inhibition titers 1:40 or greater. Geometric means of reciprocal hemagglutination inhibition titers were calculated for baseline and postvaccination samples, and the geometric mean fold rises (geometric mean of the within-patient fold increases from prevaccination and postvaccination) were also calculated. In addition to hemagglutination inhibition titers, immunostaining was performed to determine the subtype of the immunoglobulin (Ig) G antibody response for the pandemic H1N1 in the 2009–2010 cohort. Briefly, Madin-Darby canine kidney epithelial cells were infected with California 04/2009 pseudotyped virus at multiplicity of Infection=5 and then cultured for 18–24 hours on 96-well flat-bottomed plates. Cells, which now express viral proteins HA/NA, were fixed before incubation with serial dilutions of patient serum. A peroxidase-conjugated secondary antibody that is specific against total IgG, IgG1, IgG3, or IgG4 was used to identify IgG antibody subtypes.
We calculated descriptive statistics, including geometric mean titers, seroconversion rates by timing of vaccination during gestation, and seroprotection rates according to vaccine strains and patient characteristics by vaccine year and overall. For the geometric means, a titer of less than 10 was interpreted as 5.
We constructed a multivariable adjusted model for seroconversion, starting with a large pool of variables of interest and then reducing the number of variables using a backward elimination, ending with those that were statistically significant given the other terms in the model or of primary clinical interest. Predictors of seroconversion tested included both vaccine-related characteristics as well as maternal characteristics. The vaccine- related variables included: 1) nine combinations of year of administration and strain; 2) baseline hemagglutination inhibition titer values stratified into 0–20, 40, and 80 and above; and 3) whether the woman had a vaccine in the previous year. The maternal characteristics included in the final model were 1) age (stratified into less than 23, 23–29, and older than 30 years); 2) obesity (body mass index [calculated as weight (kg)/[height (m)]2] 30 or greater); and 3) education (less than high school, high school degree, more than high school). Statistical models accounted for the fact that women had multiple vaccines administered at the same time (eg, multiple strains in the inactivated seasonal vaccine and, in year 4, an additional monovalent H1N1 vaccine) by using generalized linear models as implemented in proc genmod of SAS 9.2. We started out by fitting an unstructured three-by-three correlation matrix in which it was assumed that there was a common correlation between any two vaccine strains administered at the same time but another correlation between the pandemic vaccine and each of the two seasonal Brisbane vaccines in year 4. The resulting pattern indicated, as expected, a positive correlation between the strains administered at the same time, but based on small sample sizes, we noted a slightly negative correlation between the pandemic and seasonal vaccine. To assure that the results were not unduly influenced by this implausible negative correlation between the pandemic and seasonal vaccines, for the final model, we set these correlations to zero.
Two hundred eighty-one patients were enrolled, and 245 completed both baseline and postvaccination study visits and also had serial specimens available. Those that did not have a postvaccination specimen did not differ from the study population in any of the dimensions summarized in Table 1 (data not shown). In addition, six human immunodeficiency virus-infected pregnant patients were eliminated from the current analysis. The description of the 239 patients is summarized in Table 1. Each year, the goal was to complete enrollment before the onset of the peak clinical flu season. In fact, most patients were vaccinated by the end of November (year 1: 89.2%; year 2: 84.3%; year 3: 93.0%; year 4 seasonal : 92.3%; year 4 H1N1: 66.7%). In total, only eight patients reported a possible influenza-like illness (defined as a febrile illness with either cough or sore throat) at the time of the postvaccination blood draw and only one of these cases was confirmed by a health care provider.
