Influenza Virus Vaccination of Patients With Chronic Lung Disease

Influenza Virus Vaccination of Patients With Chronic Lung Disease* Geoffrey]. Gorse, MD; Esther E. Otto, BSN; Carlos C. Daughaday, MD, FCCP; Frances K. Newman, MS; Christopher S. Eickhoff, BSc; Douglas C. Powers, MD;f and Rodney H. Lusk, MD

Study objectives: To evaluate the safety of, and mucosal and systemic immune responses induced by two influenza virus vaccine regimens in subjects with COPD. Design: Single-center, blinded, randomized, prospective clinical trial evaluating two vaccine regimens. Setting: Outpatient clinics of St. Louis Department of Veterans Affairs Medical Center. Participants: Volunteers (age range, 42 to 88 years) had preexisting COPD with severe obstruction to airflow on average, were male, and were not receiving immunosuppressive medication. Interventions: Twenty-nine volunteers were randomly assigned to receive either bivalent live attenuated influenza A virus vaccine (CAV) or saline solution placebo intranasally. All subjects also received an IM injection of bivalent inactivated influenza virus vaccine (TVV) simultaneously. Measurements and results: Clinical status and pulmonary function measured by spirometry did not change significantly after vaccination. Using hemagglutinins (HI and H3 HA) which more closely resembled those in CAV, mean levels of anti-HA immunoglobulin A (IgA) antibodies in nasal washings increased significantly after vaccination with CAV and TVV compared to prevaccination, but they did not increase significantly after TVV and intranasal placebo. Mean levels of influenza A virusstimulated interleukin-2 and -4 produced by peripheral blood mononuclear cells in vitro increased significantly after administration of the combination vaccine regimen and to a lesser extent after TVV and intranasal placebo compared to respective prevaccination levels. The timing of the cytokine response appeared different following CAV and TVV compared to TVV and intranasal placebo. Conclusions: lntranasally administered CAV was safe when given with IM administered TVV and there may be an immunologic advantage to administration of the combination vaccine regimen compared to TVV with intranasal placebo. (CHEST 1997; 112:1221-33) Key words: cellular immunity; chronic lung disease; immune response; influenza A virus; mucosal immunity; virus vaccines Abbreviations: CAV=bivalent, cold-recombinant, live attenuated influenza A virus vaccine; ELISA =enzyme-linked immunosorbent assay; GMT = geomet1ic mean titer; HI, H2, and H3 = hemagglutinin subtypes 1, 2, and 3, respectively; HA= hemagglutinin; HAl =hemagglutination inhibition; IFN--y=interferon-gamma; Ig=immunoglobulin; IL=interleukin; Nl and N2=neuraminidase subtypes 1 and 2, respectively; NS=not significant; PBL=pelipheral blood mononuclear cells; TCID,, 0 =50% tissue culture infective dose; THl and TH2=helper T-cell subsets l and 2, respectively; TVV=tiivalent inactivated subvilion influenza vims vaccine

p atients with chronic underlying illnesses such as

COPD have an increased risk for respiratory illness-related hospitalization during influenza out*From the Section of Infectious Diseases (Drs. Gorse and Lusk), Respiratory and Critical Care Service (Dr. Daughaday), and Geriatlic Research, Education and Clinical Center (Dr. Powers ), St. Louis Department of Veterans Affairs Medical Center; and the Divisions of Infectious Diseases and Immunology (Drs. Gorse, Lusk, Ms. Otto, Ms. Newman, and Mr. Eickhoff) and Ge riatrics (Dr. Powers) , Saint Louis University School of Medicine, St. Louis. 1 Currently at the Glennan Center for Geriatrics and Gerontology, Eastern Virginia Medical School, Norfolk, Va. Supported by a Department of Veterans Affairs Medical Research Grant. Manuscript received Octobe r 1, 1996; revision accepted April 8, 1997. Reprint requests: Geoffrey]. Gorse, MD, Division of Infectious Diseases and Immunology . Saint Louis University Health Sciences Center, 3635 Vista Ave (FDT-8N), St. Louis, MO 63110

breaks independent of age, and morbidity and mortality in excess of the already higher underlying rates associated with old age. 1-6 In a recent study, the clinical predictor of pneumonia and influenza-related hospitalization with the highest odds ratio (7.2) was preexisting lung disease.6 Despite drawbacks in immunogenicity, including suboptimal stimulation of secretory and cellular immune responses, trivalent inactivated influenza virus vaccine has reduced rates of hospitalization and pneumonia in patients with chronic diseases by 30 to 60% depending on age and frailty of the population. 1 •6 -9 Higher rates of protection are needed particularly in high-risk patients. Live attenuated influenza A virus vaccines have been safe in healthy children and young adults, in older adults with chronic diseases, and in the healthy elderly. 10-22 There are possible advantages in immuCHEST I 112 I 5 I NOVEMBER, 1997

1221

nogenicity for live attenuated influenza virus vaccines.ll-14·16 - 18,20,23-25 Secretmy anti-hemagglutinin (HA) immunoglobulin (Ig) A antibody and antiinfluenza A virus cytotoxic T-cell responses w ere better in patients with COPD after immunization with monovalent live attenuated compared to inactivated influenza A virus vaccines, and in chronically ill, elderly nursing home residents after immunization with bivalent live attenuated influenza A virus coadministered with parenteral inactivated virus vaccines compared to inactivated virus vaccine alone.1 1- 14 ·20 These observations are important since anti-HA IgA antibodies in respiratory secretions and cellular responses have correlated with protection from influenza A virus infection, 16·26 ·27 although serum anti-HA IgG and hemagglutination inhibition (HAl ) antibodies also correlate with protection.l 7,26-2s Live attenuated influenza A virus vaccines have provided protective efficacy from infection with wild type virus which was as good as or better than inactivated virus vaccines in healthy children and adults .l7·27·29.3° Intranasal monovalent live attenuated influenza A virus vaccine coadministered with parenteral trivalent inactivated influenza virus vaccine was more effective than trivalent inactivated virus vaccine alone in preventing laboratmy-documented influenza A virus infection and outbreakassociated respiratory and influenza-like illnesses in elderly nursing home residents. 10 The better anti-influenza A virus mucosal antibody and cytotoxic T-cell responses induced b y vaccination regimens, which include live attenuated virus vaccine compared to inactivated virus vaccine alone, suggest that memory T -cell responses to live virus vaccination may be manifested by a more complex cytokine production pattern . Cytokines are knovm to influence T- and B-cell responses to viral infection. In vitro interleukin-2 (IL-2) production by helper T-cell subset 1 (THl ) lymphocytes is correlated with cell proliferation and activation of cytotoxic T-cell responses.3l,32 Antigen-specific lymphocyte proliferation and IL-2 production have increased after influ enza A virus vaccination of older adults.24 ,33-35 Interferon-gamma (IFN-'Y) isproduced b yTHl and CDB+ T-cells, such as cytotoxic T-cells, after an influenza virus infe ction. It has antiviral effects, and can downregulate B- and helper T-ce LI subset 2 (TH2 ) lymphocytes responding to IL-4 and IL-2.36-38 IL-4 is produced b y TH2 cells, is important for immune regulatory events in mucosal effector tissues, and can serve as a late s ignal that augments generation of antigen-specific cytotoxic T -cell activity.39-45 Therefore, it was hypothesized in the current study that live attenuated virus vaccine coadministered \vith inactivated virus vaccine would affect 1222

influenza virus antigen-stimulated cytokine production by peripheral blood mononuclear cells (PBLs) differently than inactivated virus vaccine given alone. The current study syste matically evaluated bivalent live attenuated influenza A virus vaccine in patients with COPD for the first time. A regimen including live virus vaccine coadministered with inactivated virus vaccine was compared to inactivated virus vaccine given with intranasal placebo. We measured safety, anti-influenza A virus antibodies in serum and nasal washings, and in vitro production of THl and TH2 cytokines b yinfluenza A virus-stimulated PBLs.

