Conceived and designed the experiments: DJF AWH CAM PJB. Performed the experiments: RGO RH DW LP ES SG AE CC SK. Analyzed the data: DJF AWK CAM CJH SK HG PJB. Contributed reagents/materials/analysis tools: CAM SK RO. Wrote the paper: DJF AWH CAM SK HG PJB.
The authors have declared that no competing interests exist.
Population-based febrile respiratory illness surveillance conducted by the Department of Defense contributes to an estimate of vaccine effectiveness. Between January and March 2011, 64 cases of 2009 A/H1N1 (pH1N1), including one fatality, were confirmed in immunized recruits at Fort Jackson, South Carolina, suggesting insufficient efficacy for the pH1N1 component of the live attenuated influenza vaccine (LAIV).
To test serologic protection, serum samples were collected at least 30 days post-vaccination from recruits at Fort Jackson (LAIV), Parris Island (LAIV and trivalent inactivated vaccine [TIV]) at Cape May, New Jersey (TIV) and responses measured against pre-vaccination sera. A subset of 78 LAIV and 64 TIV sera pairs from recruits who reported neither influenza vaccination in the prior year nor fever during training were tested by microneutralization (MN) and hemagglutination inhibition (HI) assays. MN results demonstrated that seroconversion in paired sera was greater in those who received TIV versus LAIV (74% and 37%). Additionally, the fold change associated with TIV vaccination was significantly different between circulating (2011) versus the vaccine strain (2009) of pH1N1 viruses (ANOVA
Among military recruits in 2011, serum antibody response differed by vaccine type (LAIV vs. TIV) and pH1N1 virus year (2009 vs. 2011). We hypothesize that antigen drift in circulating pH1N1 viruses contributed to reduce vaccine effectiveness at Fort Jackson. Our findings have wider implications regarding vaccine protection from circulating pH1N1 viruses in 2011–2012.
Crowded living quarters and stress can increase the potential for respiratory infections among military service members and lead to respiratory disease outbreaks
Since 1996, the Department of Defense (DoD) has conducted population-based febrile respiratory illness (FRI) surveillance at military recruit training centers (RTCs) across the United States
In the early months of 2011, FRI surveillance evidenced a sharp rise in pH1N1 cases among LAIV-vaccinated recruits after the second week of training at the U.S. Army RTC at Fort Jackson, South Carolina, suggesting reduced effectiveness for the pH1N1 component
All recruits at Fort Jackson and male recruits at MCRD-PI received the 2010–2011 LAIV, while female recruits at MCRD-PI were vaccinated with TIV, as required by local protocol. At Cape May, recruits were vaccinated with TIV.
Population-based FRI surveillance conducted between December 2010 and March 2011 noted laboratory-confirmed influenza rates (per 100-person weeks) among influenza-vaccinated military recruits at the three RTCs as: 0.15, Fort Jackson; 0.06, MCRD-PI; and 0.05, Cape May. During this time, influenza A/H1N1 2009, A/H3N2, and B viruses circulated in CDC region 4, with A/H1N1 2009 virus predominating (Centers for Disease Control and Prevention [CDC] FluView. 2010–2011 influenza season week 10;
In this study, a total of 540 trainees were enrolled including 201 from Fort Jackson, 259 from MCRD-PI, and 80 from Cape May. The average recruit numbers at the respective sites during this period were 9000 at Fort Jackson, 4000 at MCRD–PI, and 550 at Cape May. From 260 participants who reported no fever during the preceding 4–5 weeks of basic training and no influenza vaccination the previous year, a subset of 142 serum pairs were selected for serologic testing. These included the first 50 enrollees from Fort Jackson and MCRD-PI, and 42 from Cape May who met the aforementioned criteria. Baseline sera were obtained from the Department of Defense Serum Repository (DoDSR) in Silver Spring, MD. Post-vaccination sera were compared with matched baseline (pre-vaccination) samples drawn on average 133 days pre-vaccination (range 2–498 days). The mean ages of study participants were 22.1 years for the LAIV group and 20.9 years for the TIV recruits (
Samples from the three RTC sites were compared within TIV and LAIV-vaccinated groups. Among the paired sera studied, 78 were from LAIV-vaccinated individuals and 64 from TIV-vaccinated recruits. Madin-Darby canine kidney (MDCK) cell-generated influenza viruses A/CA/7/2009 H1N1 (2009 pH1N1), A/Perth/16/2009 H3N2 (H3N2), and A/CA/17/2011 H1N1 (2011 pH1N1) titered to a consistent concentration that gave 75% cytopathic effect (CPE) were utilized in the MN assays.
