- Email: [email protected]
Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60
PII: S0264-410X(%)00030-8
Vaccine, Vol. 14, No. 13, pp. 1214-1216, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights resewed Printed in Great Britain 0264-41 OX/96 $15+0.00
Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60 Ann R. Falsey*“f and Edward
E. Walsh*
The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in a randomized placebo-controlled, double-blind study of 64 healthy adults over age 60. Vaccination was well tolerated with no significant acute side-efects. Twenty-nine of 33 vaccinees (87%) showed a greater than or equal to fourfold rise in serum IgG to the Fprotein of RSV at 8 weeks post vaccination. Twenty of 33 vaccine recipients (61%) had a greater than or equal to fourfold rise in serum neutralizing titer to group A andlor group B RSV. Response to vaccination was inversely correlated with pre-immunization serum neutralizing titers. Active surveillance throughout the ensuing winter identtfied three RSV infections in the placebo group and none in the vaccine group. Thus, PFP-2 wasfound to be safe and immunogenic in healthy older adults. Copyright 0 1996 Elsevier Science Ltd. Keywords: Vaccine; respiratory syncytial virus; elderly
Although traditionally recognized as the most common cause of lower respiratory tract disease in young children, respiratory syncytial virus (RSV) has also been shown to be a cause of serious illness in elderly adults’-‘. RSV is a predictable cause of disease each winter in older persons living in the community, in nursing homes, or attending senior daycare programss13. Similar to influenza, a well-recognized pathogen in the elderly, peaks of RSV activity in the community are associated with excess rates of mortality in persons over age 65i4. Immunity to RSV is incomplete and reinfection throughout life is common”. The precise roles of humoral and cell-mediated immunity have not been well defined; yet there is evidence that serum neutralizing immunoglobulins have a protective effect16. Aging is accompanied by diverse changes in the immune system including diminished B and T cell function”“*. Therefore, the increased morbidity associated with RSV infection in elderly persons may be in part due to declining immunity. PFP-2, a candidate RSV vaccine which consists of the purified fusion glycoprotein of RSV, has undergone clinical trials in seropositive children and young adults and has been found to be safe and immunogenic 19q2o . The purpose of this study was to *University of Rochester School of Medicine and Dentistry and Infectious Disease Unit, Rochester General Hospital, 1425 Portland Avenue, Rochester NY 14621, USA. tTo whom correspondence should be addressed. (Received 16 December 1995; revised 25 January 1996; accepted 25 January 1996)
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Vaccine 1996 Volume 14 Number 13
evaluate the safety and immunogenicity healthy adults over the age of 60.
MATERIALS
of PFP-2 in
AND METHODS
Vaccine Purified fusion protein (PFP-2) was produced by Lederle Praxis Biologicals of Rochester, NY. The fusion protein (F) of A2 strain RSV, grown in Vero cells, was purified by ion-exchange chromatography and adsorbed to alum. The vaccine contains ~2% of the RSV attachment glycoprotein (G). A single dose of 50 pugof protein was chosen based on previous data in young adults and children. Vaccine was administered by intramuscular deltoid injection in a volume of 0.5 ml. Placebo recipients received 0.5 ml normal saline. Study population Sixty-four healthy, independently living adults over the age of 60 years were recruited and enrolled in the study during August and September, 1994. Subjects were excluded if they were receiving immunosuppressive therapy or had known immunosuppressive conditions, active infections, metastatic malignancy, severe chronic obstructive pulmonary disease requiring oxygen, end stage renal failure, or unstable cardiac conditions. Study design Volunteers were assigned to vaccine or placebo groups in a randomized, double-blind fashion. All
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh
immunologic and clinical assessments were performed blinded. Subjects were given a diary card to complete during the first 3 days following vaccination to assess any acute adverse effects (fever, systemic symptoms, local reactions). In addition, each volunteer was contacted by telephone at 72 h post vaccination by the investigators. Serum samples were collected prior to vaccination and at 4 and 8 weeks post-vaccination. A final blood sample was taken at the end of the RSV season in April of 1995. Nasal wash specimens were collected prior to immunization and at 4 weeks post-vaccination. Surveillance for respiratory illnesses in the study cohort was done during the subsequent winter season. Between 1 December 1994 and 30 April 1995, subjects were asked to contact study personnel if they developed signs or symptoms of a respiratory illness (fever and/or nasal congestion, cough, sore throat, dyspnea, or wheezing). In addition, volunteers were contacted weekly by telephone to ascertain whether respiratory illnesses had occurred. If a subject was identified as having a possible respiratory infection, the subject was seen by the study nurse. A directed history and physical examination, a nasopharyngeal viral culture, and a 4-week convalescent serum were obtained for each illness. Virus isolation Nasopharyngeal specimens obtained during illnesses were inoculated onto HEp-2, WI-38, and primary rhesus monkey kidney cells for viral isolation. Cultures were observed for 10 days for cytopathic effect. Viral growth was confirmed with virus specific monoclonal antibodies (Baxter Healthcare, West Sacramento, CA). In addition, all terminal cultures were screened for RSV infection by indirect immunofluorescent assay (IFA) with RSV-specific monoclonal antibodies. Immunologic assays Enzyme immunoadsorbent assay (EIA). Serum IgG levels to the F and G proteins of RSV were determined using immunoaffinity purified F from group A virus and G from group A and B viruses as the coating antigens (F, G,, Ga)“. Each serum sample was incubated in serial twofold dilutions from 1:800 to 1:1024000 in 96-well plates (Costar, Immunolon I), coated with 25 ng of antigen. Bound IgG was detected with alkaline phosphatase conjugated rabbit anti-human IgG followed by substrate. IgG titer was defined as the highest dilution with an optical density 20.10 and at least twice the bicarbonate control value. Evidence of natural RSV infection in PFP-2 recipients during the surveillance period was defined as a greater than or equal to fourfold rise in G, and/or G, titers in post illness or end of season sera. Neutralization. Microneutralization assays were performed using a modification of a method previously reported by Anderson et aLz2 Briefly, serial twofold dilutions of serum (1:50 to 1:256000) in MEM with 5% fresh rabbit complement were incubated with ca 100 plaque forming units (p.f.u.) of RSV [either Long strain (group A virus) or CH18537 (group B virus)]. After incubation at room temperature for 30 min, HEp-2 cells
Table 1
Characteristicsof
Mean age&.D. Female Ideal body weight Smokers Number of chronic conditions
vaccine and placebo groups Vaccine (fi33)
Placebo (1\1=31)
P
7026.1 70% 123% 12%
67r4.5 65% 122% 6% 1.2*1.1
0.03 N.S. N.S. N.S. 0.08
1.7rtl.2
at 1.5 x lo4 per 100 ~1 were added and plates incubated at 37°C at 5% CO, for 4 days. Plates were fixed with 80% acetone/phosphate-buffered saline (PBS) and developed, using RSV-specific mouse monoclonal antibodies to the F and P proteins followed by horseradish peroxidasegoat anti-mouse IgG and substrate. End point neutralization titer was defined as the highest dilution producing a 50% reduction in color development when compared to controls without serum. All assays were standardized with well characterized adult control sera.
