T-Cell Responses in Adults During Natural Respiratory Syncytial Virus Infection.

Background: The pathogenesis of respiratory syncytial virus (RSV) in older adults may be due to age-related T-cell immunosenescence. Thus, we evaluated CD4 and CD8 T-cell responses during RSV infection in adults across the age spectrum. Methods: Peripheral blood mononuclear cells collected during RSV infection in adults, age 26-96 years, were stimulated with live RSV and peptide pools representing F, M, NP, and G proteins and analyzed by flow cytometry. Results: There were no significant age-related differences in frequency of CD4+ T cells synthesizing interferon (IFN)γ, interleukin (IL)2, IL4, IL10, or tumor necrosis factor (TNF)α or in CD8+IFNγ+ T cells. IL4+CD4+ T-cell numbers were low, as were IL13 and IL17 responses. However, in univariate analysis, CD4 T-cell IFNγ, IL2, IL4, IL10, and TNFα responses and CD8+IFNγ+ T cells were significantly increased with more severe illness requiring hospitalization. In multivariate analysis, viral load was also associated with increased T-cell responses. Conclusions: We found no evidence of diminished RSV-specific CD4 or CD8 T-cell responses in adults infected with RSV. However, adults with severe disease seemed to have more robust CD4 and CD8 T-cell responses during infection, suggesting that disease severity may have a greater association with T-cell responses than age.

Intermittent compression pump for nonhealing wounds in patients with limb ischemia. The Mayo Clinic experience (1998-2000).

BACKGROUND: The aim of this retrospective observational study was to review the use of an intermittent pneumatic compression device on nonhealing wounds in patients with critical limb ischemia at Mayo Clinic Rochester. METHODS: The setting was a community and referral multidisciplinary wound care clinic. The authors analysed 107 patients, median age 73, with critical limb ischemia and active ulcers started using a compression device between 1998 and 2000; 101 patients had lower extremity ulcers, and 25% had a history of amputation, and 64% had diabetes. Of all the wounds, 64% were multifactorial in etiology, and 60% had associated transcutaneous oxygen tension levels below 20 mmHg. Patients were typically asked to use the device at home on the affected limb(s) for 6 hours daily. The main outcome criterion was complete wound healing with limb preservation. RESULTS: The median follow-up after initiation of treatment was 6 months. Complete wound healing with limb preservation was achieved by 40% of patients with TcPO(2) levels below 20 mmHg; by 48% with osteomyelitis or active wound infection; by 46% with diabetes treated with insulin; and by 28% with a previous amputation. Half of all amputations occurred in patients with prior amputations. Seven patients discontinued the device because of pain experienced with its use. CONCLUSIONS: Patients with critical limb ischemia and nonhealing wounds at high risk of amputation can achieve complete wound healing and limb preservation by using an intermittent pneumatic compression device.

Detection of IgA and IgG but not IgE antibody to respiratory syncytial virus in nasal washes and sera from infants with wheezing.

BACKGROUND AND OBJECTIVE: The capacity of respiratory syncytial virus (RSV) to stimulate an IgE antibody response and enhance the development of atopy and asthma remains controversial. Nasal washes and sera from 40 infants (20 with wheezing, 9 with rhinitis, and 11 without respiratory tract symptoms) were obtained to measure IgE, IgA, and IgG antibody to the immunodominant, F and G, virion proteins from RSV. STUDY DESIGN: Children (aged 6 weeks to 2 years) were enrolled in the emergency department during the mid-winter months and seen at follow-up when they were asymptomatic. All nasal washes were tested for RSV antigen. Determinations of antibody isotypes (IgE, IgA, and IgG) to RSV antigens were done in nasal washes and sera by using an enzyme-linked immunosorbent assay. In a subset of nasal washes, IgE to RSV was also evaluated by using a monoclonal anti-F(c)E antibody-based assay. RESULTS: Fifteen patients with wheezing, two with rhinitis, and one control subject tested positive for RSV antigen at enrollment. Thirteen patients with wheezing were <6 months old, and most (77%) were experiencing their first attack. Among the children with positive test results for RSV antigen, an increase in both nasal wash and serum IgA antibody to RSV-F(a) and G(a) was observed at the follow-up visit. However, there was no evidence for an IgE antibody response to either antigen. CONCLUSION: Both IgA and IgG antibodies to the immunodominant RSV-F(a) and G(a) antigens were readily detected in the nasal washes and sera from patients in this study. We were unable to demonstrate specific IgE antibody to these antigens and conclude that the production of IgE as a manifestation of a T(H)2 lymphocyte response to RSV is unlikely.