The proportions of LAIV and TIV vaccinees for the range of post-vaccination titers were plotted for the vaccine strain H3N2 and 2009 pH1N1 viruses and the circulating 2011 pH1N1 virus (
Virus | A/CA/7/2009 H1N1 | A/Perth/16/2009 H3N2 | A/CA/17/2011 H1N1 |
# Pre-vac. titer ≥40 (%) | 17 (22%) | 17 (22%) | 8 (10%) |
GMT Pre-vac. (95% CI) | 10.5 (8.4–13.1) | 12.5 (10.0–15.7) | 7.8 (6.3–9.8) |
# Post-vac. titer ≥40 (%) | 40 (51%) | 48 (62%) | 25 (32%) |
GMT post-vac. (95% CI) | 30.9 (23.1–41.4) | 47.2 (35.2–63.1) | 17.7 (1323.7) |
Seroconversion (%) |
27 (35%) | 37 (47%) | 22 (28%) |
Fold change (95% CI) |
2.2 (1.7–2.9) | 2.8 (2.1–3.7) | 1.5 (1.1–2.0) |
CI, confidence interval; GMT, geometric mean titer; LAIV, live attenuated influenza vaccine.
Seroconversion is defined as a 4-fold increase in titer from pre-vaccine to post-vaccine titer.
Fold change adjusted for pre-vaccine seroprotection levels.
Virus | A/CA/7/2009 H1N1 | A/Perth/16/2009 H3N2 | A/CA/17/2011 H1N1 |
# Pre-vac. titer ≥40 (%) | 10 (16%) | 19 (30%) | 5 (8%) |
GMT pre-vac. (95% CI) | 9.7 (7.6–12.4) | 18.0 (14.0–23.0) | 7.4 (5.8–9.5) |
# Post-vac. titer≥40 (%) | 47 (73%) | 62 (97%) | 39 (61%) |
GMT post-vac. (95% CI) | 77.9 (56.5–107.4) | 227.5 (165.0–313.8) | 39.4 (28.5–54.3) |
Seroconversion (%) |
47 (73%) | 54 (84%) | 41 (64%) |
Fold change (95% CI) |
5.6 (4.1–7.6) | 10.2 (7.6–13.8) | 3.4 (2.5–4.7) |
CI, confidence interval; GMT, geometric mean titer; TIV, trivalent inactivated influenza vaccine.
Seroconversion is defined as a 4-fold increase in titer from pre-vaccine to post-vaccine titer.
Fold-change adjusted for pre-vaccine seroprotection.
The baseline titer-adjusted fold change in MN titer associated with TIV vaccination was significantly different between the 2011 and 2009 pH1N1 viruses. To illustrate these differences, we modeled the logistic regression odds of a 4-fold conversion against 2011 and 2009 pH1N1 relative to conversion against the H3N2 strain (
Sera samples from a randomly selected subset of TIV (n = 10) and LAIV (n = 18) vaccinees were analyzed by surface plasmon resonance (SPR) for post-vaccination serum antibody binding to properly folded, functional oligomeric recombinant HA1 peptide against the 2009 pH1N1 strain. The maximum resonance unit (max RU) values for the serum antibody binding antibodies to rHA1 from LAIV (
To further evaluate the quality of the antibodies elicited after vaccination, antibody affinity maturation as measured by SPR were utilized to calculate the antibody dissociation off-rates for individual sera in TIV- and LAIV-vaccinated subjects (
To examine whether decreased serologic response from the A/CA/7/2009 H1N1 vaccine strain to circulating pH1N1 viruses isolated from recruits training in the southeastern United States in early 2011 correlated with antigenic drift, the HA1 genes from previous circulating and contemporaneous representative viruses were sequenced and a phylogenetic tree constructed using the neighbor-joining method (CLUSTAL W) of the MegAlign program (Lasergene software suite; DNASTAR Inc., Madison, WI) (
The phylogenetic tree was constructed using the neighbor-joining method (CLUSTAL W method) of the MegAlign program (Lasergene software suite, DNASTAR Inc., Madison, WI). The tree represents amino acids 111 through 360 of the mature HA protein. Reference strains for the six circulating subclades (2–7) are shown in bold text, with their defining mutations shown to the right. The vaccine strain, A/CA/7/2009, is shown in bold and italicized. Isolates collected by NHRC from recruits who had been vaccinated greater than 14 days are italicized. Strains from the same geographical location and time of collection from the GISAID database are included in the tree as well as two samples collected from the same recruit training sites in 2009. The scale indicates the distance created by a one amino-acid difference between sequences. The number of amino acid changes between subclades is denoted by a delta symbol (Δ).