Nasal ZgA. Nasal washes were collected from subjects by instilling 5 cm3 of sterile PBS into each nostril. Specimens were frozen at -20°C until testing. RSVspecific IgA in untreated nasal washes was measured by EIA. Methods were the same as for the serum EIA, except that the starting dilution was I:2 and IgA was detected with alkaline phosphatase conjugated goat u-human IgA. The total protein content of each sample was measured using the micro BCA Procedure (Pierce, Rockford, IL). The mean protein content for all samples was calculated and each RSV-specific IgA value was corrected to the mean.
Data analysis Proportions were compared using x2 and Fisher’s exact tests as appropriate. Linear regression analysis was used to correlate variables and comparisons of mean log, titers were done using t-test. All P values were two sided.
RESULTS Sixty-four subjects were recruited and vaccinated. All volunteers completed the study. The mean age of subjects was 68 (range: 60-80); 21 were male and 43 female. Thirty-three participants were randomized to receive vaccine and 3 1 received placebo. Vaccine recipients were slightly older and had, on average, more underlying diseases than did the placebo recipients (Table I).
Acute safety Vaccination was well tolerated by both groups and no significant difference in systemic or local symptoms was noted when placebo and vaccine groups were compared (Table 2). Pain at the injection site was more common in the vaccine group (12 vs 3%; PzO.37); however, this difference was not statistically significant. The maximum temperature recorded by the one vaccine recipient with fever was 38.O”C (oral).
Vaccine 1996 Volume 14 Number 13 1215
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh
Table2
Acute side-effects Number of subjects reporting Vaccine Placebo (N=31) (r&33)
Reaction Local
Redness 1 cm Swelling 1 cm Pain
2
0
1 4
1 1
Temp 238°C Headache Myalgias
1 3 0
: 3
Table3
mean Log, titers of PFP-2 and placebo
Systemic
Post-vaccination
groups Week post-immunization 4 0
8
Placebo (N=3 1) EIA 12.3*1.2 13.2k1.6 MNA 13.1*1.9 MNB
12.4&l .3 13.9e1.8 13.Ok1.8
12.4il.3 13.2i1.7 N.D.
PFP-2 (N=33) EIA 12.6il.5 13.3i1.4 MNA 12.5i1.6 MNB
15.6~~1.ga 15.2il.l’ 13.8*1 .8e
15.7*1.8* 14.5*1 .2d N.D.
EIA=enzyme immunoabsorbent assay for IgG to F; MNA= microneutralization assay to A virus (Long strain); MNB= microneutralization assay to B virus (CH18537 strain); ND=not determined. “0 vs 4 weeks, PcO.001. bO vs 8 weeks, PcO.001. “0 vs 4 weeks, PcO.001. ‘0 vs 8 weeks, EO.003. ‘0 vs 4 weeks, PcO.001
Immunogenicity Vaccination with PFP-2 resulted in a statistically significant increase in the mean antibody titer to F as measured by EIA at 4 and 8 weeks (Table 3). No significant increase in antibody to the G protein was noted in any vaccinee. Seventy-six percent of PFP-2 recipients achieved a greater than or equal to fourfold rise in F EIA titers at 4 weeks, and by 8 weeks 87% had a significant rise in F antibody. Vaccination was also associated with a significant increase in serum neutralizing activity to group A (Long strain) virus at both 4 and 8 weeks (Table 3). Of those who responded with a significant rise in neutralizing titer to group A virus, mean titers did not drop significantly at the end of the RSV season (32 weeks) when compared with the mean titers at 8 weeks (14.7 f 1.6 vs 14.6 f 1.2). Neutralizing titers to group B virus (CH18537) were measured at 4 weeks and also showed a significant increase in the PFP-2 vaccinated group (Table 3). Four weeks after immunization, 16 of the 33 vaccinees (48%) had a greater than or equal to fourfold rise in neutralizing titers to Long strain RSV (group A) and 15 of 33 subjects (45%) showed a greater than or equal to fourfold rise in neutralizing titers to CH18537 strain (group B virus). Eleven vaccinees had a significant rise in neutralizing activity to both viruses. Notably, the four individuals who had a significant increase in neutralizing titers to group B virus only had high baseline neutralizing titers to A group virus (15.14, 13.64, 14.64, 14.64), which were all above the group mean baseline titer of 12.6* 1.5. Additionally, four of five subjects who responded only with an increase in neutralizing titers to
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Vaccine 1996 Volume 14 Number 13
group A virus had neutralizing titers to group B virus at or above the mean. Therefore, 20 of 33 vaccinees (61%) had a significant increase in serum neutralizing activity against RSV. Most individuals who had a significant rise in EIA titers also had an increase in serum neutralizing activity against RSV. Of the 29 individuals with a greater than or equal to fourfold rise in EIA titers, 20 (70%) had a greater than or equal to fourfold rise in group A and/or group B neutralizing titers, while 10 others had at least a twofold increase in neutralizing activity. Only three vaccinees with a vigorous EIA failed to develop an increase in neutralizing titers. Five of 33 vaccine recipients (15%) showed a greater than or equal to fourfold rise in nasal RSV-specific IgA against F protein; three had an eightfold rise, and two had a fourfold rise. No RSV-specific IgA rises were detected in the placebo group. No significant differences in the age, sex, percent ideal body weight, smoking status, chronic conditions, or medications was noted between vaccine responders and nonresponders as measured by a greater than or equal to fourfold neutralizing response to group A virus. However. antibody response was inversely correlated with pre-immunization neutralizing titers (Figure 1). The baseline neutralizing titers of the responders were significantly lower than nonresponders (12.4 * 1.3 vs 14.1f 0.9, WO.001). Additionally, when individual neutralizing titers to group A virus were examined at 4 weeks post vaccination all but three vaccine recipients had a titer 14.00.