Response of C-reactive protein and serum amyloid A to influenza A infection in older adults.

Influenza epidemics are associated with significant morbidity and mortality in the elderly, with a substantial proportion of deaths due to cardiovascular events. Elevations of acute-phase proteins have been associated with an increased risk of atherosclerotic events. Therefore, serum amyloid A (SAA) and C-reactive protein (CRP) were measured during influenza illness and 4 weeks later in 7 young persons, 15 elderly outpatients, and 36 hospitalized adults. Striking elevations were seen in mean acute SAA and CRP levels in all groups, but hospitalized patients had the highest levels (SAA, 503 vs. 310 microg/mL [P=.006]; CRP, 120 vs. 34 microg/mL [P<.001]). The presence of dyspnea, wheezing, and fever was also associated with high CRP levels. Influenza infection is associated with significant elevations of SAA and CRP levels in elderly patients, especially those who require hospitalization. It is possible that direct effects of CRP may exacerbate preexisting atherosclerotic lesions and may help explain cardiovascular events associated with acute influenza.

Reverse transcription polymerase chain reaction (RT-PCR) for diagnosis of respiratory syncytial virus infection in adults: use of a single-tube "hanging droplet" nested PCR.

Rapid diagnosis of Respiratory Syncytial virus (RSV) infection is difficult in elderly persons due to the low quantities of virus shed. Therefore, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect viral RNA in respiratory secretions. A single-tube nested RT-PCR that used primers from a conserved F gene sequence was developed using a "hanging droplet" to physically separate outer and inner primer pairs during the first round of the PCR reaction. This was accomplished by placing the inner primers in a 5 microL droplet on the underside on the reaction tube cap and mixing after the first round of PCR. As few as 0.05 pfu of virus could be detected and gave positive results with RSV strains that represented the major groups and subgroups of RSV grown in tissue culture. The nested PCR was approximately 100-fold more sensitive than standard single primer PCR reactions and equivalent to standard two-tube nested PCR. Viral RNA was detected in nasopharyngeal samples from 12 of 15 culture positive illnesses and in 5 of 17 culture-negative, seropositive illnesses despite specimen volumes less than 1 microL in some samples. The method was also positive in 14 of 25 elderly volunteers inoculated with a live attenuated RSV vaccine candidate, only one of whom was culture positive. Use of a nested RT-PCR significantly improves the ability to detect RSV in respiratory samples and should improve the ability to rapidly diagnose RSV infection in adults, especially in the elderly.

Respiratory syncytial virus infection in adults.

Respiratory syncytial virus (RSV) is now recognized as a significant problem in certain adult populations. These include the elderly, persons with cardiopulmonary diseases, and immunocompromised hosts. Epidemiological evidence indicates that the impact of RSV in older adults may be similar to that of nonpandemic influenza. In addition, RSV has been found to cause 2 to 5% of adult community-acquired pneumonias. Attack rates in nursing homes are approximately 5 to 10% per year, with significant rates of pneumonia (10 to 20%) and death (2 to 5%). Clinical features may be difficult to distinguish from those of influenza but include nasal congestion, cough, wheezing, and low-grade fever. Bone marrow transplant patients prior to marrow engraftment are at highest risk for pneumonia and death. Diagnosis of RSV infection in adults is difficult because viral culture and antigen detection are insensitive, presumably due to low viral titers in nasal secretions, but early bronchoscopy is valuable in immunosuppressed patients. Treatment of RSV in the elderly is largely supportive, whereas early therapy with ribavirin and intravenous gamma globulin is associated with improved survival in immunocompromised persons. An effective RSV vaccine has not yet been developed, and thus prevention of RSV infection is limited to standard infection control practices such as hand washing and the use of gowns and gloves.