A survey to measure seroresponses to seasonal influenza vaccine was conducted among military recruits at three RTCs in the eastern and southeastern United States March 2011, following intense transmission of pH1N1 in January, including one death, among vaccinated recruits in South Carolina
Military recruits are homogeneously healthy and young. As a result of crowding and a challenging training environment, recruits experience higher respiratory disease rates than non-recruits
Both TIV and LAIV have been shown to be effective in children and adults, although multiple studies in children aged 6 months to 18 years have demonstrated that LAIV provides a greater seroresponse than TIV
Our results show differential immune response between LAIV and TIV consistent with previous reports. The quantitative and qualitative robustness of T and B cell memory responses to viral antigens are mediated by previous exposure and/or vaccination. Recently, a study in children ages 6–35 months found that LAIV conferred broader heterotypic αβ and γδ T cell immunity against conserved influenza peptides than TIV
Our results provided evidence for modest antigenic drift in pH1N1 viruses from the southeastern United States in 2011. There were significant differences in vaccine-induced serum antibody neutralization in the vaccine strain 2009 pH1N1 as compared to the circulating 2011 pH1N1. Contemporaneously collected viruses from nationwide surveillance were rooted against the vaccine and other circulating strains to infer divergence in the HA surface protein.
Recruits arrive at the RTCs from regions across the United States, resulting in the potential seeding of diverse viruses. This was demonstrated in our study as isolates from four of six subclades (Groups 2, 3, 6, and 7) were found at Fort Jackson during the first 2 months of 2011. Interestingly, only one of these, Group 3, circulated widely during the outbreak in January 2011.
In the spring of 2009, distinct spatial heterogeneity existed within pH1N1 viruses, resulting in strong regional founder effects. During this first wave, multiple phylogenetically distinct pH1N1 clades emerged globally
Analysis of the HA genome elucidated a number of mutations in 2011 pH1N1 viruses. The S183P mutation in the Fort Jackson viruses has been shown
The outbreak of pH1N1 in vaccinated recruits occurred during a time when influenza A/H3N2 and B viruses and multiple subclades of A/pH1N1 circulated in the Fort Jackson. However, only one (Group 3) was evident during the outbreak. This circulating virus had important HA mutations that mediate antibody binding. Moderate antigenic divergence between circulating and vaccine influenza strains likely contributed to the outbreak of pH1N1 among recruits at Fort Jackson in the early weeks of 2011.
At Fort Jackson, the protective threshold was breached in LAIV vaccinees infected with antigenically divergent subclade 3 pH1N1 viruses. Increased TIV vaccination in the Fort Jackson recruit population could induce higher antibody titers and protective immunity. We speculate that TIV vaccination would increase overall vaccine effectiveness, thereby providing a herd immunity effect across the population.
The effectiveness of seasonal influenza vaccines varies by season. The risk of periodic influenza epidemics as a result of antigenic drift may best be ameliorated through the development of a universal influenza vaccine
Recruits undergoing basic combat training at Fort Jackson were vaccinated intranasally prior to the first week of training with the LAIV FluMist (MedImmune, LLC, Gaithersburg, MD) per manufacturer's instructions. Each 0.2 prefilled dose contained 106.5–7.5 fluorescent focus units of live attenuated influenza virus reassortants of each of the three strains recommended by WHO for the 2010–2011 season: A/CA/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and B/Brisbane/60/2008. Prior to the first week of recruit training at MCRD-PI, female recruits were vaccinated with the TIV influenza vaccine AFLURIA (CSL Biotherapies, King of Prussia, PA) per manufacturer's instructions. The AFLURIA influenza vaccine was standardized for the 2010–2011 influenza season and formulated to contain 45 mcg HA per 0.5 mL dose in the recommended ratio of 15 mcg HA for each of the three influenza strains recommended for the 2010–2011 Northern Hemisphere influenza season. Male recruits at MCRD-PI were vaccinated with the MedImmune LAIV FluMist, as previously described. Recruits at Cape May were vaccinated with the Fluzone TIV (Sanofi Pasteur, Inc., Swiftwater, PA) per manufacturer's instructions. Each 0.5-mL dose of Fluzone contained a total of 45 mcg of influenza virus HA equally distributed among the three components of the 2010–2011 influenza vaccine.