Respiratory surveillance Of the possible 1024 person weeks of surveillance, 908 (89%) were available for study and all volunteers had an end-of-winter-season serum sample analyzed by EIA to detect asymptomatic RSV infection. Thirty-two of the 64 subjects reported 41 illnesses during the 5 month surveillance period. The number of respiratory illnesses reported was similar in the vaccine and placebo groups. Twenty-six of the 41 illnesses (63%) were evaluated clinically and most were upper respiratory infections (URI). No hospitalizations, pneumonia, or death occurred in either group. None of the vaccine recipients had identifiable RSV infection in contrast to three serologically confirmed infections in the placebo group. Serologic evidence of infection was defined as a greater than or equal to fourfold rise in antibody to the G protein which was not effected by vaccination. Two of these infections were associated with clinical illness, one URI, the other bronchitis. The third infection, identified by a greater than fourfold rise in RSV antibody in the end-of-season serum sample, was in an individual who did not report an illness. RSV was not cultured from any subject during illness evaluation.
DISCUSSION RSV infection in the elderly has been shown to cause substantial morbidity and mortality and thus prevention of this infection by vaccination is highly desirable. This study demonstrates that PFP-2 was well tolerated and
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh Neutralization
Titers of Individual
Vacinees l
0
Pre Vaccination Post Vaccination
l8 T 17 1G 15 Titer
(log 2)
,‘, I
10
T”“I,,,,I,,,,I,,,,i,,,,l,,,,l,,,,i 0
5
10
1.5
20
25
30
35
Subjert
Figure 1 Pre- and post-immunization
neutralizing
titers (IogJ to Group A virus of individual
immunogenic in persons over age 60. Although advancing age has been associated with diminished responses to a number of vaccines18, the 87% response by EIA and 61% response by neutralization are comparable to trials of PFP in healthy young adults and seropositive children ’ 9q20.In addition, the response rates of older adults in our study to PFP are also similar to response rates of older persons to inactivated influenza vaccines23. A “protective” level of serum neutralizing antibody for RSV has not been clearly established. However, evidence that serum antibody has a beneficial effect comes from a number of sources. Animal and human data suggest that circulating neutralizing antibody and mucosal antibodies may modify disease severity and be required to prevent reinfection. Passive transfer of neutralizing antibodies to the two major surface proteins of RSV (F and G) protects rodents against RSV challenge, as does immunization with these glycoproteins24,25. Human studies show that levels of neutralizing antibodies are inversely correlated with severity of illness in infants26. In a study of experimental reinfection of young adult volunteers, Hall et al. showed that neutralizing F and G antibody levels correlated with protection, although even at the highest levels of antibody 25% of subjects could still be infected27. In addition, recent studies on the benefits of IgG prophylaxis for RSV infections in high risk children also suggest humoral immunity is important28. Relatively little is known about the immune status to RSV in the elderly adult. Agius and colleagues found that the institutionalized elderly were capable of mounting a brisk serum IgA and IgG response to RSV infection29. In an earlier study, we found no association between EIA titers of serum IgG and severity of infection in nursing home patients3’. There was a trend towards lower neutralizing titers in those with more severe illness; however, statistical significance was not achieved. This study was hampered by the fact that sera were obtained after patients were identified as ill and may not have represented a true pre-illness baseline. All adults have had multiple RSV infections and have neutralizing antibody to RSV. However, it is possible that advancing age which is associated with declining immune function as well as less frequent exposures to respiratory viruses, results in decreasing serum neutralizing antibody titers to RSV. Data from our laboratory indicate that the mean serum neutralizing antibody titers
PFPQ vaccinees
of healthy older adults are significantly lower than those of young adults (unpublished data). In our study, response to vaccination was best correlated with pre-immunization neutralizing titers. Similar results have been noted with influenza vaccine studies where response rates of vaccinees decreased with increasing hemagglutination inhibition antibody titers31*32.It is possible that there is a maximal level of immunity which can be induced either by natural infection or vaccination after which no additional benefit is possible. In our present study, it was notable that, although only 48% of subjects had a greater than or equal to fourfold rise in neutralizing titers to group A virus, all but three vaccinees had a titer 14.00 postvaccination (Figure 2). Interestingly, in a previous study the mean baseline neutralization titers of older persons who became infected with RSV was 12.4 f 2.2 compared to 14.2& 2.2 in matched controls without infection (unpublished data). In summary, PFP-2 was found to be a safe and immunogenic RSV vaccine in healthy persons over age 60. Frail older persons with underlying heart and lung disease as well as those living in nursing homes are groups which are at highest risk for severe RSV disease. Since immune function may be influenced by a number of chronic conditions it will be important to assess PFP-2 immunogenicity in debilitated elderly adults in addition to the healthy elderly33. The frail elderly represent a rapidly growing segment of the population who are at risk for serious sequelae from viral respiratory illnesses and PFP-2 vaccine may prove to be useful in preventing RSV infection in this age group.
ACKNOWLEDGEMENTS This study was supported by a grant from Lederle Praxis Biologicals. We thank Joanne Prives for transcription assistance, Maria Formica for technical assistance, and Michelle Christenson for help in conducting the study.