MATERIALS AND METHODS

Vaccines The bivalent, cold-recombinant, li ve attenu ated influenza A virus vaccine (CAY) (Wyeth -Ayerst; Philadelphia) was derived from cold-adapted influenza A/Ann Arbor/6/60 (H2N2) virus and grown in th e allantoic cavity of eggs as described. 46 •47 The resulting cold-recombinant viruses contained sixgenes that code for internal proteins fi·om the donor cold-adapted strain and genes that code for HA and neuraminidase from influenza iVKawasaki/9/86 (H1 N1) and A/Beijing/353/89 (H3N2) viruses. Just prior t o s tarting this study, a 0.8-mL dose of CAY was found to contain w ru plague-forming units of influenza A/Kawasaki/ 9186 (H1Nl ) cold-recombinant virus and 106 -~ plague-formin g units of the influenza A/Beijing/353/89 (H3N2 ) cold-recombinant virus. Two years prior to starting the current study, the amount of each virus strain in the vaccine lot was determin ed bv the vaccine manufacturer to be 107 ·4 50% tissue culture infective doses (TClD 50 ) per 0.8 mL. The CAV was stored at - 70°C in th e intetvening time and, afte r th awing, was administered intranasally as nose drops, 0.4 mL in eac h n aris, as descti bed. t9,2o The trivalent inactivated subvi ri on influe nza virus vaccine (TVV) contained 15 f.Lg of HA from each ofthree influenza virus strains per 0.5-mL dose. All 29 subjects received the TVV recommended for th e 1994 to 1995 influenza season (A/Texas/ 36/91 [H1Nl]. A/Shangdong/9/93 [H3N2]. and B/Panama/45/90) (\Nyeth-Ayerst). Selection of Subjects and Clinical Studies Volunteers with a histOty o f COPD were re cruited in the outpati ent clinics of the Department of V eterans Affairs Medical Center, St. Louis. A m edical hist01y including respiratory symptoms, aphysical examination, and clinical laborat01y tests (serum albu min, alanine amino transferase and choles terol, and CBC counts) were performed prior to immunization . Criteria for llows: ( 1) a hist01y of exclusion from p articipation were as fo hypersensitivity to influenza virus vaccines and eggs; (2) receipt of influenza virus vaccine < 6 months prior to enrollment in the study; (3) incompetence to give written informed consent; (4) current administration of immunosuppressive che motherapy; (5) hematologic malignancy not in remission; and (6) a blood hemoglobin concentration < 11 g!dL. Eligible subjects were randomly ass igned to receive either CAV or saline solution placebo intranasally. All subjects received TW at the same tim e by IM inj ection i n the deltoid muscle. Subjects and study personnel were blinded to vaccination assignment with th e exception of the Clinical Investigations

study nurse who administered the vaccines. Enrollment and vaccination of 12 subjects occurred b etween O ctober and December 1994 and of 17 subjects between Aptil and June 1995. Subjects were not enrolled during the 1994 to 1995 influenza season. All subjects returned to the outpatient clinic three times between each of days 1 to 5, 7 t o 10, and 21 to 28 after immunization for clinical evaluation. Possible adverse reactions to the vaccines (eg, runny nose, sore throat, s neezing, hoarseness, earache, red eyes, nasal congestion, cough, increased shortness of breath, increased cough and sputum production, fever, auscultatory pulmonary changes, and local injection site pain and tenderness) were assessed at aech visit. Pulmonary function tests consisting o f basic spirometry were performed in the clinic using a portable spirometer (MicroLab Model 3000; Spirometries Inc; Auburn, Maine) before and 7 to 10 days after vaccination in all subjects. Each time spirometry was performed, several trials were done and the best effort was reported. Spirometry consisted of the FEV1, FVC, and FEV/ FVC (percent).19 ·48 -51 Volunteers were categorized as having COPD if they had an FEV/ FVC (percent) < 70%. The percent predicted FEV1 was used to grade the severity of obstruction to airflow according to standard criteria.s 1 A possibly significant change in severity of obstruction to airflow between study visits was defin ed as a decrease of at least 20%.s1 Spirometry was repeated 21 to 28 days after vaccination when changes in obstruction to airflow or respiratory symptoms occurred on day 7 to 10 compared t o before vaccination. Serum, heparinized whole blood, and nasal washes (20 mL of norm al saline s olution) for immunologic assays were obtained before immunization, and at 7 to 10 and 21 to 28 days after immunization. On 2 days during the first 7 t o 10 days after immunization , nasal and oropharyngeal secretion swab specimens were obtained for virus isolation, asdescribed.ll.l 9 -52 \.Yhen influenza A virus was isolated, it was assessed for th e presence of revertant virus with the capability of growth at 39°C and the virus titer was determined as the TCID 5 ofmL, as described.19 -53 Serologic Testing Serum HAl Antibodies: Serum aliquots were pretreated with receptor destroying enzyme to remove nonspecific inhibitors. Serum HAl antibodies were then m easured by using whole virus homologous to the vaccine strains in a standard microtiter assay (Centers for Disease Control; Atlanta). 54 Serum and Nasal Wash Anti-HA Antibody Assays: After sonication, each nasal wash specimen was concentrated as described14 Aliquots of serum were depleted of IgG b ybinding to recombinant protein G ptior to measuring anti-HA lgA antibody titers using a commercial kit (Quik-Sep Kit; Isolab Inc; Akron, Ohio) . An end-point enzyme-linked immunosorbent assay (ELISA) was employed as described t o determine the antiinfluenza virus HA serum and nasal wash I gA natibody titers in specimens obtained b efore and after vaccination using homologous purified HA.13.1 4 .19.zo.ss.s6 Purified HA from influenza AI Taiwan/1/86 (H1N1) and A/Beijing/353/89 (H3N2) viruses (gifts of Joseph Messina; Wyeth-Ayerst Research; Radnor, Pa; and Maurice Harmon; Connaught Laboratories, Inc; Swiftwater, Pa) were used in the ELISA. The nasal wash I gA nati-HA titers were corrected to a total lgA concentration of500 mg!L based on the total I gAconcentration in each specimen as described. 14 Cellular Immune Testing

PELs were separated from heparinized whole blood b yFicoliPaque (Pharmacia; Piscataway, NJ) density gradient centrifuga-

tion as described .57 Live influenza A virus stimulation of PELs was carried out using influenza A/Taiwan/1/86 (H1N1 ), A/Beijing/353/89 (H3N2), or A/Shangdong/9/93 (H3N2) virus-infected autologo us PELs as described.1 1·12 PELs were then added at 106 cells per w ell to 24-well tissue culture plates (Becton Dickinson and Co; Lincoln Park, NJ) in RPMI 1640 medium (Bio-Whittaker Inc; Walkersville, Md) supplemented with heat-inactivated pooled human AB serum (Advanced Biotechnologies; Columbia, Md) a t a 10% concentration (volume/volume), L-glutamine, and antibiotics. For stimulation of PELs by heat-inactivated virus, uninfected PELs suspended in supplemented RPMI 1640 medium were added at 106 cells per w ell. Each of the three heat-inactivated influenza A viruses were added to the PBL cell cultures in aliquots containing an infectious titer prior to inactivation of 5 x l0·5 TCID 50 . Control wells contained PELs and supplemented medium without influenza A virus. Cell supernatant fluids were collected from PBL cultures established with mouse monoclonal anti-IL-2 receptor antibody (5 1-Lg/mL fin al concentration; PharMingen; San Diego, Calif) after incubation at 37°C in 5% C0 2 for 72 h. Cell supernatant fluids from other cell cultures without antibody to IL-2 receptor were collected after 168 h of incubation at 37°C in 5% C02 . Cell supernatant fluids were clmifled of debris by low-speed centtifugation, stored at - 70°C, and assayed later using commercially available ELISA kits for cytokine detection follO\ving the manufacturers ' procedures (Interleukin-2 Enzyme Immunoassay Kit; lmmunotech Inc; Westbrook, Maine; Cytoscreen Immunoassay Kit for human IFN-"( and Cytoscreen Ultra Sensitive Immunoassay Kit for human IL-4; BioSource International; Camarillo, Calif). IL-2 levels meas ured in cell supernatant fluids collected after 72 h of incubation, and IFN-'Y and IL-4 levels measured in cell supernatant fluids collected after 168 h of incubation were optimal. Statistical Methods

Geometric mean r eciprocal antibody titers, and arithmetic means o fclinical l aboratory data, cytokin e levels, and spirometric measures of pulmonary function with SEs are reported. Mean data within groups measured b efore and after vaccination were compared u sing the \Vilcoxon signed r anks nonparametric test. Means of unpaired data between groups were compared using the Mann-Whitney U test for independent samples. Fisher's Exact Test was employed for two-by-two comparisons .