Evaluation of the live attenuated cpts 248/404 RSV vaccine in combination with a subunit RSV vaccine (PFP-2) in healthy young and older adults.

The safety and immunogenicity of the live attenuated cold-passaged, temperature-sensitive (cpts) 248/404 respiratory syncytial virus (RSV) A2 and the RSV A2 purified F glycoprotein (PFP-2) vaccine candidates were evaluated in a placebo-controlled trial in 60 healthy young adults and 60 healthy elderly subjects using simultaneous and sequential (cpts 248/404 followed by PFP-2) vaccination schedules. Both vaccines were well tolerated. The cpts 248/404 vaccine was moderately infectious in both young and old volunteers, but was highly restricted in replication in those who were infected. After both vaccines, RSV neutralizing antibody (neut Ab) titers increased fourfold in 22% of young subjects and in 16% of elderly subjects. Of those with low levels of RSV neut Ab (titer <9), 10/12 (83% of) young subjects and six/eight (75% of) elderly subjects had a >/=four fold rise in neut Ab titer. Young and elderly subjects immunized simultaneously had similar serum IgG and IgA postimmunization titers to RSV F (IgG, 16.4 vs 16.2, IgA 11.6 vs 12. 5, respectively) as did those who were immunized sequentially (IgG 17.4 vs 17.0, IgA 13.0 vs 13.5). In both age groups, sequential immunization elicited higher postimmunization RSV F IgG and IgA titers than simultaneous immunization. Further studies that combine the PFP-2 subunit vaccine with a less attenuated RSV vaccine should be performed.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Respiratory syncytial and other virus infections in persons with chronic cardiopulmonary disease.

Respiratory syncytial virus (RSV) has been increasingly recognized as an important cause of serious respiratory illness in some adult populations, including those with underlying cardiopulmonary diseases. However, the precise incidence and the clinical impact of RSV in this group are unknown. Therefore, the incidence and clinical impact of RSV infection in persons with chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF) who reside in the community were prospectively evaluated over two consecutive winters in 134 persons. Eight RSV (incidence of 4.3 per 100 subject-winters), 13 influenza A (incidence of 7.0 per 100 subject-winters), seven rhinovirus, nine coronavirus, and two parainfluenza virus infections were identified. The clinical illnesses associated with RSV and influenza A virus were similar, causing both upper and lower respiratory signs and symptoms. The clinical impact was significant as three of eight RSV-infected subjects were hospitalized compared with six of 13 influenza A-infected persons and zero of seven rhinovirus-infected persons.

Comparison of respiratory syncytial virus humoral immunity and response to infection in young and elderly adults.

Little information about immunity to respiratory syncytial virus (RSV) and disease pathogenesis in elderly persons exists. Humoral immunity to RSV was assessed in 41 young, 56 healthy elderly, and 49 frail elderly adults by measuring baseline RSV specific IgG by enzyme immunoassay (EIA) and microneutralization assay (MNA) in serum. A comparison of the immune response of 11 young and 28 elderly persons with natural RSV infection was also performed. Despite significant differences in age and functional status, no decreases in RSV antibody levels by either EIA or MNA were noted in the elderly compared with the young. Mean baseline MNA titers expressed as log2 were 10.5 +/- 1.1 for the young, 10.5 +/- 1.5 for the healthy elderly, and 10.9 +/- 1.6 for the frail elderly. The frail elderly who attend a daycare had the highest RSV titers to F by EIA at 16.6 +/- 2.0, compared with 15.4 +/- 1.4 and 15.1 +/- 1.4 in the healthy elderly and young, respectively. This finding may reflect recent infection due to their communal setting or increased production of non-neutralizing antibody. The immune response of older persons to RSV infection was as vigorous as the younger subjects, with 79% having a >/=fourfold rise in MNA titers compared to 64% in the young. These data suggest that the severe clinical manifestations of RSV in the elderly are not due to a significant defect in humoral immunity.