Geometric mean titers prevaccination and postvaccination are presented in Table 2 according to vaccination year, strain, and timing of vaccination; this table provides descriptive information without formal statistical testing. Averaged over all strains, geometric mean titers were higher postvaccination compared with prevaccination regardless of the gestation time period when a woman was vaccinated. Vaccination in the late postpartum and late third trimester resulted in the largest postvaccination compared with prevaccination titer ratios. During our study period, three viral strains appeared in two successive flu seasons: Wisconsin H3N2 2006/2007 and 2007/2008, Brisbane H3N2 2008/2009 and 2009/2010, and Brisbane H1N1 2008/2009 and 2009/2010. There were no notable differences in geometric mean titers postvaccination according to timing or year of vaccination for these repeating strains. The effect of timing of vaccination on seroconversion is presented in Table 3, which summarizes for each pregnancy time period the number of women, the average seroconversion rates, and the odds ratio based on a generalized linear model adjusting for the other terms in the model; data are summarized separately for all trivalent inactivated influenza vaccine strains combined and for monovalent pandemic H1N1. Averaged over all strains, the crude rate of seroconversion was lowest in the first trimester (54.8%) and immediate postpartum (54.8%) and highest in the late third trimester (69.6%) and late postpartum (69.4%). The low rate of seroconversion in trimester 1 was even more dramatic for pandemic H1N1 (28.6%). However, this was based on a very small number of patients. There were no statistically significant differences in odds of seroconversion according to time of vaccination (P=.23).
Relations between immunologic and patient characteristics and odds of seroconversion are presented in Table 4. Baseline antibody titer was strongly related to odds of seroconversion (P<.001) with the odds of seroconversion falling dramatically with increasing baseline antibody level. Women who received an influenza vaccination in the previous year had significantly (P=.03) lower odds of seroconversion than those who did not. Seroconversion rates also varied significantly (P<.001) among the nine vaccine strains administered during our study period; 2006/7 A Wisconsin had the lowest seroconversion rates (37%), the 2007/8 Solomon Islands the highest seroconversion rates (78%), and all the others were in the narrow range 56–65%. There were no other significant predictors of seroconversion among the factors examined. As noted previously, differences between trimesters were not significant (P=.23). In the multivariable adjusted model, maternal obesity did not reach statistical significance (P=.16); however, obese women (odds ratio 0.68, 95% confidence interval 0.41–1.14) had slightly although nonsignificantly lower odds of seroconversion.
Seroprotection rates (prevaccination and postvaccination titers greater than 1:40) are summarized in Figures 1 and 2; these figures provide descriptive information without formal statistical testing. Higher baseline (prevaccination) seroprotection rates were observed when the same antigen was used in consecutive years. Overall, for H3N2 strains, the seropotection rates postvaccination varied from 65% to 95%. Overall for H1N1 strains, the seroprotection rates postvaccination varied from 75% to 98%.
Only 16% of the patients receiving the monovalent H1N1 vaccine had background levels of antibody to the pandemic H1N1 strain allowing us to measure IgG antibody class without interference from pre-existing antibodies. Peroxidase-conjugated secondary antibodies specific for total IgG, IgG1, IgG3, or IgG4 were used to evaluate Ig class switching. Immunostaining is more sensitive than the hemagglutination inhibition method to detect antibody responses, and some patients with negative vaccine responses by hemagglutination inhibition assay showed clearly detectable staining postvaccination (data not shown). The results (data not shown) demonstrated an overwhelming IgG1 response to the vaccine at all vaccination time points assessed (both antepartum and postpartum). No change in IgG subtype preference was observed at any time point.
We assessed seroconversion rates to trivalent inactivated influenza vaccine through four consecutive flu vaccination seasons (starting October 2006 through January 2010) as well as to the pandemic 2009 monovalent H1N1 vaccine and observed rates generally similar to what has been reported among nonpregnant adults.17 Pregnant women demonstrated adequate immunologic responses to inactivated influenza vaccines throughout pregnancy and postpartum. When comparing seroconversion rates in the different gestational time periods with postpartum rates, the highest vaccine response rates were observed in the late third trimester (34 weeks of gestation or greater) and the lowest response rates were observed in the first trimester (less than 13 weeks of gestation) and in the immediate postpartum period (within 72 hours of delivery); however, these differences were not statistically significant. It is unlikely that any of the observed antibody responses were confounded by exposure to wild-type circulating virus because almost all patients were vaccinated before the onset of peak flu season and only eight patients reported a possible influenza-like illness at the time of their postvaccination visit.