REFERENCES 1
Fransen, H., Sterner, G., Forsgren, M. et al. Acute lower respiratory illness in elderly patients with respiratory syncytial virus infection. Acta Med. Stand. 1967, 182, 323-329
Vaccine 1996 Volume 14 Number 13 1217
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh 2 3 4
5
6 7
8
9
10
11
12
13
14 15
16 17 18
19
Garvie, D.G. and Gray, J. Outbreak of respiratory syncytial virus infection in the elderly. Br. Med. J. 1980, 281, 1253-1254 Mathur, U. and Bentley, D.W. Concurrent respiratory syncytial virus and influenza A infections in the institutionalized elderly and chronically ill. Ann. Int Med. 1980, 93, 149-152 Morales, F., Calder, M.A., Inglis, J.M., Murdoch, P.S. and Williamson, J. A study of respiratory infections in the elderly to assess the role of respiratory syncytial virus. J. Infect. 1983, 7, 236-247 Sorvillo, F.J., Huie, S.F., Strassberg, M.A., Butsumyo, A., Shandera, W.X. and Fannin, S.L. An outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly. J. Infect 1984, 8, 252-256 Hart, J. An outbreak of respiratory syncytial virus infection in an old people’s home. J. Infect. 1984, 8, 259-261 Falsey, AR., Treanor, J.J., Betts, R.F. and Walsh, E.E. Viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings. J. Am. Geriatr. Sot. 1992, 40, 115-I 19 Falsey, AR., Cunningham, C.K., Barker, W.H. et al. Respiratory syncytial virus and influenza A infections in hospitalized elderly. J. Infect. Dis. 1995, 172, 389-394 Vikerfors, T., Grandien, M. and Olcen, P. Respiratory syncytial virus infections in adults. Am. Rev. Respir. Dis. 1987, 136, 561-564 Kimball, A.M., Foy, H.M., Cooney, M.K., Allan, I.D., Matlock, M. and Plorde, J.J. Isolation of respiratory syncytial virus and influenza viruses from the sputum of patients hospitalized with pneumonia. J. Infect. Dis. 1983, 147, 181-184 Wald, T.G., Miller, B.A., Shult, P., Drinka, P., Langer, L. and Gravenstein, S. Can respiratory syncytial virus and influenza A be distinguished clinically in older persons?. J. Am. Geriatr. Sot. 1995,43,170-174 Falsey, A.R., McCann, R.M., Hall, W.J. et a/. Acute respiratory tract infection in daycare centers for older persons. J. Am. Geriatr. Sot. 1995, 43, 30-36 Zaroukian, M.H., Kashyap, G.H. and Wentworth, B.B. Case report: respiratory syncytial virus-a cause of respiratory distress and pneumonia in adults. Am. J. Med. Sci. 1988, 295, 218-227 Fleming, D.M. and Cross, K.W. Respiratory syncytial virus or influenza?. Lancet 1993.342. 1507-l 510 Johnson, K.M., Bloom, H.H., ‘Mufson, M.A. and Chanock, R.M. Natural reinfection of adults by respiratory syncytial virus. N. En@. J. Med. 1962, 26, 66-71 Hemming, V.G. and Prince, G.A. Respiratory syncytial virus: babies and antibodies. Infect Agts. Dis. 1992, 1, 24-32 Salzman, R.L. and Peterson, P.K. lmmunodeficiency of the elderly. Rev. Infect. Dis. 1987, 9, 1127-1139 Powers, D.C. Immunological principles and emerging strategies of vaccination for the elderly. J. Am. Geriatr. Sot. 1992, 40, 81-94 Tristam, D.A., Welliver, R.C., Mohar, C.K., Hogerman, D.A., Hildreth, S.W. and Paradiso, P. lmmunogenicity and safety of respiratory syncytial virus subunit vaccine in seropositive children 18-36 months old. J. Infect. Dis. 1993, 167, 191-195
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Paradiso, P.R., Hildreth, S.W., Hogerman, D.A. et al. Safelyand immunogenicty of a subunit respiratory syncytial virus vaccine in children 24 to 48 months old. Pediatr. Infect. Dis. J. 1994,13, 792-798 21 Hendry, M.R., Burns, J.C., Walsh, E.E., Graham, B.S., Wright, P.F. and Hemming, V.G. Strain-specific antibody responses in infants undergoing primary infection with respiratory syncytial virus. J. infect. Dis. 1988, 157, 640-847 22 Anderson, L.J., Hierholtzer, J.C., Bingham, P.G. and Stone, Y.O. Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J. C/in. Micro. 1985, 2, 1050-1052 23 Treanor, J.J., Mattison, H.R., Dumyati, G. et a/. Protective efficacy of combined live intranasal and inactivated influenza A virus vaccines in the elderly. Ann. hf. Med. 1992,117,625-633 24 Walsh, E.E., Schlesinger, J.J. and Brandriss, M.W. Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies. Infect. Immun. 1984, 43,756-758 25 Walsh, E.E., Hall, C.B., Briseili, M., Brandriss, M.W. and Schlesinger, J.J. Immunization with the glycoprotein subunits of respiratory syncytial virus to protect cotton rats against viral infection. J. Infect. Dis. 1987, 155, 1198-1204 26 Glezen, W.P., Paredes, A., Allison, J.E., Taber, L.H. and Frank, A.L. Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody. J. Pediatr. 1981, 98, 708-715 27 Hall, C.B., Walsh, E.E., Long, C.E. and Schnabel, KC. Immunity and frequency of infection with respiratory syncytial virus. J. Infect. Dis. 1991, 163, 693-698 28 Groothius, J.R., Simoes, A.F., Levin, M.J. et a/. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. N. Engl. J. Med. 1993, 329, 1524-l 530 29 Agius, G., Dindinaud, G., Biggar, R.J. et al. An epidemic of respiratory syncytial virus in elderly people: clinical and serological findings. J. Med. Viral. 1990, 30, 117-127 30 Falsey, A.R. and Walsh, E.E. Humoral immunity to respiratory syncytial virus infection in the elderly. J. Med. Vim/. 1992, 36, 39-43 31 Wenzel, R.P., Hendley, J.O., Sande, M.A. and Gwaltney, J.M. Revised (1972-1973) bivalent influenza vaccine: serum and nasal antibody responses to parenteral vaccination. J. Am. Med. Assoc. 1973, 226,43w38 32 Beyer, W.E., Palanche, A.M., Batjet, M. and Masurel, N. Antibody induction by influenza vaccines in the elderly: a review of the literature. Vaccine 1989, 7, 385-394 33 Gross, P.A., Quinnan, G.V., Wekster, M.E., Setia, U. and Douglas, R.G. Relation of chronic disease and immune response to influenza vaccine in the elderly. Vaccine 1989, 7, 303-308 20
Vaccine, Vol. 14, No. 13, pp. 1214-1216, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights resewed Printed in Great Britain 0264-41 OX/96 $15+0.00
Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60 Ann R. Falsey*“f and Edward
E. Walsh*
The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in a randomized placebo-controlled, double-blind study of 64 healthy adults over age 60. Vaccination was well tolerated with no significant acute side-efects. Twenty-nine of 33 vaccinees (87%) showed a greater than or equal to fourfold rise in serum IgG to the Fprotein of RSV at 8 weeks post vaccination. Twenty of 33 vaccine recipients (61%) had a greater than or equal to fourfold rise in serum neutralizing titer to group A andlor group B RSV. Response to vaccination was inversely correlated with pre-immunization serum neutralizing titers. Active surveillance throughout the ensuing winter identtfied three RSV infections in the placebo group and none in the vaccine group. Thus, PFP-2 wasfound to be safe and immunogenic in healthy older adults. Copyright 0 1996 Elsevier Science Ltd. Keywords: Vaccine; respiratory syncytial virus; elderly
