Live Attenuated Influenza A Virus Vaccines in Children: Results of a Field Trial

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 150, NO.6 DECEMBER by The University of Chicago. All rights reserved /84/ $01.00 Live Attenuated Influenza A Virus Vaccines in Children: Results of a Field Trial Robert B. Belshe, Lee P. Van Voris, Julie Bartram, and Frances K. Crookshanks From the Center for Vaccine Development, Department ofmedicine, Marshall University School ofmedicine; and Research Service, Huntington Veterans Administration Medical Center, Huntington, West Virginia One hundred three young children were inoculated intranasally with either influenza AI California/10/78 cold-recombinant vaccine ( /0 tissue culture infective doses [TCIDsol per child), CR-37 (HINI), or influenza A/Washington/897/80 cold-recombinant vaccine (10 6 s. TCIDsoper child), CRA8 (H3N2). The vaccine was welltolerated. Of the 51 children vaccinated with CR-37 (HINI), 45 were initially seronegative for this virus; 33 of the 45 became infected with the vaccine virus, as indicated by a fourfold rise in antibody titer or by shedding of vaccine virus. Of the 52 children vaccinated with CR-48 (H3N2), six were initially seronegative and all were infected; 46 were initially seropositive and 25 of these children developed fourfold rises in antibody titer. An outbreak of influenza A (H3N2 type predominated) occurred in Huntington one to three months after the children were vaccinated. Significantly fewer febrile illnesses occurred in the CR-48 (H3N2) vaccine group than among the CR-37 (HIN1) vaccine group. Hemagglutination inhibition antibody (titer, ~I :32) to H3N2 was significantly associated with protection from illness and infection with influenza A/H3N2. Cold-recombinant influenza A viruses have been evaluated previously in several clinical trials for their potential as live attenuated vaccines [1-7]. Recent cold-recombinant influenza vaccines have been genotyped and found to consist of reassortants containing novel "donor" hemagglutinin and neuraminidase genes with the six genes that code for the internal proteins derived from the parent cold-adapted virus, influenza AIAnn Arbor/6/60 [8]. Reassortant viruses with six genes from the cold-adapted parent exhibit genetic stability after prolonged replication in children; Received for publication April 16, 1984, and in revised form July 10, Informed consent was obtained from the parents of study participants. The protocol was approved by the Institutional Review Board of Marshall University and by the Clinical Review Committee of the National Institute of Allergy and Infectious Diseases. This research was supported by contract no. NOI-AI from the National Institute of Allergy and Infectious Diseases. The authors acknowledge the assistance of Lynn Plagge, Sally Wells, Arlene Napier, Carol Berry, Pamela Feaganes, Carol Stout, Betty Burk, and Cheryl Stokes in conducting this investigation. Please address requests for reprints to Dr. Robert B. Belshe, Department of Medicine, Marshall University School of Medicine, Huntington, West Virginia they do not cause adverse reactions, and only a small quantity of vaccine virus is required to infect most seronegative children [7]. Two studies [5, 7] have suggested that these vaccines would be effective in preventing influenza. Children who had been vaccinated intranasally with a cold-recombinant HINI virus (influenza A/ California/10/78, CR-37) were protected from experimental challenge with high-dose CR-37 (HINt) six months after primary vaccination [7]. In Wright's study [5], children who had participated in safety and infectivity studies with CR-29 (H3N2 reassortant derived from influenza A/Alaska/6/77 and cold-adapted influenza A/Ann Arbor/6/60) were protected from infection when influenza A/Bangkok (H3N2) circulated and caused naturally acquired infection in the community. In addition to the desirable characteristics of the cold-recombinant vaccines, these observations suggested that cold-recombinant vaccines should be evaluated in an efficacy field trial. Materials and Methods Vaccines. Two cold-recombinant influenza A vaccines were evaluated in this study: CR