Our multivariable model (Table 4) examined both maternal and vaccine characteristics as potential predictors of seroconversion. Only baseline antibody titer and prior year vaccination were strongly related to odds of seroconversion. Seroconversion rates also varied significantly among the nine vaccine strains administered during our study period. Women older than 30 years and obese women were observed to have nonsignificant but lower odds of seroconversion. Obesity, an increasing prevalent global health problem had emerged as a probable independent risk factor for pandemic 2009 H1N1 influenza disease severity.18 Obesity has been linked to diminished hepatitis vaccine responsiveness19 possibly mediated by immunologic alterations related to adipocyte dysfunction20 or alternatively as a result of improper vaccination technique.21
We used influenza vaccine as our model because of its routine use during pregnancy. However, it is not an ideal model to study primary immunologic responses because of the likelihood of significant past exposure to the vaccine antigen and related strains either from prior vaccinations or from prior wild-type infections, or both. Neutralizing antibodies from previous exposures may block access to B cells or deliver suppressive signals. In fact, in our cohort, a high level of circulating baseline antibodies was the strongest predictor of diminished vaccine responsiveness.
Our study has focused on the third trimester of pregnancy because this has been recognized as a time of immunologic vulnerability. Suppression of T cell activation has been suggested to be the basis of increased disease susceptibility or increased disease severity to certain infections including Listeria monocytogenes,22Plasmodium falciparum,23Varicella zoster,24 seasonal influenza,7 and most recently the novel H1N1 influenza, or both.1–6 Alterations in B cell function have been less well studied during pregnancy; however, significant suppression of B cell lymphopoiesis has been reported25 and steroid hormones have been implicated in changes of B cell function26 including possible changes in isotype switching.27
The availability of patients who received the monovalent H1N1 vaccine afforded us the unique opportunity to measure vaccine responses in a naïve population without background antibody interference. Although we enrolled only a very small number of first-trimester H1N1 vaccinees, our data suggest the possibility of a diminished first-trimester immune response, which warrants further investigation. Despite the existing clinical recommendations for influenza vaccination throughout gestation,7 women in the first trimester continue to be excluded from participation in clinical trials of pregnancy-related influenza vaccine immunogenicity.14 Among our H1N1 vaccinees, we were also able to assess IgG class switching. Immunoglobulin class switching is strongly influenced by the cytokine milieu,28 which changes during pregnancy in a predictable fashion.29 Th1 cytokines interferon-γ and interleukin-12 drive a switch to the IgG1 subtype, whereas Th2 cytokines such as interleukin-4 direct a switch to IgG2 and IgG4. As pregnancy progressed, if we had observed a shift away from IgG1 to other subtypes, this would have provided indirect support for a shift from Th1 to Th2 dominance, which has been posited to occur. In addition, transport across the placenta varies by class (IgG1>IgG4>IgG3>IgG2) and a switch in IgG class could potentially influence the protection afforded to the newborn.30 We did not observe a change in IgG subtype; at all gestational time points tested, IgG1 overwhelmingly dominated the response.
In summary, our observational cohort study provides practical guidance to clinicians faced with the need to counsel pregnant and postpartum patients about the benefits of influenza vaccination and also further elucidates our understanding of the immunologic alterations that characterize normal gestation. Vaccine responsiveness to inactivated influenza vaccine antigens was demonstrated throughout gestation with no diminution seen in the third trimester, a time strongly associated with increased influenza-related morbidity and mortality. Although our study was not designed and powered to identify the ideal time to vaccinate women during pregnancy, our data do suggest the possibility of lower seroconversion rates in the first trimester as well as in the immediate postpartum period. In addition, obesity may also be associated with lower seroconversion rates. Future studies specifically designed to assess the gestational age effect on vaccine responsiveness and among obese pregnant women are warranted by our observations and would help to refine influenza and other vaccination recommendations for pregnant and postpartum women.
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