Although traditionally recognized as the most common cause of lower respiratory tract disease in young children, respiratory syncytial virus (RSV) has also been shown to be a cause of serious illness in elderly adults’-‘. RSV is a predictable cause of disease each winter in older persons living in the community, in nursing homes, or attending senior daycare programss13. Similar to influenza, a well-recognized pathogen in the elderly, peaks of RSV activity in the community are associated with excess rates of mortality in persons over age 65i4. Immunity to RSV is incomplete and reinfection throughout life is common”. The precise roles of humoral and cell-mediated immunity have not been well defined; yet there is evidence that serum neutralizing immunoglobulins have a protective effect16. Aging is accompanied by diverse changes in the immune system including diminished B and T cell function”“*. Therefore, the increased morbidity associated with RSV infection in elderly persons may be in part due to declining immunity. PFP-2, a candidate RSV vaccine which consists of the purified fusion glycoprotein of RSV, has undergone clinical trials in seropositive children and young adults and has been found to be safe and immunogenic 19q2o . The purpose of this study was to *University of Rochester School of Medicine and Dentistry and Infectious Disease Unit, Rochester General Hospital, 1425 Portland Avenue, Rochester NY 14621, USA. tTo whom correspondence should be addressed. (Received 16 December 1995; revised 25 January 1996; accepted 25 January 1996)
1214
Vaccine 1996 Volume 14 Number 13
evaluate the safety and immunogenicity healthy adults over the age of 60.
MATERIALS
of PFP-2 in
AND METHODS
Vaccine Purified fusion protein (PFP-2) was produced by Lederle Praxis Biologicals of Rochester, NY. The fusion protein (F) of A2 strain RSV, grown in Vero cells, was purified by ion-exchange chromatography and adsorbed to alum. The vaccine contains ~2% of the RSV attachment glycoprotein (G). A single dose of 50 pugof protein was chosen based on previous data in young adults and children. Vaccine was administered by intramuscular deltoid injection in a volume of 0.5 ml. Placebo recipients received 0.5 ml normal saline. Study population Sixty-four healthy, independently living adults over the age of 60 years were recruited and enrolled in the study during August and September, 1994. Subjects were excluded if they were receiving immunosuppressive therapy or had known immunosuppressive conditions, active infections, metastatic malignancy, severe chronic obstructive pulmonary disease requiring oxygen, end stage renal failure, or unstable cardiac conditions. Study design Volunteers were assigned to vaccine or placebo groups in a randomized, double-blind fashion. All
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh
immunologic and clinical assessments were performed blinded. Subjects were given a diary card to complete during the first 3 days following vaccination to assess any acute adverse effects (fever, systemic symptoms, local reactions). In addition, each volunteer was contacted by telephone at 72 h post vaccination by the investigators. Serum samples were collected prior to vaccination and at 4 and 8 weeks post-vaccination. A final blood sample was taken at the end of the RSV season in April of 1995. Nasal wash specimens were collected prior to immunization and at 4 weeks post-vaccination. Surveillance for respiratory illnesses in the study cohort was done during the subsequent winter season. Between 1 December 1994 and 30 April 1995, subjects were asked to contact study personnel if they developed signs or symptoms of a respiratory illness (fever and/or nasal congestion, cough, sore throat, dyspnea, or wheezing). In addition, volunteers were contacted weekly by telephone to ascertain whether respiratory illnesses had occurred. If a subject was identified as having a possible respiratory infection, the subject was seen by the study nurse. A directed history and physical examination, a nasopharyngeal viral culture, and a 4-week convalescent serum were obtained for each illness. Virus isolation Nasopharyngeal specimens obtained during illnesses were inoculated onto HEp-2, WI-38, and primary rhesus monkey kidney cells for viral isolation. Cultures were observed for 10 days for cytopathic effect. Viral growth was confirmed with virus specific monoclonal antibodies (Baxter Healthcare, West Sacramento, CA). In addition, all terminal cultures were screened for RSV infection by indirect immunofluorescent assay (IFA) with RSV-specific monoclonal antibodies. Immunologic assays Enzyme immunoadsorbent assay (EIA). Serum IgG levels to the F and G proteins of RSV were determined using immunoaffinity purified F from group A virus and G from group A and B viruses as the coating antigens (F, G,, Ga)“. Each serum sample was incubated in serial twofold dilutions from 1:800 to 1:1024000 in 96-well plates (Costar, Immunolon I), coated with 25 ng of antigen. Bound IgG was detected with alkaline phosphatase conjugated rabbit anti-human IgG followed by substrate. IgG titer was defined as the highest dilution with an optical density 20.10 and at least twice the bicarbonate control value. Evidence of natural RSV infection in PFP-2 recipients during the surveillance period was defined as a greater than or equal to fourfold rise in G, and/or G, titers in post illness or end of season sera. Neutralization. Microneutralization assays were performed using a modification of a method previously reported by Anderson et aLz2 Briefly, serial twofold dilutions of serum (1:50 to 1:256000) in MEM with 5% fresh rabbit complement were incubated with ca 100 plaque forming units (p.f.u.) of RSV [either Long strain (group A virus) or CH18537 (group B virus)]. After incubation at room temperature for 30 min, HEp-2 cells
Table 1
Characteristicsof
Mean age&.D. Female Ideal body weight Smokers Number of chronic conditions
vaccine and placebo groups Vaccine (fi33)
Placebo (1\1=31)
P
7026.1 70% 123% 12%
67r4.5 65% 122% 6% 1.2*1.1
0.03 N.S. N.S. N.S. 0.08
1.7rtl.2
at 1.5 x lo4 per 100 ~1 were added and plates incubated at 37°C at 5% CO, for 4 days. Plates were fixed with 80% acetone/phosphate-buffered saline (PBS) and developed, using RSV-specific mouse monoclonal antibodies to the F and P proteins followed by horseradish peroxidasegoat anti-mouse IgG and substrate. End point neutralization titer was defined as the highest dilution producing a 50% reduction in color development when compared to controls without serum. All assays were standardized with well characterized adult control sera.