Human monoclonal antibodies isolated from spontaneous Epstein-Barr virus-transformed tumors of Hu-SPL-SCID mice and specific for fusion protein display broad neutralizing activity toward respiratory syncytial virus.

Two human monoclonal antibodies, RF-1 and RF-2, specifically recognize the fusion protein of the human respiratory syncytial virus (RSV). These were isolated from spontaneous tumors in SCID mice reconstituted with human splenocytes and boosted with fusion protein. The tumors consisted of Epstein-Barr virus-transformed human B cells in animals with antigen-specific antibody titers>105. The binding affinity of RF-1 and RF-2 to the fusion protein is 1010 and 109 M-1, respectively. The antibodies bind specifically to a conformational epitope of the fusion protein on RSV-infected HEp-2 cells. Both antibodies display virus-neutralizing properties in vitro at concentrations varying between 8 and 1000 ng/mL. Virus neutralization applies to a broad variety of wild and laboratory-adapted virus strains belonging to both virus types A and B. These antibodies are potential candidates for passive immunotherapy of severe RSV infections.

A simple and reproducible method for collecting nasal secretions in frail elderly adults, for measurement of virus-specific IgA.

The standard method for collection of respiratory secretions, by use of a nasal wash (NW) to measure virus-specific IgA, is problematic in frail elderly adults. Therefore, a simplified collection approach using a nasal swab (NS) is described. NW and NS samples were collected from healthy young and frail elderly adults, and IgA titers to respiratory syncytial virus (RSV) fusion and attachment glycoproteins were determined by enzyme immunoassay. Correlation between IgA titers in NW and NS was excellent for each of the antigens (correlation coefficients,.71-.93). In addition, NS results were reproducible when frail elderly subjects were sampled several weeks apart and were nearly equivalent to results from NW samples. The ability to sample nasal secretions by use of an NS when an NW is not technically feasible will facilitate the study of mucosal immunity to RSV as well as the study of mucosal response to candidate RSV vaccines in frail elderly populations.

Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein.

Five monoclonal antibody (MAb) neutralization escape mutants of respiratory syncytial virus (RSV) were produced by growing the Long strain RSV (group A virus) in the presence of a neutralizing, group cross-reactive MAb specific for the attachment protein (G). Four viruses (RSV-2, -6, -14 and -15) had amino acid replacements clustered within a highly conserved centrally located 13 amino acid region (position 164-176). Reactivity with group A-specific MAbs and with polyclonal anti-G serum was maintained and growth kinetics were unaffected. An additional virus (RSV-3) had four amino acid substitutions in the cytoplasmic tail and transmembrane region of G, and had restricted growth and formed small syncytia. Immunofluorescent and Western blot analysis indicated that G protein was not membrane associated and had reduced incorporation into the virion, thereby escaping neutralization by L9 and polyclonal anti-G serum. The predominant form of G produced by RSV-3 was found in infected cell supernatants, consistent with the size of secreted G.

Relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults.

The relationship of serum antibody to respiratory syncytial virus (RSV) and risk of RSV infection was prospectively evaluated in frail elderly persons. Baseline blood samples from 22 subjects who developed symptomatic RSV infection during the 26-month study and from 22 control subjects who did not become infected with RSV were compared. The mean serum IgG titer to RSV fusion protein was significantly lower in the RSV-infected group than in the controls (15.4 +/- 1.6 vs. 16.4 +/- 1.8, P = .05), as were the neutralizing titers to group A RSV (12.4 +/- 2.2 vs. 14.2 +/- 2.2, P = .008) and group B virus (9.1 +/- 2.1, vs. 10.3 +/- 1.5, P = .01). These results suggest that older adults with low titers of serum neutralizing antibody may be at greater risk of developing symptomatic RSV infection than those who have high antibody titers.

Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in the institutionalized elderly.