2 Efficacy ofinfluenza A Vaccine 835 (HINI) and CR-48 (H3N2). CR-37 (HINI) was composed of the hemagglutinin and neuraminidase of influenza A/California/IO/78, and CR-48 (H3N2) contained the hemagglutinin and neuraminidase derived from influenza A/Washington/ 897/80. Each vaccine contained the six genes coding for internal proteins from the donor coldadapted influenza AIAnn Arbor/6/60. Derivation of the donor virus and cold-recombinant viruses have been described elsewhere [8-10]. The CR-37 (HINI) vaccine (lot E-167) contained TCIDso/ml and the CR-48 (H3N2) vaccine (lot E-179) contained TCIDso/ml. The vaccines were diluted 10-fold in Leibowitz's medium (L-15) without ph indicator, snap frozen, and stored at -70 C until used. One-half milliliter was administered to each vaccinee; thus, the quantity of vaccine administered to each volunteer was TCIDsoofCR-37 (HINI) or TCIDsoofCR-48 (H3N2). This quantity was at least 100x the minimal virus dose required to infect seronegative children. The titer of frozen vaccine did not change during the study. Patient population and vaccination of volunteers. Children residing in Huntington, West Virginia and its environs participated in the study. Healthy children, 12 to 72 months of age, were enrolled in this study during routine visits to their pediatricians for health maintenance. Each child had normal urinalysis, complete blood cell count, chest roentgenogram, and a negative tine test before intranasal administration of a vaccine. Children were assigned to either the CR-37 (HINI) or CR-48 (H3N2) vaccine groups according to a table of random numbers. Neither the children's mothers nor the vaccine study nurses knew which vaccine was administered. The vaccine key was available only to one of the investigators, and it was not consulted until the study was completed. Each child was examined by a pediatrician before administration of the vaccine. A swab sample of respiratory secretions for the isolation of viruses was obtained before administration of the vaccine. In this way, children with a viral infection at the time of immunization were identified. Each child was placed in the recumbent position and vaccine was instilled one drop at a time in alternate nares until 0.5 ml had been administered. Children were kept in the recumbent position for 0.5 min to ensure contact of the vaccine with the nasopharyngeal mucosa. Vaccinations were administered from October 18; 1982, through December 10, Adverse reactions to vaccine. Study personnel visited each child three and six days after vaccination to examine the child for possible adverse reactions to the vaccines and to obtain nasal and throat swab specimens for isolation ofvaccine viruses. In addition, each vaccinee's mother was asked to mark a list of potential adverse reactions daily during the week after the vaccination and to notify study personnel if and when adverse reactions occurred. The list included measurement of fever and observation for runny nose, cough, earache, sore throat, red eyes, and congestion. When any symptoms were noted by the mother, the study personnel visited the child to make independent observations of the possible adverse reaction. Long-term follow-up. Long-term follow-up of vaccinees for respiratory illness also was performed. Each mother was asked to notify the study personnel when her child became ill with any type of acute respiratory illness. We contacted each mother monthly to reinforce our desire for longterm follow-up. A team of study personnel consisting of a registered nurse and a licensed practical nurse visited each vaccinee in his or her home to examine the child at the time of each reported illness and to obtain specimens for the isolation of viruses. Serological tests and isolation of viruses. Serum specimens were obtained from each vaccinee before administration of either CR-37 (HINI) or CR-48 (H3N2), at four weeks postvaccination (preinfluenza A outbreak), and four to six months (postinfluenza A outbreak) after vaccine administration. The sera were stored at - 20 C until testing for antibody to HINI or H3N2. Antibody levels were determined with use of HAl [11]. Each pre- and postvaccine serum pair was tested by the hemagglutinin-specific ELISA assay, as previously descri bed [12]. Swab specimens were obtained by rubbing the throat and nasal mucosa with a cotton-tipped applicator and then agitating the applicator in vealinfusion broth. The veal-infusion broth was transported on wet ice to the laboratory for inoculation onto tissue culture cells. Throat swab specimens were not frozen before testing for viruses. Each specimen was inoculated onto

3 836 Belsheet al. primary rhesus monkey kidney or Madin-Darby canine kidney cells with trypsin overlay, HEp-2 cells, and MRC-5 cells. Viruses were identified as previously described [13]. Statistical tests. For two-by-two comparisons, X 2 analyses were done. Distributions were compared with use of the Kolmolgorow-Smirnov statistic, D. [14]. Significance in the difference in proportions was calculated with Williams correction, Gadj [15]. Student's t test was used to compare the characteristics of the two vaccine populations. Results Characteristics of the study population. One hundred three children were vaccinated with either CR-37 (HINl) or CR-48 (H3N2). Of the 51 children given the CR-37 (HINl) vaccine, seven were months of age, two were months of age, 20 were months of age, 15 were months of age, and seven were months of age. Among the 52 children in the CR-48 (H3N2) vaccine group, ten were aged months, ten were months, 16 were months, 11 were months, and three were months of age. There was no significant difference in the age distribution between the two groups (D =.2; P, not significant). Other factors, including the number of siblings (mean, 1.0 and 1.1, CR-37 (HINl) vs. CR-48 (H3N2) group, respectively); economic status (mean income $14,000- Table 1. $20,000/year, both groups); urban residence (800/0 vs, 720/0); attendanceat day care (480/0 vs. 57%); or number who had been breastfed (52% vs. 48%) were not significantly different (Student's t test or X 2 ; P, not significant), Clinical response to cold-recombinant vaccines. Fever (oral temperature, >100 F) occurred in five of the 103 children during the six days after the intransal administration of a vaccine. Three children who had fever were shedding another virus (one each of parainfluenza, adenovirus, and enterovirus). Two children had fever but did not shed a virus. One child (maximal oral temperature, F, for one day) had an antibody response to the CR-37 (HINl) vaccine. The other child (maximal oral temperature, F, one day; afebrile on all other days) did not have an antibody response to the CR-48 (H3N2) vaccine. Antibody response to vaccines. For evaluation of the results of the field trial, children were divided into two groups according to the vaccine received and further subdivided into categories (A H) according to their initial antibody status and their antibody response to the respective vaccine (table 1). Among the 51 children given CR-37 (HINl) vaccine, 45 were initially HAl-antibody negative (categories A plus B). Thirty-two (category B) of these 45 manifested a fourfold rise in HAl-antibody response, and one other had a fourfold rise in antibody titer (by ELISA) but no HAItiter rise. The geometric mean HAl-antibody titer HAl-antibody status before and response after intranasal vaccination with CR-37 or CR-48. Geometric mean reciprocal antibody titer to respective hernagglutininl Vaccine, initial HAI- Infected with Category/ antibody status* vaccine virus t no. of children Prevaccine Postvaccine CR-37 (HINl) Seronegative No A/12 <4 <4 Seronegative Yes B/33 < Seropositive No C/ Seropositive Yes 0/ CR-48 (H3N2) Seronegative No E/O Seronegative Yes F/6 < Seropositive No G Seropositive Yes H NOTE. Children vaccinated with HINI, n = 51; with H3N2, n = 52. * Seronegative = HAl <4; seropositive = HAl ~4 when tested against antigen in the respective vaccine. t Fourfold HAl-antibody response and/or fourfold ELISA antibody response and/or shed vaccine virus in respiratory secretions. +Prevaccine antibody titers to the heterologous vaccine were not significantly different.

4 Efficacy ofinfluenza A Vaccine 837 rose from <1:4 to 1:11.8 among the group who received CR-37 (HINl) and who were infected by the vaccine (category B). Of the six children who were seropositive to HINI and received the CR-37 (HINl) vaccine (categories C plus D), three manifested a fourfold rise in HAl-antibody titer to HINI when vaccinated intranasally; no additional children in this category had ELISA-antibody titer increases. Among the CR-48 (H3N2) vaccine group, only six of 52 were initially seronegative, a result indicating that the majority of children in our area had previously been infected with an H3N2 virus. All six of these children manifested a fourfold antibody response to vaccination. The antibody response to H3N2 was large in relation to the antibody response among the CR-37 (HINl) vaccinees; the geometric mean HAl-antibody titer rose from <1:4 to 1:45.3. Among the children who were seropositive for H3N2, 26 of 46 manifested a fourfold antibody titer increase (16 had a fourfold HAl-antibody titer increase, and 9 others had a fourfold ELISA-antibodY titer increase). The geometric mean HAl-antibody titer rose from 1:20.9 to 1:47.7 among this group (category H). Naturally acquired respiratory illness among the vaccine groups. An outbreak of influenza A oc- curred in our community approximately one to three months after the children were vaccinated. This natural experiment allowed us to gather information on the possible efficacy of cold-recombinant influenza A vaccines. Influenza A was first isolated in the community on January 19, 1983, and the last isolation was made on March 31, During the outbreak in Huntington, 790/0 of the influenza A viruses isolated were of H3N2 antigenic type, and 21% were HINI viruses. From January 19 to March 31, 103illnesses were reported by 72 of the 103 children (table 2). Most of the illnesses were afebrile upper-respiratorytract infections and occurred in similar numbers for both groups. However, most of the febrile disease occurred in the CR-37 (HINt) group. Thirteen febrile illnesses occurred in the CR-37 (HINl) vaccine group; significantly fewer febrile