RESULTS

Patient Population

Sixteen subjects received CAV and TVV and 13 subjects received TVV and intranasal placebo. All 29 subjects were male and were evaluable for safety and immunogenicity. Demographic characteristics and mean prevaccination clinical laboratory test results were not different between groups. For the entire 29-subject study population, the mean age was 65.2±2.1 years (range, 42 to 88 years), mean total WBC count was 7,710:±:298 cells/J-LL, lymphocytes constituted a mean percentage of the total WBC count of 22.7%:±:1.4%, mean serum albumin level was 4.3:±:0.07 gldL, and mean total cholesterol level was 222.8 ±12.4 mgldL. The proportions of subjects with underlying medical illnesses w ere not different except for a higher proportion with a history of liver CHEST I 112 I 5 I NOVEMBER, 1997

1223

disease who received CAV and TVV (5 of 16 subjects) compared to those who received TVV and intranasal placebo (0 of 13 subjects) (p<0.05). Overall among the 29 subjects, 6 (21%) subjects had underlying renal disease, 19 (66%) had underlying heart disease, ll (38%) had underlying neurologic disease, 6 (21%) had underlying diabetes mellitus, 28 (97%) reported smoking tobacco products, and 26 (90%) reported using alcohol in the past. Safety of Vaccines Clinical Evaluation: The proportions of subjects in the two vaccine groups with changes in lower respiratory tract symptoms after vaccination did not differ. Increased cough, shortness of breath, and/or sputum production compared to before vaccination were reported by four and six subjects on days 7 to 10 and 21 to 28 after vaccination, respectively, in CAV and TVV recipients, and by six and two subjects, respectively, among recipients of TVV and intranasal placebo. An improvement in one or more of these symptoms compared to before vaccination was reported by t\vo and three subjects on days 7 to 10 and 21 to 28 after vaccination, respectively, among CAV and TVV recipients, and by one and two subjects, respectively, among recipients of TVV and intranasal placebo. Reports of new upper respiratory tract symptoms compared to before vaccination also did not differ bet\veen the vaccine groups (day 7 to 10 postvaccination: five recipients of CAV and TVV compared with five recipients of only TVV; day 21 to 28 postvaccination: five recipients of CAV and TVV compared with one recipient of only TVV). There were no changes in findings on auscultation of the lungs and no febrile reactions.

Pulmonary Function Analysis: The two vaccine groups did not differ in terms of the distribution of subjects by severity of airflow obstruction before and on day 7 to 10 after vaccination. Both groups had preexisting severe obstruction to airflow and mean pulmona1y function did not change significantly during the study (Table 1). Among the seven subjects in each vaccine group who had spirometry performed 21 to 28 days after vaccination because of a worsening in respiratory symptoms or pulmonary function 7 to 10 days after vaccination, mean spirometric values did not change significantly compared to before vaccination (Table 1). Proportions of subjects with changes in obstruction to airflow after vaccination did not differ between the two groups. Among recipients of CAV and TVV, five subjects had a one-category improvement in obstruction to airflow after vaccination and three subjects experienced a one-category decrease. In each of the three CAV and TVV recipients with a one-categmy decrease, the decrement was only 16% from the baseline percent predicted FEV 1 . One of these three subjects had no change in symptoms. One reported a nonproductive cough, mildly increased shortness of breath, and malaise on day 7 to 10 after vaccination, and increased sputum production thereafter not requiring additional treatment. His percent predicted FEV1 returned to baseline on day 21 to 28 after vaccination. Influenza A virus grew from nasopharyngeal throat swabs on days 2 and 7 after vaccination in the third subject (see below). This subject was 75 years old and had a history of ischemic cardiomyopathy with compensated congestive heart failure, insulin-dependent diabetes mellitus, and mild chronic renal insufficiency in addition to COPD. He received an oral antibiotic for symp-

Table !-Spirometric Measures of Lung Function Before and After Vaccination With CAV Coadministered With TVV or TVV With Intranasal Placebo (TVV Alone) Mean Values:+:SE All Subjects Given CAV and TVV (n=l6) or TVV Alone (n=13)

Subjects With Hepeat Spirometry 21 to 28 d Postvaccination* (CAV and TVV: n=7; TVV alone: n=7)

Vaccine Group

Day of Study

Percent Predicted FEV,

FEV/FVC.%

Percent Predicted FEV 1

FEV/FVC.%

CAV and TVV

Prevaccinatiun Day 7-10 1 , Day 21-28' Prevaccination Day 7-10 Day 21-28

43.8:+::4.7 44.1 :+:: 4.9 NA* 43.8:+::7.5 41.2:+::7.0 NA*

60.4 :+::2.9 60.6:+::3.3 NA 56.0:+::5.3 57.1:+::4.2 NA

31>.4::'::6.6 31.9:+::5.8 33.0:+::6.9 38.6:+:: 11.1 33.6:+::10.0 37.7:+:: 10.4

.54.2:+::2.9 52.7 :+::3.4 59.4:+::0.07 53.0:+::4.1 49.2 :+:: 4.8 50.8:+::4.4

TVV alone

*NA=not applicable; seven subjects in each vaccine group had a wurse niug iu respiratory symptoms or pulmonary li.tnction at day 7 to 10 and had repeat spirometly per!rJrm e d at day 21 to 28. Hesults for this subgroup at all study time points are shown on the right half of the Table. 1 Seven to 10 days postvaccination. 'Twe nty-one to 28 days postvaccination.

1224

Clinical Investigations

toms of mildly increased shortness of breath and purulent sputum production on day IO after vaccination. His symptoms resolved by I6 days after vaccination and the percent predicted FEV 1 returned to baseline by 2I to 28 days after vaccination. Without a change in category of obstruction to airflow, two recipients of CAV and TW had 24% and 23% decreases and three experienced a >20% improvement in percent predicted FEV 1 7 to 10 days after vaccination compared to before vaccination. In the two with >20% decreases, the spirometric changes were attributed to differences in the administration of bronchodilator medication prior to spirometry in one subject, and to symptoms of seasonal allergic rhinitis in the other. Among TW and intranasal placebo recipients, one subject experienced a one-category improvement in obstruction to airflow after vaccination and none experienced a decrease in category. Without a change in category of obstruction to airflow, two subjects experienced at least a 20% reduction and one a 40% improvement in percent predicted FEV 1 7 to IO days after vaccination compared to before vaccination. In the two subjects with at least a 20% decrease in percent predicted FEV 1 , one experienced no change in respiratory symptoms and the other subject reported an earache and sore throat 7 to IO days after vaccination. His symptoms resolved and the percent predicted FEV 1 returned to baseline by the 2I to 28-day follow-up visit.