The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in an open label study in 37 frail institutionalized persons over age 65. Vaccination was well tolerated without significant side-effects. Thirty-six of 37 volunteers completed the study. Nineteen of 36 (53%) vaccinees had a greater than or equal to fourfold increase in IgG to F protein at 4 weeks and 17 (47%) had a greater than or equal to fourfold rise in neutralizing titers to either group A or B virus. Although response rate to PFP-2 vaccine in the frail elderly was somewhat diminished compared to results in the healthy elderly, the vaccine was well tolerated and relatively immunogenic.

Severity of respiratory syncytial virus infection is related to virus strain.

The relationship between respiratory syncytial virus (RSV) strain and disease severity was assessed in 265 hospitalized infants over a 3-year period (1988-1991). A severity index of clinical and physiologic parameters was used to grade illness severity. Multivariate analysis of 134 infants infected with group A RSV strains and 131 infants infected with group B strains indicated that prematurity, underlying medical conditions, group A RSV infection, and age < or =3 months were independently associated with severe disease. Odds ratios for severe disease for these risk factors were 1.83, 2.84, 3.26, and 4.39, respectively. Among infants without underlying medical conditions, group B RSV infection rarely required ventilatory support, in contrast to group A infections (1/90 vs. 13/107; P < .006), and had significantly lower severity indices (mean +/- SD, 0.6 +/- 9 vs. 1.3 +/- 1.9; P = .05). Results confirm earlier findings that group A RSV infection results in greater disease severity than group B infection among hospitalized infants.

Respiratory syncytial virus infection in the elderly.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and children throughout the world. Respiratory syncytial virus infections in the elderly represent reinfections in the hosts who have had many prior episodes. Thus, RSV infections are usually not considered serious in adults, since reinfections are generally known to result in mild disease. Nevertheless, in adults, as in children, the infection has been reported to cause altered airway resistance and exacerbation of chronic obstructive lung disease. In people over 60 years of age, RSV usually causes mild nasal congestion, but can also result in fever, anorexia, pneumonia, bronchitis, and even death. Diagnosis of RSV infection in the elderly by the standard methods used in children is not as successful as in the latter group. This may be due to a combination of factors such as shorter shedding phase, lower viral titers, and dry mucosa. An alternative, rapid, and direct viral diagnostic method, the polymerase chain reaction, has recently been introduced in the diagnosis of RSV infections.

Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60.

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-effects. Twenty-nine of 33 vaccinees (87%) showed a greater than or equal to fourfold rise in serum IgG to the F protein 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 and/or group B RSV. Response to vaccination was inversely correlated with pre-immunization serum neutralizing titers. Active surveillance throughout the ensuring winter identified three RSV infections in the placebo group and none in the vaccine group. Thus, PFP-2 was found to be safe and immunogenic in healthy older adults.

Humoral, mucosal, and cellular immune response to topical immunization with a subunit respiratory syncytial virus vaccine.

The efficacy of topical immunization with the respiratory syncytial virus (RSV) fusion (F) protein was tested in mice using cholera toxin B chain (CTB) as an adjuvant. The dose of CTB required for the adjuvant effect (as measured by local and systemic antibody stimulation) and protection from viral challenge was > or = 5 micrograms. With this dose, mice immunized intranasally with CTB plus F protein were highly protected from viral replication in the upper and lower respiratory tract. This protection was associated with the induction of mucosal IgA and serum IgG and neutralizing antibody. Addition of parenteral immunization with F protein to the topical vaccination provided protection from viral challenge nearly equivalent to immunization with live RSV intranasally. Topical and parenteral immunization with F protein was not associated with induction of splenic cytotoxic T cells, in contrast to live virus given intranasally.

Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit.

Local IgA and IgG antibodies against respiratory syncytial virus (RSV) were induced in the respiratory tract of mice following intranasal vaccination with an RSV chimeric FG glycoprotein and cholera toxin B (CTB) as a mucosal adjuvant. Local antibody production was not induced following parenteral immunization with FG administered in alum adjuvant. While both vaccination protocols induced serum antibodies against RSV and protected the lower respiratory tract from RSV infection, only intranasal FG/CTB afforded protection of the upper respiratory tract. These data suggest that vaccination via the mucosal route may be superior to vaccination by a parental route in providing complete protection against RSV.