illnesses (five) were reported by the CR-48 (H3N2) vaccine group (X 2 = 4.5, P <.05). The occurrence of febrile disease was also significantly lower among the categories of children infected by the CR-48 (H3N2) vaccine (categories F and H) when compared with those who were infected by CR-37 (categories Band D, 3 of 31 vs. 12 of 36, X 2 = 5.4, P<.05). The number of isolates of influenza A virus was Table 2. Illnesses, isolation of viruses, and antibody response to influenza A among vaccinees during a two month influenza A outbreak. Fourfold HAIantibody rise to No. of No. of antigen/no. tested No. febrile illnesses* afebrile illnesses Vaccine/category in group (viruses isolatedjl (viruses isolated) t HINI H3N2 CR-37 (HINl), n = 51 A (HINl, H3N2) 4/12 1/12 B (2-H3N2, HINl, flu B, 25 (2-adeno, H3N2) 7/30 7/30 adeno) C /2 1/2 D /3 0/3 CR-48 (H3N2), n = 52 E 0 F 6 2 (H3N2) 10 (HtNt) 2/6 2/6 G 21 2 (H3N2, RSV) 18 (adeno) 1/ H 25 1 (HtNt) t8 (adeno) 6121t 0121 t * Significantly more febrile illnesses in the CR-37 (HtNt) categories than in the CR-48 (H3N2) categories (P <.05). t One each unless indicated. Unless indicated, viruses were not isolated. All children were tested for virus shedding during illness. t Significantly more antibody titer rises to HtNI (difference in significance of proportions test with Williams correction, Gadj = 8.1; P <.Ot). Significantly fewer rises in antibody titer in category H compared with categories A-D (0 of 21 vs. 9 of 47, X 2 = 4.6, P <.05) or compared with all other categories (0 of 21 vs. 14 of 74, X 2 = 4.2; P <.05).

5 838 Belshe et al. small in both vaccine groups. Ten children who were participating in the field trial shed influenza A during the outbreak in Huntington. This number was too small for a meaningful test of significance when comparing the two vaccine groups. In addition to isolation of viruses, the number of influenza infections occurring in the study population was determined by a fourfold rise in HAl antibody titer. Paired sera bracketing the outbreak of influenza A were available from 95 of the 103 children (table 2). Children who had previously been naturally infected with influenza A H3N2 and who manifested a booster antibody response to CR-48 (H3N2) vaccine (category H) were significantly protected from infection with H3N2 when compared with CR-37 (HI Nl) vaccinees (X 2 = 4.6, P <.05) or all other vaccinees (X 2 = 4.2, P <.05). Of the 30 vaccinees who were infected by CR-37 (HINI) vaccine virus (category B), seven had a further fourfold antibody response to HINI, and seven had a fourfold antibody response to H3N2 during the influenza A outbreak. However, significantly more children who received CR-48 (H3N2) and had manifested a booster antibody response to H3N2 (category H) were infected by HINI than by H3N2 (6 of 21 vs. 0 of 21, Gadj = 8.1, P<.01). The effect of serum HAl antibody directed against influenza A/HINI or influenza A/H3N2 on the subsequent development of naturally occurring influenza A infection among the vaccinees, regardless of the type of vaccine administered, was evaluated (table 3). Significantly more H3N2 isolates and fourfold antibody responses to H3N2 occurred during the influenza A outbreak among children who possessed little antibody (~I:16) to H3N2 compared with children with higher (~I :32) antibody levels (X 2 = 6.1, P <.05 and X 2 = 11.7, P<.01, respectively). In contrast, no such relation between antibody level and HINI infection was Table 3. Relation of postvaccine antibody titer to infection with influenza A virus and illness, regardless of vaccine administered. No. shedding No. with fourfold indicated virus antibody increase/ Reciprocal postvaccine during outbreak no. tested No. of febrile antibody titer to antigen No. in group HINl* H3N2t HINl* H3N2t illnesses during outbreak HINI <4 60 3* 3 14/56* 7/ /1 0/ /12 3/ /10 1/ /10 3/ /2 0/2 0 ~ /1 0/1 0 NT H3N2 < /2 2/2t t 3/11 5/ /21 4/ /27 2/ I 6/21 1/21 4 ~ /10 0/10 1 NT * No significant difference in shedding of HI N 1 viruses or fourfold rise to HI N 1 by children with antibody titers ~ I: 16 compared with children who possessed antibody titers ~1:32 (4 of 87 vs. 1 of 15, X 2 <1, P = not significant; and 18 of 79 vs. 1 of 13, X 2 = 1.55, P = not significant). t Significantly more children shed H3N2 or had fourfold H3N2 antibody rise who had antibody titers ~ 1:16 compared with those with antibody titers ~1:32 (5 of 37 vs. I of 65, X 2 = 6.1, P <.05, and 11 of 34 vs. 3 of 58, X 2 = 12.3, P <.01, respectively). + No significant reduction in febrile respiratory illnesses among children with ~ I: 16 antibody titer to HI N 1 or H3N2 compared with children with ~1 :32 antibody titers. (febrile, HINI antibody ~16 vs. ~16, 16 of 87 vs. 2 of 15, X 2 <1, P = not significant; febrile, H3N2 antibody ~16 vs. ~16, 10 of 37 vs. 8 of 65, X 2 = 3.5, 0.1 > P>.05).