Nasal Shedding of Virus Among the I6 recipients of CAV, one had influenza A (H3N2) virus isolated in titers of 102 and 103 TCID 5 ofmL from upper respiratory tract secretions on both days 2 and 7 after vaccination, respectively. The influenza A viruses retained the temperaturesensitive phenotype of the CAV strains. No influenza A (HINI) virus was detected in either of the two specimens in which influenza virus was isolated.

Anti-influenza A Virus Antibody Responses to Vaccination Among subjects who received CAV and TVV, the reciprocal geometric mean anti-HA IgA antibody titers in nasal wash specimens increased significantly 7 to 10 days after vaccination against HI HA, and at both 7 to IO days and 2I to 28 days after vaccination against H3 HA compared to before vaccination against the respective antigens (p:50.05, Wilcoxon signed ranks test) (Table 2). No significant increases in mean anti-HA IgA antibody titers in nasal washings occurred after vaccination with only TW compared to prevaccination levels (Table 2). The proportion of subjects with any rise in anti-H3 HA IgA

antibody in nasal washings after vaccination was significantly higher among CAV and TW recipients compared to recipients of TVV and intranasal placebo (p<0.05, Fisher's Exact Test; Table 2). The magnitude of the prevaccination reciprocal anti-HA IgA antibody titer in nasal washings was not statistically associated with a postvaccination increase in anti-HA antibody titer in nasal washings (p=not significant [NS], Fisher's Exact Test). There were no significant differences in prevaccination mean anti-HA IgA antibody titer in nasal washings, comparing across the nvo vaccine groups for each HA antigen. A more stringent criterion for a significant change in nasal wash anti-HA IgA antibody in each subject would be a fourfold increase in titer. Using the criterion of a fourfold rise in titer does not allow one to distinguish responses benveen the two vaccine groups as well. Fourfold increases in nasal wash anti-H3 HA IgA antibody titer were observed in 4 of I6 recipients of CAV and TW and in 2 of I3 recipients of the TW and intranasal placebo. The correction of ELISA-measured antibody titers for total IgA concentration in the specimen makes this type of analysis less straightforward. Reciprocal geometric mean titers (GMT) of HAl and anti-HA IgA and IgG antibodies in serum increased significantly after vaccination in both vaccine groups against both HINI and H3N2 viruses compared to prevaccination levels (Tables 2 and 3). The proportions of subjects with fourfold increases in serum antibody titer did not differ statistically between vaccine groups. The magnitude of the prevaccination serum HAl antibody titer was not statistically associated with a postvaccination increase in nasal wash anti-HA IgA antibody level (p=NS, Fisher's Exact Test). A larger proportion of subjects who received CAV and TW experienced a fourfold rise in serum anti-HA IgA antibody titer and a rise in nasal wash anti-HA IgA antibody titer of any magnitude concomitantly (anti-HI HA: 9 [56%] of I6 subjects, and anti-H3 HA: 9 [56%] of I6 subjects) compared to recipients of TW and intranasal placebo (anti-HI HA: 6 [46%] of I3 subjects, and anti-H3 HA: 3 [23%] of I3 subjects). There were no significant differences in prevaccination mean anti-HA or HAl serum antibody titers comparing across the two vaccine groups for each viral antigen, except for a significantly higher prevaccination mean anti-HI HA serum IgA antibody titer in the subjects who received TVV and intranasal placebo compared to the recipients of CAV and TVV (p<0.05, MannWhitney U test). The subject from whose upper respiratory tract secretions CAV was grown had prevaccination reciprocal HAl antibody titers against influenza A/ShangCHEST I 112 I 5 I NOVEMBER, 1997

1225

Table 2-Nasal Wash and Serum IgA Antibody GMT to Influenza A Virus Purified Hl and H3 HAs Before and After Vaccination With CAV Coadministered With TVV or TVV With Intranasal Placebo (TVV Alone), and Numbers of Subjects With Increases in Antibody Titer After Vaccination asal Wash IgA Antibody to HA Reciprocal GMT of Antibody [No. of Subjects With Any Rise in Antibody Titer Postvaccination (Percent of Total)]

Vaccine Group, No.

Influenza A Virus HA

Prevaccination

CAV and TVV, 16

Hl/86

40.2*

H3/89

28.4

H1/86

71.5

H3/89

39.9

TVV Alone, 13

11

Day 7-10 Postvaccination

Day 21-28 Postvaccination

53.4* [12 (75)] 36.3 1 [11 (69)] 102.5 [8 (62)] 37.3 [6 (46)]

53.1 [9 (56)] 48.5 1 [13 (81)] 11 86.2 [4 (31)] 35.3 [3 (23)] 11

Semm IgA Antibody to HA Reciprocal GMT of Antibody [No. of Subjects With a ~4-fold Ri se in Antibody Titer Postvaccination (Percent of Total)]

No. With Rise in Tite r Postvaccination at Either Time Point (%)

Prevaccination

13 (81)

79.3h*

14 (88)1

198.1,

9 (69)

210.8,

6 (46)§

242.2~

Day 7-10 Postvaccination

Day 21-28 Postvaccination

171.31 [8 (50)] 430.5 [9 (56)] 398.9, [6 (46)] 413.0, [5 (38)]

203.7** [9 (56)] 396.21 [8 (50)] 613.1 ~ [7 (54)] 461.4' [5 (38)]

No. With ~4-fold

Rise in Titer Postvaccination at Either Time Point (Percent of Total) 10 (63) 10 (63) 8 (62) 7 (54)

Note: Hl/86, influenza A/Taiwan/1/86 (H l Nl); H3/89, influenza NBeijing/353/89 (H3N2). *p<0.05, Wilcoxon signed ranks test, mean value postvaccination higher than prevaccination. 1p=0.05, Wilcoxon signed ranks test, mean value postvaccination higher than prevaccination. 1p < 0.05, Wilcoxon signed ranks test, mean value postvaccination higher than prevaccination. §p
dong/9/93 (H3N2) and influenza A!faiwan/1186 (HlNl) of 32 and 512, respectively. In this subject, there was a fourfold increase in HAl antibody titer against the H3N2 virus, and fourfold increases in anti-HI and anti-H3 HA serum IgA antibody titer after vaccination. His prevaccination nasal wash anti-HI and anti-H3 HA nasal wash antibody levels were 26 and 51, respectively. The anti-I-ll HA level was below the GMT against Hl HA for the CAV and TVV group as a whole before vaccination, but there were other subjects from whom virus was not grown whose prevaccination nasal wash anti-HA IgA antibody levels were lower than those of this subject. The anti-HI and H3 HA nasal wash antibody levels in the subject from whom CAV was recovered increased postvaccination , but less than fourfold. Production of Cytokines IL-2 was produced at significantly higher levels to all three heat-inactivated influenza A virus antigens after vaccination with CAV and TVV compared to prevaccination (Fig I ), and was highest at day 21 to 28. Mean IL-2 production by PBLs was significantly higher after vaccination compared to prevaccination in response to only two of the three heat-inactivated 1226

influenza A virus antigens in the TVV alone group. The level of statistical significance of the differences between prevaccination and postvaccination levels for these two antigens was lower for the TVV alone group (p<0.05, Wilcoxon signed ranks test) compared to the CAV and TVV group (p
Table 3-Serum Geometric Mean Reciprocal HAl and Anti-HA IgG ELISA Antibody Titers (GMT) to Influenza AI Taiwan/1/86 (HINI), A/Beijing/353189 (H3N2), A/Shangdong/9193 (H3N2) Virus Antigens Before and After Vaccination With CAV Coadministered With TVV, or TVV With Intranasal Placebo (TVV Alone), and Numbers of Subjects With Fourfold Increases in Antibody Titer Postvaccination No. With ~4fold Rise in Serum HAl or anti-HA IgG ELISA HAl Antibody IgG Antibody to HA Measured by ELISA Antibody Tite r Postvaccination Influenza A (Percent of Day 21-28 Day 7-10 Day 21-28 Vaccine Virus Day 7-10 Total) Antigen Prevaccination Postvaccination Postvaccination Prevaccination Postvaccination Postvaccination Group, No. Reciprocal GMT of Serum Antibody [No. of Subjects with ~4- fold Rise in Antibody Titer Postvaccination (Percent of Total)]