The proposal for serologic draw was reviewed and approved as a public health response, non-research activity by the U.S. Army Public Health Command Public Health Review Board, the Institutional Review Board (IRB) at the Naval Health Research Center (NHRC), and the Armed Forces Health Surveillance Center. Participation for blood draw was voluntary, and consent obtained verbally per observation by local investigators. Per the proposal approved by the NHRC IRB, all samples were de-identified. Concurrently, throat and nasal swabs were collected under ongoing NHRC protocol NHRC.1999.0002 approved by the NHRC IRB from recruits who presented with an FRI and consented, in writing, to be swabbed. This project has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research.
The serosurvey was conducted by systematically enrolling recruits 4–5 week's post-influenza vaccination. Recruits completed a short case report form (CRF) that included information on previous vaccination, age, sex, and signs and symptoms. Influenza vaccination history (date, vaccine type) was abstracted from medical records at each training center. Blood was drawn in 10-ml serum separator tubes (BD Biosciences, Franklin Lakes, NJ) and allowed to clot for 30 minutes. Tubes were centrifuged for 10 minutes, and then frozen at between −20°C and −80°C prior to shipment to NHRC for analysis. Baseline sera were provided from the DoDSR, which maintains serum specimens collected from service members for periodic HIV testing and operationally required pre- and post-deployment blood draws
Concurrently, throat and nasal swabs were collected from recruits who presented with an FRI. At RTCs, denominator data for all FRIs are collected and 10–20 patients are randomly selected for sampling each week throughout the year. FRI was defined by fever (>38.0°C), sore throat, and/or cough. Enrollees provided a CRF containing demographic and medical history data. Swabs were collected in universal transport medium (Copan Diagnostics, Inc., Murrieta, CA), stored at 4°C, and then shipped to NHRC for real-time RT-PCR (rRT-PCR), viral isolation, and genetic characterization.
Influenza viruses were propagated in MDCK cells to high titer, and TCID50 determined using the Reed-Muench method. Negative and positive control sera for A/CA/7/2009 (2009 pH1N1) and A/Perth/16/2009 (H3N2) were obtained from the 2010–2011 WHO Influenza Reagent Kit. Positive control ferret antisera for A/CA/17/2011 (2011 pH1N1) were provided by CDC, Atlanta, Georgia. All control sera were treated with receptor-destroying enzyme (RDE), heat inactivated at 56°C for 30 minutes, and then diluted to a 1∶10 concentration. Serum antibody MN assays were performed according to described procedures
The HI assays were performed according to the WHO protocol for serological diagnosis of influenza virus infection using standardized 0.75% guinea pig red blood cells (GPRBC). Serum samples were treated with RDEs overnight at 37°C, and heat inactivated the following day. Serum samples, including negative and positive controls, were initially diluted 1∶10 with PBS and then serially diluted 2-fold from 1∶10 to 1∶1280. Influenza viruses were adjusted to contain 4 HA units/25 µl and added to wells. GPRBC were added to all wells, and incubated at room temperature for 1 hour. Antibody titer for the particular virus was determined as the highest serum dilution showing complete inhibition for each serum sample tested.
Differences between groups were examined for statistical significance using Student's t-test. An unadjusted
Population pre- and post-vaccination GMTs were calculated for the three viruses (2009 pH1N1, 2011 pH1N1, H3N2) for each vaccination type (TIV and LAIV). Additionally, adjusted geometric mean fold change was estimated for each virus and vaccination type, after adjustment for starting titer as previously described
Linear regression was used to evaluate differences in the geometric increases in HI and MN titers for different viruses and different types of vaccination. Logistic regression allowed differences in the odds of a 4-fold rise in titer to be evaluated between vaccination types and virus strains while adjusting for pre-vaccine levels. For this comparison, the rate of 4-fold rise in titer against H3N2 was used as a reference.