6 Efficacy ofinfluenza A Vaccine 839 found. The number of children developing a further fourfold antibody rise to H3N2 was inversely related to the HAl antibody titer to H3N2. However, there was no relation between postvaccine H3N2 antibody titer and development ofhini antibody. Although the majority of febrile illnesses occurred among children with low antibody levels to H3 N2 (~I: 16) compared with children who possessed high H3N2 antibody levels (~I :32), this relation did not achieve statistical significance (X 2 = 3.5, 0.1 > P>.05). Discussion Both cold-recombinant vaccines were well tolerated by children and serious adverse reactions did not occur. The vaccines were administered in the three months preceding the influenza A outbreak, a desirable time and sequence for an efficacy trial. The outbreak of influenza A provided a natural experiment for the evaluation of vaccine efficacy. When this trial was designed, it was anticipated that only one subtype ofinfluenza, if any, would circulate in the community. Two subtypes were used to vaccinate children since it could not be predicted whether HINI or H3N2 would cause infections in 1983, and each group of vaccinees would serve as a control group for the other. This outbreak of influenza A was a modest one and occurred during a year in which only small excess mortality due to pneumonia and influenza was reported by the Centers for Disease Control. Although the majority of isolates from the community of Huntington were H3N2 strain, the frequent occurrence of antibody titer rises in our study population indicated that HINI viruses were also prevalent in the population of young children. Simultaneous outbreaks of HINI and H3N2 viruses have been reported previously [17], and the occurrence of two viruses in the community simultaneously made analysis of the data complex. Nevertheless, significantly fewer febrile illnesses occurred in the CR-48 (H3N2) group and significantly fewer H3N2 infections were demonstrated among this same group. Both vaccines were antigenic but to different degrees. The H3N2 vaccine induced high levels of antibody titers in both the seronegative and seropositive volunteers. However, among the CR-37 (HINI) vaccine recipients, only low levels of antibody were stimulated, and the rate of seroconversion was low (730/0). Other HINI antigens may induce higher antibody titers, but the rate of antibody stimulation was similar (730/0-79%) in other studies [5, 7]. Increasing the inoculum did not increase the proportion of seronegative children infected by CR-37 (HINI) [7]; however, multiple doses ofvaccine may result in more satisfactory antibody titers and a higher rate of seroconversion among HINI vaccine recipients. In a previous study [7], we found that children were significantly protected from high-dose CR-37 (H 1N1) vaccine challenge by a previous dose of the same vaccine. Booster antibody responses were stimulated in 38% of vaccinees challenged six months after the primary vaccination, but shedding of vaccine virus did not occur with booster vaccination. The reduced rate of antibody stimulation by HINI vaccine viruses appears to be a direct effect ofthe hemagglutinin in the vaccine, since the six internal genes of the CR-48 (H3N2) and CR-37 (HINI) vaccines are identical. Perhaps the HI hemagglutinin of the influenza A/California/IO/ 78 virus is less efficient at attaching to human cells than is the H3 hemagglutinin. Similarly, it has been suggested that influenza/a HINI viruses cause less serious disease than do influenza A/H3N2 viruses during natural infection [18]. The CR-37 (HINI) vaccine virus induced only low levels of antibody compared with CR-35 (influenza A/Hong Kong/HINI) in Wright's study [5]. A comparison (using homologous tissue culture-grown antigens) of postvaccination HAl antibody titers in children vaccinated with CR-35 (HINl) or CR-37 (HINI) revealed that CR-35 (HINI) vaccinees developed significantly higher antibody titers to either influenza A/California/IO/78 (HINI) antigen (1:21.8) or influenza A/ Hong Kong/77 (HINI) antigen (1:37.3) than did CR-37 (HINI) vaccinees (1:8.7 and 1:11.3, respectively; R. B. B. and Peter Wright, unpublished observation). Therefore, HI hemagglutinins appears to vary in their capacities for antibody stimulation. Intranasal vaccination with influenza A vaccines should be pursued. The donor strain may be overly attenuated for production of HINI vaccines, and different donor strains with varying levels of attenuation may have to be developed for use with different hemagglutinins. The booster antibody responses and efficacy in reducing febrile diseases