CAV and TVV, 16

TVV alone, 13

Hl/86

103*

1-!3

50*

Hl/86

98 1

1-!3

30* 1

128 [1(6)] 61 [1(6)] 218 1 [3(23)] 75 1 [3(23)]

217* [2(13)] 145* [6(38)] 256 1 [6(46)] 159* [8(62)]

190* 1 1,522 1 152 836 1

349 1 [7(44)] 2,348 [4(25)] 209 [2(15)] 1,280 [3(23)]

381* [8(50)] 3,044 1 [6(38)] 232 [3(23)] 1,961 f [6(46)]

10 (63) 9 (56) 6 (46)

8 (62)

Note: 1-!1/86: influenza A/Taiwan/1/86 (H1N l ) whole virus used in HAl assay and purified H1 HA in anti-HA IgG antibody ELISA; 1-!3: influenza A/Shangdong/9/93 (H3N2) whole virus used in HAl assay, purified H3 1-!A from influenza A/Beijing/353/89 (H3N2) virus used in anti-H3 HA IgG antibody ELISA. *p<0.01 , Wilcoxon signed ranks test, mean value postvaccination higher than prevaccination. 1 p<0.05, \Vilcoxon signed ranks test, mean value postvaccination higher than prevaccination.

shown). H1N1 and H3N2 influenza A virus-stimulated mean IL-4levels were significantly higher 21 to 28 days after vaccination with CAV and TVV compared to prevaccination mean levels (Fig 3). As in the case of IL-2, the mean IL-4 levels were highest in response to all influenza A virus antigens at day 21 to 28 for the CAV and TVV recipients. Among subjects who received TVV and intranasal placebo, mean IL-4 levels increased significantly after vaccination compared to prevaccination only upon stimulation with influenza A (H1N1) virus and only at the 7- to 10-day time point, which was earlier than that obseived for the recipients of CAY and TVV (Fig 3). Influenza A (H3N2) virus-stimulated mean IL-4 levels did not increase significantly after vaccination with only TVV compared to prevaccination, and IL-4 levels declined by the day 21 to 28 measurement (Fig 3). The mean IL-4 level induced by the influenza A/93 (H3N2) virus antigen at 21 to 28 days after vaccination with CAV and TVV was significantly higher than that 21 to 28 days after TVV and intranasal placebo (p<0.01, Mann-Whitney U test) . IL-2, IFN-')1, IL-4 levels induced by all three influenza A viruses in vitro were each higher after vaccination compared to prevaccination in the subject from whose upper respiratory tract secretions CAV was grown. Increases in nasal wash anti-HA IgA antibody titer were not statistically associated with concomitant twofold increases in influenza A virus-stimulated

IL-2, IFN-')1, or IL-4 production (p=NS, Fisher's Exact Test). Fourfold increases in serum anti-HA IgG antibody or serum HAl antibody titers were not statistically associated ·with concomitant hvofold increases in influenza A virus-stimulated IL-2, IFN-')1, or IL-4 production (p= NS, Fisher's Exact Test). There were no differences between the hvo vaccination groups in the proportions of subjects with at least a twofold increase in each cytokine level postvaccination compared to prevaccination for each influenza virus antigen used to stimulate PBL, respectively. An association behveen the proportion of all subjects with a twofold increase in IL-2 and a concomitant twofold increase in IFN -')I level after vaccination was observed for all three influenza virus antigens and this achieved statistical significance in the case of in vitro stimulation with the influenza A/93 (H3N2) virus antigen (Table 4). An association behveen the proportion of all subjects with a twofold increase in IL-4 and a concomitant less-than-hvofold increase or decrease in IFN-')1 level after vaccination was observed for all three influenza virus antigens and this achieved statistical significance in the case of influenza A/89 (H3N2) virus antigen (Table 4). Twofold increases in influenza virus antigen-stimulated IL-2 production were not statistically associated vvith concomitant changes in IL-4 production (p=NS, Fisher's Exact Test). No statistical differCHEST I 112 I 5 I NOVEMBER, 1997

1227

"HINl/86 o HJN2/89 • H3N2/93 o medium control

.... ..,;

250

+I -a;

200

c-o:::::i'

ISO

...."'

- CAV t TVV --- TVV alone

" HINI/86 o H3N2/89 • HJN2/93 o medium control

• P< 0.05 t P
- CAV t TVV -- - TVValone • P=0.06 I P<0.05

2500

>

.5~

-"'en :::t a.

-!- 100 ~

-=..c:

...

~

50 0

Prdaccination

Day 7-10 Post-Vaccination

Day 21-28 Post-Vaccination

Study Day FIGURE l. Mean (±SE) cytokine levels measured by ELISA in supernatant fluids of PBLs stimulated by influenza A/Taiwan/ 1/86 (H1N1) (triangles), influenza NBeijing/353/89 (H3N2) (open circles), and influenza NShangdon!V9/93 (I-I3N2) (closed circles), or unstimulated (medium control} (boxes) are shown as a function of study day for each vaccine group (solid lines = CA V coadministered 'Nith TVV; dashed lines=TVV coadministered with intranasal placebo). Mean (± SE) IL-2 levels produced by PBLs, which were cultured with anti-IL-2 receptor antibody and stimulated with each of the three heat-inactivated influenza A viruses, or incubated in medium as a control, are shown (asterisk: p
ences in these associations between cytokine responses were observed between the two vaccination groups.

DISCUSSION

The major findings in this study were the safety of bivalent CAV when coadministered with TVV in subjects with underlying COPD, and the suggestion that the levels of anti-HA IgA antibodies in nasal washings and influenza virus-stimulated IL-2 and IL-4 production were enhanced by coadministration of CAV with TVV compared to TVV and intranasal placebo. Mean pulmonaq function did not change after vaccination with CAV. There was also a lack of significant differences in clinical symptoms between the two vaccine groups. There were signihcantly increased mean levels of IgA antibody to both Hl 1228

Pre-Vaccination

Day 7-10 Post-Vaccination

Ooy 21-28 Post-Vaccination

Study Day FIGURE 2. Mean (±SE) IFN-"1 levels produced by PBLs, which were stimulated with each of three heat-inactivated influenza A viruses, or incubated in medium as a control, are shown (asterisk: p=0.06; dagger: p
and H3 HA postvaccination compared to prevaccination in nasal washes of subjects who received both CAV and TVV, but not among recipients ofTVV and intranasal placebo, despite an increase in mean serum anti-HA IgA antibodies postvaccination in both groups. This indicates that the mucosal IgA antibody response among CAV and TVV recipients was most likely due to the CAV component. The use of purifled Hl HA from influenza Affaiwan/l/86 (HlNl) virus, which is antigenically more similar to the influenza A/Kawasakl/9/86 (HlNl) virus in CAV, to detect anti-Hl HA antibodies, and purified H3 HA from influenza A/Beijing/353/89 (H3N2) virus, which is homologous to the CAV strain, to detect anti-H3 HA antibodies by ELISA may have biased against the detection of anti-Hl and H3 HA antibodies induced by influenza Affexas/ 36/91 Hl HA and A/Shangdong/9/93 H3 HA present in TVV. There is antigenic variation between the two HlNl strains and the two H3N2 strains reflected by a fourfold difference in the HAl titer of ferret antiserum raised against influenza Affaiwan/1/86 virus when the antiserum is tested against influenza A!fexas/36/91 virus, and at least a fourfold difference in the titer of ferret antiserum raised against A/Shangdong/9/93 virus when the antiserum is tested against the influenza A/Beijing/353/89 virus antigen.58-60 However, assays using the Hl HA of influClinical Investigations