Steady-state equilibrium binding of post-immunization 2009 pH1N1 human vaccine sera was monitored at 25°C using a ProteOn SPR biosensor (Bio-Rad Laboratories, Inc., Hercules, CA), as previously described
Steady-state equilibrium binding of pre- and post-H1N1 human vaccine sera was monitored at 25°C using a ProteOn SPR biosensor (Bio-Rad). Antibody off-rate constants, which describe the stability of the complex (i.e., the fraction of complexes decaying per second), were determined directly from plasma sample interaction with properly folded, pH1N1-functional HA1 globular domain and HA2 stalk domain proteins
Ribonucleic acid was extracted from combined throat and nasal swabs using the QIAamp RNA Mini Kit (Qiagen, Valencia, CA) following manufacturer's instruction. rRT-PCR assays were used to detect influenza A and B viruses and to subtype A viruses as either H1, pH1, or H3, as previously described
Virus isolation was performed in MDCK cells or R-Mix Too Shell Vials (Diagnostic Hybrids, Inc. [DHI], Athens OH). In shell vials, medium was aspirated and 1 mL of R-Mix Refeed media (DHI) containing 100 units/mL penicillin, 100 ug/mL streptomycin, and trypsin (bovine origin) at 1.33 ug/mL, was added. Vials were inoculated with 0.2 mL of swab extract and then centrifuged at 2100 rpm (RT) for 1 hour. After centrifugation, vials were incubated for 48 hours, washed with sterile PBS twice, and fixed in acetone. Immunofluorescence assays were conducted using D3 Ultra DFA Respiratory Virus Screening Reagent (DHI). Viruses were replicated and amplified in MDCK flasks, at 80–90% confluency. Upon CPE approximately 75% of the susceptible cells were harvested and centrifuged at 2000 rpm, at 4°C, for 5 minutes. The TCID50 for each of the viruses were calculated to obtain 200 TCID50/50 µL.
Primers for sequencing influenza HA protein were provided by the CDC and are available upon request. An initial amplicon was produced with a paired primer set in a 25 uL aqueous reaction containing 1× Colorless GoTaq Flexi Reaction Buffer (Promega Corporation, Madison, WI), 0.2 mM each dNTP (Promega), 1.5 mM MgCl2 (Promega), 0.6 uM concentration of each primer, 0.5 U GoTaq DNA Polymerase (Promega), and 5 uL of template. Products were mixed 5∶1 with loading dye (Sigma-Aldrich, St. Louis, MO) and run for 90 min at 125 V on 2% agarose (Bio-Rad, Hercules, CA) gels with ethidium bromide (Sigma-Aldrich). Cycling conditions were an initial 60 seconds at 96°C followed by 25 cycles of 10 seconds, 96°C; 5 seconds, 50°C; 4 minutes, 60°C, and a final hold at 40°C. Gels were visualized in an ultraviolet light box. Amplicons were excised and purified using the QIAquick gel extraction kit (Qiagen) according to manufacturer's instructions. Amplicons were then used as template in sequencing reactions containing 1× BigDye Terminator v3.1 Buffer (Applied Biosystems, Foster City, CA), 1 uL of BigDye Terminator Ready Reaction Mix v. 3.1, 0.2 uM forward or reverse primer, and 5 uL of amplicon in a 20 uL aqueous reaction. Reactions were performed on an iCycler PCR machine (Bio-Rad). Sequencing products were purified using Performa DTR Gel Filtration Cartridges (Edge BioSystems, Gaithersburg, MD) and sequenced on a 3130 Genetic Analyzer (Applied Biosystems).
Sequencing, genome assembly, and closure reactions were performed as described
We thank the medical staffs at Fort Jackson, the Marine Corps Recruit Depot, Parris Island, and the Coast Guard Training Center at Cape May for their assistance in the conduct of this study and Dr. Alexander Klimov for provision of the ferret anti-serum against A/CA/17/2011 H1N1. We are grateful to Ms. Damaris Padin and Ms. Larivhie Delacruz for coordinating sample collection and shipment from NHRC and the J. Craig Venter Institute, Rockville, MD. Thanks to Ms. Daisy Cabera, Mr. Robert Coon, and Mrs. Melinda Balansay-Ames for supervision of laboratory activities at NHRC. We are indebted to Drs. Timothy K. Uyeki and Timothy H. Burgess for critical review.
The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of Defense, or the U.S. Government. Approved for public release; distribution is unlimited. The proposal for serologic draw was reviewed and approved as a public health response, non-research activity by the U.S. Army Public Health Command Public Health Review Board, the Institutional Review Board (IRB) at the Naval Health Research Center (NHRC), and the Armed Forces Health Surveillance Center. Participation for blood draw was voluntary, and consent obtained verbally per observation by local investigators. Per the proposal approved by the NHRC IRB, all samples were de-identified. Concurrently, throat and nasal swabs were collected under ongoing NHRC protocol NHRC.1999.0002 approved by the NHRC IRB from recruits who presented with an FRI and consented, in writing, to be swabbed. This project has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research.