Nasal ZgA. Nasal washes were collected from subjects by instilling 5 cm3 of sterile PBS into each nostril. Specimens were frozen at -20°C until testing. RSVspecific IgA in untreated nasal washes was measured by EIA. Methods were the same as for the serum EIA, except that the starting dilution was I:2 and IgA was detected with alkaline phosphatase conjugated goat u-human IgA. The total protein content of each sample was measured using the micro BCA Procedure (Pierce, Rockford, IL). The mean protein content for all samples was calculated and each RSV-specific IgA value was corrected to the mean.
Data analysis Proportions were compared using x2 and Fisher’s exact tests as appropriate. Linear regression analysis was used to correlate variables and comparisons of mean log, titers were done using t-test. All P values were two sided.
RESULTS Sixty-four subjects were recruited and vaccinated. All volunteers completed the study. The mean age of subjects was 68 (range: 60-80); 21 were male and 43 female. Thirty-three participants were randomized to receive vaccine and 3 1 received placebo. Vaccine recipients were slightly older and had, on average, more underlying diseases than did the placebo recipients (Table I).
Acute safety Vaccination was well tolerated by both groups and no significant difference in systemic or local symptoms was noted when placebo and vaccine groups were compared (Table 2). Pain at the injection site was more common in the vaccine group (12 vs 3%; PzO.37); however, this difference was not statistically significant. The maximum temperature recorded by the one vaccine recipient with fever was 38.O”C (oral).
Vaccine 1996 Volume 14 Number 13 1215
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh
Table2
Acute side-effects Number of subjects reporting Vaccine Placebo (N=31) (r&33)
Reaction Local
Redness 1 cm Swelling 1 cm Pain
2
0
1 4
1 1
Temp 238°C Headache Myalgias
1 3 0
: 3
Table3
mean Log, titers of PFP-2 and placebo
Systemic
Post-vaccination
groups Week post-immunization 4 0
8
Placebo (N=3 1) EIA 12.3*1.2 13.2k1.6 MNA 13.1*1.9 MNB
12.4&l .3 13.9e1.8 13.Ok1.8
12.4il.3 13.2i1.7 N.D.
PFP-2 (N=33) EIA 12.6il.5 13.3i1.4 MNA 12.5i1.6 MNB
15.6~~1.ga 15.2il.l’ 13.8*1 .8e
15.7*1.8* 14.5*1 .2d N.D.
EIA=enzyme immunoabsorbent assay for IgG to F; MNA= microneutralization assay to A virus (Long strain); MNB= microneutralization assay to B virus (CH18537 strain); ND=not determined. “0 vs 4 weeks, PcO.001. bO vs 8 weeks, PcO.001. “0 vs 4 weeks, PcO.001. ‘0 vs 8 weeks, EO.003. ‘0 vs 4 weeks, PcO.001
Immunogenicity Vaccination with PFP-2 resulted in a statistically significant increase in the mean antibody titer to F as measured by EIA at 4 and 8 weeks (Table 3). No significant increase in antibody to the G protein was noted in any vaccinee. Seventy-six percent of PFP-2 recipients achieved a greater than or equal to fourfold rise in F EIA titers at 4 weeks, and by 8 weeks 87% had a significant rise in F antibody. Vaccination was also associated with a significant increase in serum neutralizing activity to group A (Long strain) virus at both 4 and 8 weeks (Table 3). Of those who responded with a significant rise in neutralizing titer to group A virus, mean titers did not drop significantly at the end of the RSV season (32 weeks) when compared with the mean titers at 8 weeks (14.7 f 1.6 vs 14.6 f 1.2). Neutralizing titers to group B virus (CH18537) were measured at 4 weeks and also showed a significant increase in the PFP-2 vaccinated group (Table 3). Four weeks after immunization, 16 of the 33 vaccinees (48%) had a greater than or equal to fourfold rise in neutralizing titers to Long strain RSV (group A) and 15 of 33 subjects (45%) showed a greater than or equal to fourfold rise in neutralizing titers to CH18537 strain (group B virus). Eleven vaccinees had a significant rise in neutralizing activity to both viruses. Notably, the four individuals who had a significant increase in neutralizing titers to group B virus only had high baseline neutralizing titers to A group virus (15.14, 13.64, 14.64, 14.64), which were all above the group mean baseline titer of 12.6* 1.5. Additionally, four of five subjects who responded only with an increase in neutralizing titers to
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Vaccine 1996 Volume 14 Number 13
group A virus had neutralizing titers to group B virus at or above the mean. Therefore, 20 of 33 vaccinees (61%) had a significant increase in serum neutralizing activity against RSV. Most individuals who had a significant rise in EIA titers also had an increase in serum neutralizing activity against RSV. Of the 29 individuals with a greater than or equal to fourfold rise in EIA titers, 20 (70%) had a greater than or equal to fourfold rise in group A and/or group B neutralizing titers, while 10 others had at least a twofold increase in neutralizing activity. Only three vaccinees with a vigorous EIA failed to develop an increase in neutralizing titers. Five of 33 vaccine recipients (15%) showed a greater than or equal to fourfold rise in nasal RSV-specific IgA against F protein; three had an eightfold rise, and two had a fourfold rise. No RSV-specific IgA rises were detected in the placebo group. No significant differences in the age, sex, percent ideal body weight, smoking status, chronic conditions, or medications was noted between vaccine responders and nonresponders as measured by a greater than or equal to fourfold neutralizing response to group A virus. However. antibody response was inversely correlated with pre-immunization neutralizing titers (Figure 1). The baseline neutralizing titers of the responders were significantly lower than nonresponders (12.4 * 1.3 vs 14.1f 0.9, WO.001). Additionally, when individual neutralizing titers to group A virus were examined at 4 weeks post vaccination all but three vaccine recipients had a titer 14.00.