"H1Nl/86 o H3N2/89 • H3N2/93 o medium control

Pre-Vaccination

-CAV+ TVV --- TVV alone • P
Day 7-10 Post-Vaccination

·t

Day 71-78 Post-Voccinotian

Study Day FIGURE 3. Mean (±SE) IL-4 levels produced b y PBLs, which were stimulated with autologous PBLs infected with each of the three influenza A viruses, or unstimulated in medium alone are shown (asterisk: p< 0.05, postvaccination value higher than respective prevaccination value, Wilcoxon signed ranks test). Cell supernatant fluids were harvested after 168 h for later IL-4 assay. Samples were available for measurement of IL-4 from 15 recipients of CAY and TYY, and 12 recipients of TVV and intranasal placebo. A higher mean IL-4 level stimulated by each influenza virus antigen, respectively, among recipients of CAY and TYY compared to TVV and intranasal placebo was observed at day 21 to 28, reaching statistical signiflcance in the case of the H3N2193 antigen (dagger: p < 0.01 ). This indicates, perh aps, at least a later peak in the IL-4 response following CAY and TVY compared to TVV and intranasal placebo.

enza A!faiwan/l/86 (HlNl) and the H3 HA of influenza A/Beijing/353/89 (H3N2) were capable of detecting significant increases in anti-Hl and anti-H3 HA IgA and IgG antibody titers in serum, which were most likely stimulated by the Hl and H3 HA present in TVV. The current study is an important step beyond previous studies that evaluated monovalent live attenuated influenza A virus vaccines in patients with COPD.l3,19,20,24,6l Lack of significant changes in clinical parameters after vaccination in the current study suggests that bivalent live attenuated influenza A virus vaccine is also safe in subjects with COPD, at least when coadministered with TVV. It is possible that TVV induced a rapid anamnestic immune response to influenza virus antigens and in so doing reduced the likelihood of adverse reactions due to CA V. Administration of CA V alone would better address the safety of this product in patients with COPD. The isolation of CAV from culture of respiratmy tract secretions of only one subject is unexplained by the level of his prevaccination antibody titers, although his anti-Hl HA antibody titer in nasal washings before vaccination was below the mean for the group. He was also capable of mounting antibody

and cytokine responses to vaccination. His older age and multiple underlying chronic health problems may have predisposed him to having higher, detectable levels of virus replication in his nasal secretions. In earlier studies of patients with COPD, monovalent live attenuated influenza A virus vaccines induced anti-HA antibody in nasal secretions _l3- 20 The mucosal antibody responses to the CAV vaccine in this study were as good as or better than those seen in chronically ill, elderly nursing home residents who were vaccinated with the same bivalent live attenuated virus vaccine. 14 Immunogenicity of CAV was demonstrable in the current study despite the low rate of virus recovery from cultures of respiratory secretions. Since CAV was undetectable by conventional culture techniques in all but one subject, viral replication in culture-negative subjects must have occurred at low levels and for a short duration of time after immunization, if at alL While unlikely, the possibility that intranasal inoculation of CAV after first inactivating it could have induced a similar immune response cannot be ruled out by our study. Besides the effects of the intranasal route of administration and the live viruses in CAV, another possible reason for better immunogenicity with the combined regimen could be an antigen dose effect, since in essence a "double dose" of vaccine was given to recipients of CAV and TVV compared to the recipients of TVV and intranasal placebo. In a study that did not utilize intranasal vaccination, Keitel et al62 reported that increasing the dose of antigen given IM, up to nine times the standard dose, resulted in increased frequencies of rises in anti-HA IgG and IgA antibodies in nasal washings of elderly subjects. Anti- HA IgA antibodies in respiratory tract secretions have correlated with protection from influenza A virus infection, 16-26-27 although other factors, such as serum anti-HA IgG and HAl antibodies and cellular responses, are also important contributors to protection from influenza A virus infection _l7,26-28 Monovalent live attenuated influenza A virus vaccine given alone and bivalent CAV coadministered with TVV have induced higher levels of memory-inducible anti-influenza A virus cytotoxic T-cell activity compared to inactivated virus vaccine given alone to subjects with underlying COPD and chronically ill subjects residing in nursing homes.1 1-12 The better mucosal IgA antibody and anti-influenza A vims cytotoxic T -cell responses to CAV in chronically ill older adults suggest that CAV will contribute to higher levels of protective immunity in these patients. Increases in influenza A virus-stimulated mean IL-2 and IL-4 levels after vaccination compared to prevaccination were observed at greater levels of CHEST I 112 I 5 I NOVEMBER, 1997

1229

Table 4-Associations of Concomitant Changes in IFN-y Production With Changes in IL-2 and IL-4 Production After Vaccination, Combining All Subjects in Both Vaccine Groups Change in Level Postvaccination Compared to Cytokine Measured !)revaccination IL-2 IL-4*

2:2-fold <2-fold 2:2-Jold <2-fOld

increase increase increase increase

No. of Subjects With the Indicated Change in IFN-'Y Level Postvaccination Compared to Prevaccination H3N2/89

HlNl/86

H3N2/93

2:2-fold Increase <2-fold Increase 2: 2-fold Increase <2-fold Increase ::::2-(()ld Increase <2-fold Increase 13 8 8 11

3 5 5 3

7 6 21 91

6 lO ll'

st

10' 3' 5 7

5' ll ' ll 4

Note: The virus antigens used to stimulate the PBLs i11 vitro were HlNl/86, influenza A/Taiwan/l/86 (HlN1 ); I-I3N2189, influenza A/Beijing/353/89 (H3N2); and H3N2/93, influenza A/Shangdong/9/93 (H3N2). In each subject, the increase in cytokine level compared to prevaccination may have occurred at day 7 to lO and/or day 2I to 28. The category of <2-fold increase includes subjects whose postvaccination cytokine levels increased less than 2-fOid and whose levels decreased compared to prevaccination. *Supernatant fluids were not available for IL-4 measurement from two subjects, one in each of th e two vaccine groups. 10f 15 subjects with a 2: 2-fold increase in IL-2, 10 also had a 2:2-fold increase in IFN-'Y compared to 14 subjects without a 2:2-fold increase in IL-2, of whom three had a 2:2-fold increase in IFN-'Y level; IO of 15 vs 3 of 14, p<0.05, Fisher's Exact Test. lof 13 subjects with a 2:2-fold increase in IL-4, two also had a 2:2-fold increase in IFN-'Y compared to 14 subjects without a 2:2-/0id increase in IL-4, of whom 9 had a 2:2-fold increase in IFN-'Y level; 2 of 13 vs 9 of 14, p<0.05, Fisher's Exact Test.

statistical significance, to more viral antigens, and at a later time point in subjects who received CAV and TVV compared to TVV and intranasal placebo. This may reflect a more complex immune regulatory response to the combined regimen and a longerlasting change in the T -cell memory response due to the live virus vaccine component. Mean levels of IFN -"{ production increased in both vac~ine groups to influenza A (HlNl) virus antigen stimulation at day 21 to 28 compared to respective prevaccination levels. In previous studies, influenza A virus-stimulated IFN-"{ production did not increase in chronically ill subjects after vaccination with monovalent live attenuated and inactivated influenza A virus vaccines administered alone, 12 but did increase in healthy young adults after vaccination with trivalent live attenuated influenza virus vaccine.m The association we observed between concomitant increases in IL-2 and IFN -"{ is consistent with the concept that both cytokines represent a THl cell response. The inverse relationship between changes in IFN -"{ and IL-4 production after vaccination is also consistent with a dichotomy between THl and Tl-12 responses to vaccination. However, this inverse relationship did not hold true for comparisons of changes in IL-2 and IL-4 production after vaccination, between which there was a lack of statistical associations. This may be due in part to the proposed better stimulation of both IL-2 and IL-4 production after immunization with CAV and TVV compared to TVV given alone based on comparisons of group means of cytokine levels measured prevaccination and postvaccination. The lack of a well-defined change in cytokine production after vaccination in individual subjects that correlates with biological significance, the numbers of subjects in each vaccination group, and the study 1230