7 840 Belsheet 01. among the CR-48 (H3N2) group is encouraging. Multiple doses ofvaccine may be necessary to provide more complete protection from infection. References I. Murphy BR, Holley HP, Jr, Berquist EJ, Levine MM, Spring SB, Maassab HF, Kendal AP, Chanock RM. Cold-adapted variants of influenza A virus: evaluation in adult seronegative volunteers of A/Scotland/ and A/Victorial3175 cold-adapted recombinants derived from the cold adapted AIAnn Arbor/6/60 strain. Infect Immun 1979;23: Murphy BR, Rennels MB, Douglas RG, Jr, Betts RF, Couch RB, Cate TR, Jr, Chanock RM, Kendal AP, Maassab HF, Suwanagool S, Sotman SB, Cisneros LA, Anthony WC, Nalin DR, Levine MM. Evaluation of influenza AIHong Kong/123/77 (HINl) ts-i A2 and coldadapted recombinant viruses in seronegative adult volunteers. Infect Immun 1980;29: Moritz AJ, Kunz C, Hofman H, Liehl E, Reeve P, Maassab HF. Studies with a cold-recombinant A/Victoria/31 75 (H3N2) virus. II. Evaluation in adult volunteers. J Infect Dis 1980;142: Murphy BR, Chanock RM, Clements ML, Anthony WC, Sear AJ, Cisneros LA, Rennels MB, Miller EH, Black RE, Levine MM, Betts RF, Douglas RG Jr, Massaab HF, Cox NJ, Kendal AP. Evaluation of AIAlaska/6177 (H3N2) cold-adapted recombinant viruses derived from AIAnn Arbor/6/60 cold-adapted donor virus in adult seronegative volunteers. Infect Immun 1981;32: Wright PF, Okabe N, McKeeKT, 1r, Maassab HF, Karzon DT. Cold-adapted recombinant influenza A virus vaccines in seronegative young children. J Infect Dis 1982;146: Clements ML, O'Donnell S, Levine MM, Chanock RM, Murphy BR. Dose response of AIAlaska/6177 (H3N2) cold-adapted reassortant vaccine virus in adult volunteers: role of local antibody in resistance to infection with vaccine virus. Infect Immun 1983;40: Belshe RB, Van Voris LP. Cold recombinant influenza AI California/10178 (HINl) virus vaccine (CR-37) in seronegative children: infectivity and efficacy against investigational challenge. J Infect Dis 1984; Maassab HF, Kendal AP, Abrams GO, Monto AS. Evaluation of a cold-recombinant influenza virus vaccine in ferrets. J Infect Dis 1982;146: Maassab HF. Adaptation and growth characteristics of influenza virus at 25 0 C. Nature 1967;213: Cox NJ, Maassab HF, Kendal AP. Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: nonrandom reassortment of genes during preparation of live virus vaccine candidates by recombination at 25 0 between recent H3N2 and HINI epidemic strains and cold-adapted AIAnn Arbor/6/60. Virology 1979;97: World Health Organization. The hemagglutination inhibition test for Influenza Viruses. Atlanta, Georgia: US Department of Health, Education and Welfare Procedure Manual, Centers for Disease Control, Murphy BR, Phelan MA, Nelson DL, Yarchoan R, Tierney EL, Alling DW, Chanock RM. Hemagglutininspecific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses. J Clin Microbiol 1981 ;13: Belshe RB, Van Voris LP, Mufson MA. Parenteral administration of live respiratory syncytial virus vaccine: results of a field trial. J Infect Dis 1982;145: Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research 2nd ed. San Francisco: WH Freeman and Co : Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research 2nd ed. San Francisco: WH Freeman Co., 1981: Murphy BR, Tierney EL, Barbour BA, Yolken RH, Alling OW, Holley HP Jr, Mayner RE, Chanock RM. Use of enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenzaa virus vaccines. Infect Immun 1980;29: Frank AL, Taber LH, Wells 1M. Individuals infected with two subtypes of influenza A in the same season. J Infect Dis 1983;147: Wright PF, Thompson J, Karzon DT. Differing virulence of HINI and H3N2 influenza strains. Am J Epidemiol 1980;112:814-9