Respiratory surveillance Of the possible 1024 person weeks of surveillance, 908 (89%) were available for study and all volunteers had an end-of-winter-season serum sample analyzed by EIA to detect asymptomatic RSV infection. Thirty-two of the 64 subjects reported 41 illnesses during the 5 month surveillance period. The number of respiratory illnesses reported was similar in the vaccine and placebo groups. Twenty-six of the 41 illnesses (63%) were evaluated clinically and most were upper respiratory infections (URI). No hospitalizations, pneumonia, or death occurred in either group. None of the vaccine recipients had identifiable RSV infection in contrast to three serologically confirmed infections in the placebo group. Serologic evidence of infection was defined as a greater than or equal to fourfold rise in antibody to the G protein which was not effected by vaccination. Two of these infections were associated with clinical illness, one URI, the other bronchitis. The third infection, identified by a greater than fourfold rise in RSV antibody in the end-of-season serum sample, was in an individual who did not report an illness. RSV was not cultured from any subject during illness evaluation.
DISCUSSION RSV infection in the elderly has been shown to cause substantial morbidity and mortality and thus prevention of this infection by vaccination is highly desirable. This study demonstrates that PFP-2 was well tolerated and
RSV vaccine in the elderly: A. R. Falsey and E. E. Walsh Neutralization
Titers of Individual
Vacinees l
0
Pre Vaccination Post Vaccination
l8 T 17 1G 15 Titer
(log 2)
,‘, I
10
T”“I,,,,I,,,,I,,,,i,,,,l,,,,l,,,,i 0
5
10
1.5
20
25
30
35
Subjert
Figure 1 Pre- and post-immunization
neutralizing
titers (IogJ to Group A virus of individual
immunogenic in persons over age 60. Although advancing age has been associated with diminished responses to a number of vaccines18, the 87% response by EIA and 61% response by neutralization are comparable to trials of PFP in healthy young adults and seropositive children ’ 9q20.In addition, the response rates of older adults in our study to PFP are also similar to response rates of older persons to inactivated influenza vaccines23. A “protective” level of serum neutralizing antibody for RSV has not been clearly established. However, evidence that serum antibody has a beneficial effect comes from a number of sources. Animal and human data suggest that circulating neutralizing antibody and mucosal antibodies may modify disease severity and be required to prevent reinfection. Passive transfer of neutralizing antibodies to the two major surface proteins of RSV (F and G) protects rodents against RSV challenge, as does immunization with these glycoproteins24,25. Human studies show that levels of neutralizing antibodies are inversely correlated with severity of illness in infants26. In a study of experimental reinfection of young adult volunteers, Hall et al. showed that neutralizing F and G antibody levels correlated with protection, although even at the highest levels of antibody 25% of subjects could still be infected27. In addition, recent studies on the benefits of IgG prophylaxis for RSV infections in high risk children also suggest humoral immunity is important28. Relatively little is known about the immune status to RSV in the elderly adult. Agius and colleagues found that the institutionalized elderly were capable of mounting a brisk serum IgA and IgG response to RSV infection29. In an earlier study, we found no association between EIA titers of serum IgG and severity of infection in nursing home patients3’. There was a trend towards lower neutralizing titers in those with more severe illness; however, statistical significance was not achieved. This study was hampered by the fact that sera were obtained after patients were identified as ill and may not have represented a true pre-illness baseline. All adults have had multiple RSV infections and have neutralizing antibody to RSV. However, it is possible that advancing age which is associated with declining immune function as well as less frequent exposures to respiratory viruses, results in decreasing serum neutralizing antibody titers to RSV. Data from our laboratory indicate that the mean serum neutralizing antibody titers
PFPQ vaccinees
of healthy older adults are significantly lower than those of young adults (unpublished data). In our study, response to vaccination was best correlated with pre-immunization neutralizing titers. Similar results have been noted with influenza vaccine studies where response rates of vaccinees decreased with increasing hemagglutination inhibition antibody titers31*32.It is possible that there is a maximal level of immunity which can be induced either by natural infection or vaccination after which no additional benefit is possible. In our present study, it was notable that, although only 48% of subjects had a greater than or equal to fourfold rise in neutralizing titers to group A virus, all but three vaccinees had a titer 14.00 postvaccination (Figure 2). Interestingly, in a previous study the mean baseline neutralization titers of older persons who became infected with RSV was 12.4 f 2.2 compared to 14.2& 2.2 in matched controls without infection (unpublished data). In summary, PFP-2 was found to be a safe and immunogenic RSV vaccine in healthy persons over age 60. Frail older persons with underlying heart and lung disease as well as those living in nursing homes are groups which are at highest risk for severe RSV disease. Since immune function may be influenced by a number of chronic conditions it will be important to assess PFP-2 immunogenicity in debilitated elderly adults in addition to the healthy elderly33. The frail elderly represent a rapidly growing segment of the population who are at risk for serious sequelae from viral respiratory illnesses and PFP-2 vaccine may prove to be useful in preventing RSV infection in this age group.
ACKNOWLEDGEMENTS This study was supported by a grant from Lederle Praxis Biologicals. We thank Joanne Prives for transcription assistance, Maria Formica for technical assistance, and Michelle Christenson for help in conducting the study.