design do not allow further exploration of these relationships based on this study. Little is known about the clinical significance and relative contributions of THl and TH2 cytokines in human immunity to influenza A virus infection. The IL-2 response is indicative of helper T-cell memory for influenza virus antigen, which can also be measured in part by antigen-stimulated lymphocyte proliferation. Enhanced IL-2 production after influenza virus vaccination of elderly subjects \vith split and whole inactivated influenza virus vaccines has been reported,33-35 and vaccination of chronically ill older adults with monovalent live attenuated influenza A virus vaccines has resulted in increased levels of influenza A virus-specific lymphocyte proliferation. 24 IL-2 is produced by THl cells and is known to mediate help for B-cell production of virus-specific antibodies and expansion of antiviral cytotoxic T -cell populations. 32 ·64-6 7 This suggests a facilitative role for IL-2 in vaccine-induced immune responses that are thought to be immune correlates of protection from influenza A virus infection and severe influenza illness. IL-4 is produced by TH2 cells and promotes IgGl, IgA, and IgE antibody production by B cells. It can somewhat paradoxically serve as a late signal, augmenting human anti-influenza A virus cytotoxic T-cell activity and differentiation, and murine and human cytotoxic T -cell activity in mixed leukocyte culture, despite its down-regulatory effects on IFN-"{ production. 39-45 It appears from studies of murine influenza A virus pneumonia that TH2 cytokines play an important immune regulatory role in mucosal effector tissues, that IL-4 treatment early after infection may delay clearance of influenza A virus from lungs of infected mice, and that both THl and TH2 cytokines are produced in the lung and regional Clinical Investigations

lymph nodes during influenza pneumonia. 39,41,68-7l Hence, better characterization of human cytokine responses to influenza virus vaccines, as in the current study, may contribute to a better understanding of human immunity to influenza viruses. Stimulation of anti-HA mucosal IgA antibody, THl and TH2 cytokine production, and anti-influenza A virus cytotoxic T-cells by live attenuated influenza A virus vaccines with and without coadministered inactivated virus vaccines in this and earlier studies 11 - 14·20 demonstrates a broader immune response when live virus is included in the immunization regimen. This has clinical importance since live attenuated virus vaccine is associated with equal or better efficacy compared to inactivated virus vaccine.l0·11·29·30 The protective efficacy of monovalent live attenuated influenza A virus vaccine coadministered with TVV in chronically ill elderly institutionalized subjects reported by Treanor et al 10 and our findings suggesting that the immunogenicity of CAV and TVV is superior to that of TVV administered alone are reasons to further evaluate the combined regimen in the chronically ill elderly and patients with COPD. When trivalent live attenuated influenza virus vaccine that incorporates viruses antigenically similar to the licensed inactivated virus vaccine for the same year becomes available, it will offer an additional, attractive vaccination regimen for investigational evaluation of protective efficacy in chronically ill older adults. ACKNOWLEDGMENTS: We thank Dominick Iacuzio, Carole Heilman, and Robert B. Belshe for assistance and helpful suggestions; Joseph Messina (Wyeth-Ayerst Research, Radnor, Pa) and Maurice Harmon (Connaught Laboratories, Swiftwater, Pa) for providing purified HA reagents; Heidi Israel for nursing assistance; Kathleen N. Gillespie and Sharon Homan for statistical assistance; Mark Campbell for technical laboratory assistance; Laura McDurmont for assistance \vith electronic database management; and Carolyn Novotny for expert manuscript preparation.

REFERENCES 1 Advisory Committee on Immunization Practices. Prevention and control of influenza. MMWR 1993; 42(No. RR-6 ):1-14 2 Eickhoff TC, Sherman IL, Serfling RE. Observations on excess mortality associated with epidemic influenza. JAMA 1961; 176:776-82 3 Monto AS. Influenza: quantifying morbidity and mortality. Am J Med 1987; 82(suppl 6A):20-25 4 Barker WH, Mullooly JP. Impact of epidemic type A influenza in a defined adult population. Am J Epidemiol 1980; 112:798-811 5 Glezen WP, Decker M, Perrotta DM. Survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in Houston, 1978-1981. Am Rev Respir Dis 1987; 136:550-55 6 Foster DA, Talsma AN, Furumoto-Dawson A, et al. Influenza vaccine effectiveness in preventing hospitalization for pneumonia in the elderly. Am J Epidemiol 1992; 136:296-307

7 Nichol KL, Margolis KL, Wuorenma J, et al. The efficacy and cost effectiven ess of vaccination against influenza among elderly persons living in the community. N Eng! J Med 1994; 331:778-84 8 Gross PA, Quinnan GV Jr, Weksler ME, et al. Relation of chronic disease and immune response to influenza vaccine in the elderly. Vaccine 1989; 7:303-08 9 Strassburg MA, Greenland S, Sorvillo FJ, et al. Influenza in the elderly: report of an outbreak and a review of vaccine effectiveness reports. Vaccine 1986; 4:38-44 10 Treanor JJ, Mattison HR, Dumyati G, et al. Protective efficacy of combined live intranasal and inactivated influenza A virus vaccines in the elderly. Ann Intern Med 1992; 117:625-33 11 Gorse GJ, Campbell MJ, Otto EE, et al. Increased antiinfluenza A virus cytotoxic T cell activity following vaccination of the chronically ill elderly with live attenuated or inactivated influenza virus vaccine. J Infect Dis 1995; 172:1-10 12 Gorse GJ, Belshe RB. Enhancement of anti-influenza A virus cytotoxicity follO\ving influenza A virus vaccination in older, chronically ill adults. J Clin Microbial 1990; 28:2539-50 13 Gorse GJ, Belshe RB, Munn NJ. Superiority of live attenuated compared with inactivated influenza A virus vaccines in older, chronically ill adults. Chest 1991; 100:977-84 14 Gorse GJ, Otto EE, Powers DC, et al. Induction of mucosal antibodies by live attenuated and inactivated influenza virus vaccines in the chronically ill elderly. J Infect Dis 1996; 173:285-90 15 Belshe RB, Van Voris LP, Bartram J, et al. Live attenuated influenza A virus vaccines in children: results of a field trial. J Infect Dis 1984; 150:834-40 16 Johnson PR, Feldman S. Thompson JM, et al. Immunity to influenza A virus infection in young children: a comparison of natural infection, live cold-adapted vaccine, and inactivated vaccine. J Infect Dis 1986; 154:121-27 17 Clements ML, Betts RF, Murphy BR. Advantage of live attenuated cold-adapted influenza A virus over inactivated vaccine for A/Washington/SO (H3N2) wild-type virus infection. Lancet 1984; 1:705-08 18 Clements ML, Murphy BR. Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine. J Clin Microbial 1986; 23:66-72 19 Gorse GJ, Belshe RB, Munn NJ. Safety of and serum antibody response to cold-recombinant influenza A and inactivated trivalent influenza virus vaccines in older adults with chronic diseases. J Clin Microbial 1986; 24:336-42 20 Gorse GJ, Belshe RB, Munn NJ. Local and systemic antibody responses in high-risk adults given live-attenuated and inactivated influenza A virus vaccines. J Clin Microbial 1988; 26:911-18 21 Powers DC, Sears SD, Murphy BR, et al. Systemic and local antibody responses in elderly subjects given live or inactivated influenza A virus vaccines. J Clin Microbiol1989; 27:2666-71 22 Powers DC, Fries LF, Murphy BR, et al. In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory. J Clin Microbial 1991; 29:498-505 23 Zahradnik JM , Kasel JA, Martin RR, et al. Immune responses in serum and respiratory secretions following vaccination with a live cold-recombinant (CR35) and inactivated A/USSR/77 (H1N1 ) influenza virus vaccine. J Med Virol1983; 11:277-85 24 Gorse GJ, Belshe RB. Enhanced lymphoproliferation to influenza A virus follo\ving vaccination of older, chronically ill adults with live-attenuated viruses. Scand J Infect Dis 1991; 23:7-17 CHEST I 112 I 5 I NOVEMBER, 1997

1231

25 Ennis FA, Yi-Hua Q, Schild GC. Antibody and cytotoxic T lymphocyte responses of humans to live and inactivated influenza vaccines. J Gen Virol 1982; 58:273-81 26 Hay, AJ, Belshe RB, Anderson EL, et al. Influenza viruses. In: Belshe RB, ed. Textbook of human virology. 2nd ed. St.Louis: Mosby-Year Book, 1991; 307-41 27 Clements ML, Betts RF, Tierney EL, et a!. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A \vild-type virus. J Clin Microbial 1986; 24:157-60 28 McMichael AJ, Gotch FM, Noble GR, et al. Cytotoxic T cell immunity to influenza. N Eng! J Med 1983; 309:13-17 29 Clements ML, Betts RF, Tierney EL, et a!. Resistance of adults to challenge with influenza A \vild-type virus after receiving live or inactivated virus vaccine. J Clin Microbial 1986; 23:73-76 30 Edwards KM, Dupont WD, Westrich MK, et al. A randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza A disease. J Infect Dis 1994; 169:68-76 31 Mossman TR, Coffman RL. THI and TH2 cells: different patterns of lymphokine secretion lead to different functional prope rties. Ann Rev Immunoll989; 7:145-73 32 Goldsmith MA, Greene WC. Inte rleukin-2 and the interleukin-2 receptor. In: Thomson AW, ed. The cytokine handbook. London: Academic Press, 1994; 57-80 33 McElhaney JE, Meneilly GS, Beattie BL, et al. The effect of influenza vaccination on 1L2 production in healthy elderly: implications for current vaccination practices. J Gerontal 1992; 47:M3-8 34 McElhaney JE, Meneilly GS, Lechelt KE, et al. Split-virus inlluenza vaccines: do they provide adequate immunity in the elderly? J Gerontol1994; 49:M37-43 35 McElhaney JE, Meneilly CS, Pinkoski MJ, et a!. Vaccinerelated determinants of the interleukin-2 response to influenza vaccination in healthy young and elderly adults. Vaccine 1995; 13:6-10 36 Locksley RM. Interleukin 12 in host defens e against microbial pathogens. Proc Natl Acad Sci USA 1993; 90:5879-80 37 Scott P. IL-12: initiation cytokine for cell-mediated immunity. Science 1993; 260:496-97 38 De Maeyer E, De Maeyer-Guignard J. Interferons. In: Thomson AW, ed. The cytokine handbook. London: Academic Press, 1994; 265-88 39 Baumgarth N, Brown L, Jackson D , et al. Novel features of the respiratory tract T-cell response to influenza virus infection: lung T cells increase expression of gamma interferon mRNA in vivo and maintain high levels of mRNA expression for interleukin-5 (IL-5) and IL-10. J Virol 1994; 68:7575-81 40 Boom WI-I, Liano D , Abbas AK. Heterogeneity of helper/ inducer T lymphocytes: II . Effects of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes. J Exp Med 1988; 167:1350-63 41 Murray PD, McKenzie DT, Swain SL, et al. lnterleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression. J Immunol 1987; 139:2669-75 42 Banchereau J, Rybak ME. Interleukin-4. In: Thomson AW, ed. The cytokine handbook. London: Academic Press, 1994; 99-126 43 Horohov DW, Grim JA, Smith PL, et a!. IL-4 (B cellstimulatory factor l ) regulates multiple aspects of influenzaspecific cell-mediated immunity. J Immunol 1988; 141:4217-23 44 Widmer MB, Grabstein KH. Regulation of cytolytic Tlymphocyte generation by B-cell stimulatmy factor. Nature 1987; 326:795-98 1232

45 Widme r MB , Acres RB, Sassenfeld HM , e t a!. Regulation of cytolytic cell populations from human peripheral blood by B cell stimulatmy factor I (interleukin 4). J Exp Med 1987; 166:1447-55 46 Maassab HF. Biologic and immunologic characteristics of cold-adapted influenza virus. J Immunol 1969; 102:728-32 47 Cox NJ, Maassab HF, Kendal AP. Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: non-random reassortment of genes during preparation of live virus vaccine candidates by recombination at 25° between recent H3N2 and H1N1 epidemic strains and cold-adapted A/Ann Arbor/6/60. Virology 1979; 97:190-94 48 American College of Chest Physicians. Report of the Section on Respiratmy Pathophysiology: statement on spirometJy. Chest 1983; 83:547-50 49 Gardner RM. Standardization of spirometry: a summary of recommendations from the American Thoracic Society; the 1987 update. Ann Intern Med 1988; 108:217-20 50 Knudson RJ, Lebowitz MD, Holberg CJ, et al. Changes in the normal maximal expiratory flow-volume curve with grovvth and aging. Am Rev Respir Dis 1983; 127:725-34 .51 Medical Section of the American Lung Association. Lung function testing: selection of reference values and interpretative strate~Pes. Am Rev Respir Dis 1991; 144:1202-18 52 Belshe RB, Van Voris LP, Mufson MA. Parenteral administration of live respiratmy syncytial virus vaccine: results of a field trial. J Infect Dis 1982; 145:311-19 53 Reed LJ, Muench H. Simple method of estimating 50 percent endpoints. Am J Hyg 1938; 27:493-97 54 Dowdle WR, Kendal AP, Noble CR. Influenza viruses. In: Lennette EH , Schmidt NJ, eds. Diagnostic procedures for viral, lickettsial, and chlamydia! infections. 5th ed. Washington , DC: American Public Health Association, 1979; 585-609 55 Murphy BR, Phelan MA, Nelson DL, et al. Hemagglutininspecific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses. J Clin Microbial 1981; 13:554-60 56 Murphy BR, Nelson DL, Wright PF, et al. Secretory and systemic immunological response in children infected with live-attenuated influenza A virus vaccines. Infect Immun 1982; 36:1102-08 57 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Sc
64 Scherle PA, Gerhard W. Functional analysis of influenzaspecific helper T cell clones in vivo, T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. JExp Med 1986; 164:1114-28 65 Swain SL, Dennert G, Warner JF, et al. Culture supernatants of a stimulated T-cell line have helper activity that acts synergistically with interleukin 2 in the response of B cells to antigen. Proc Nat! Acad Sci USA 1981; 78:2517-21 66 Palladino G, Scherle PA, Gerhard W. Activity of CD4+ T-cell clones of type 1 and type 2 in generation of influenza virusspecific cytotoxic responses in vitro. J Viral 1991; 65:6071-76 67 Braakman E, Treep-Van Leeuwen P, Roosnek EE, e t al. The role of IL-2 and T4+ cells in the generation of human influenza virus-specific CTL activity. Cell Immunol 1986; 100:462-73

68 Sarawar SR, Doherty PC. Concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice \vith influenza pneumonia. J Virol1994; 68:3112-19 69 Carding SR, Allan \V, McMickle A, et al. Activation of cytokine genes in T cells during primary and secondary murine influenza pneumonia. J Exp Med 1993; 177:475-82 70 Graham MB , Braciale VL, Braciale TJ. Influenza virusspecific CD4+ T h elper type 2 T lymphocytes do not promote recovery from experimental virus infection. J Exp Med 1994; 180:1273-82 71 Moran TM , Isobe H , Fernandez-Sesma A, et al. Interleukin-4 causes d elayed viral clearance in influenza virus-infected mice. JVirol 1996; 70:5230-34

CHEST I 112/5 I NOVEMBER, 1997

1233