REFERENCES 1
Fransen, H., Sterner, G., Forsgren, M. et al. Acute lower respiratory illness in elderly patients with respiratory syncytial virus infection. Acta Med. Stand. 1967, 182, 323-329
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5
6 7
8
9
10
11
12
13
14 15
16 17 18
19
Garvie, D.G. and Gray, J. Outbreak of respiratory syncytial virus infection in the elderly. Br. Med. J. 1980, 281, 1253-1254 Mathur, U. and Bentley, D.W. Concurrent respiratory syncytial virus and influenza A infections in the institutionalized elderly and chronically ill. Ann. Int Med. 1980, 93, 149-152 Morales, F., Calder, M.A., Inglis, J.M., Murdoch, P.S. and Williamson, J. A study of respiratory infections in the elderly to assess the role of respiratory syncytial virus. J. Infect. 1983, 7, 236-247 Sorvillo, F.J., Huie, S.F., Strassberg, M.A., Butsumyo, A., Shandera, W.X. and Fannin, S.L. An outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly. J. Infect 1984, 8, 252-256 Hart, J. An outbreak of respiratory syncytial virus infection in an old people’s home. J. Infect. 1984, 8, 259-261 Falsey, AR., Treanor, J.J., Betts, R.F. and Walsh, E.E. Viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings. J. Am. Geriatr. Sot. 1992, 40, 115-I 19 Falsey, AR., Cunningham, C.K., Barker, W.H. et al. Respiratory syncytial virus and influenza A infections in hospitalized elderly. J. Infect. Dis. 1995, 172, 389-394 Vikerfors, T., Grandien, M. and Olcen, P. Respiratory syncytial virus infections in adults. Am. Rev. Respir. Dis. 1987, 136, 561-564 Kimball, A.M., Foy, H.M., Cooney, M.K., Allan, I.D., Matlock, M. and Plorde, J.J. Isolation of respiratory syncytial virus and influenza viruses from the sputum of patients hospitalized with pneumonia. J. Infect. Dis. 1983, 147, 181-184 Wald, T.G., Miller, B.A., Shult, P., Drinka, P., Langer, L. and Gravenstein, S. Can respiratory syncytial virus and influenza A be distinguished clinically in older persons?. J. Am. Geriatr. Sot. 1995,43,170-174 Falsey, A.R., McCann, R.M., Hall, W.J. et a/. Acute respiratory tract infection in daycare centers for older persons. J. Am. Geriatr. Sot. 1995, 43, 30-36 Zaroukian, M.H., Kashyap, G.H. and Wentworth, B.B. Case report: respiratory syncytial virus-a cause of respiratory distress and pneumonia in adults. Am. J. Med. Sci. 1988, 295, 218-227 Fleming, D.M. and Cross, K.W. Respiratory syncytial virus or influenza?. Lancet 1993.342. 1507-l 510 Johnson, K.M., Bloom, H.H., ‘Mufson, M.A. and Chanock, R.M. Natural reinfection of adults by respiratory syncytial virus. N. En@. J. Med. 1962, 26, 66-71 Hemming, V.G. and Prince, G.A. Respiratory syncytial virus: babies and antibodies. Infect Agts. Dis. 1992, 1, 24-32 Salzman, R.L. and Peterson, P.K. lmmunodeficiency of the elderly. Rev. Infect. Dis. 1987, 9, 1127-1139 Powers, D.C. Immunological principles and emerging strategies of vaccination for the elderly. J. Am. Geriatr. Sot. 1992, 40, 81-94 Tristam, D.A., Welliver, R.C., Mohar, C.K., Hogerman, D.A., Hildreth, S.W. and Paradiso, P. lmmunogenicity and safety of respiratory syncytial virus subunit vaccine in seropositive children 18-36 months old. J. Infect. Dis. 1993, 167, 191-195
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Paradiso, P.R., Hildreth, S.W., Hogerman, D.A. et al. Safelyand immunogenicty of a subunit respiratory syncytial virus vaccine in children 24 to 48 months old. Pediatr. Infect. Dis. J. 1994,13, 792-798 21 Hendry, M.R., Burns, J.C., Walsh, E.E., Graham, B.S., Wright, P.F. and Hemming, V.G. Strain-specific antibody responses in infants undergoing primary infection with respiratory syncytial virus. J. infect. Dis. 1988, 157, 640-847 22 Anderson, L.J., Hierholtzer, J.C., Bingham, P.G. and Stone, Y.O. Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J. C/in. Micro. 1985, 2, 1050-1052 23 Treanor, J.J., Mattison, H.R., Dumyati, G. et a/. Protective efficacy of combined live intranasal and inactivated influenza A virus vaccines in the elderly. Ann. hf. Med. 1992,117,625-633 24 Walsh, E.E., Schlesinger, J.J. and Brandriss, M.W. Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies. Infect. Immun. 1984, 43,756-758 25 Walsh, E.E., Hall, C.B., Briseili, M., Brandriss, M.W. and Schlesinger, J.J. Immunization with the glycoprotein subunits of respiratory syncytial virus to protect cotton rats against viral infection. J. Infect. Dis. 1987, 155, 1198-1204 26 Glezen, W.P., Paredes, A., Allison, J.E., Taber, L.H. and Frank, A.L. Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody. J. Pediatr. 1981, 98, 708-715 27 Hall, C.B., Walsh, E.E., Long, C.E. and Schnabel, KC. Immunity and frequency of infection with respiratory syncytial virus. J. Infect. Dis. 1991, 163, 693-698 28 Groothius, J.R., Simoes, A.F., Levin, M.J. et a/. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. N. Engl. J. Med. 1993, 329, 1524-l 530 29 Agius, G., Dindinaud, G., Biggar, R.J. et al. An epidemic of respiratory syncytial virus in elderly people: clinical and serological findings. J. Med. Viral. 1990, 30, 117-127 30 Falsey, A.R. and Walsh, E.E. Humoral immunity to respiratory syncytial virus infection in the elderly. J. Med. Vim/. 1992, 36, 39-43 31 Wenzel, R.P., Hendley, J.O., Sande, M.A. and Gwaltney, J.M. Revised (1972-1973) bivalent influenza vaccine: serum and nasal antibody responses to parenteral vaccination. J. Am. Med. Assoc. 1973, 226,43w38 32 Beyer, W.E., Palanche, A.M., Batjet, M. and Masurel, N. Antibody induction by influenza vaccines in the elderly: a review of the literature. Vaccine 1989, 7, 385-394 33 Gross, P.A., Quinnan, G.V., Wekster, M.E., Setia, U. and Douglas, R.G. Relation of chronic disease and immune response to influenza vaccine in the elderly. Vaccine 1989, 7, 303-308 20