key: cord-0683487-dwkjwst1
authors: Turner, Ronald B.; Felton, Alma; Kosak, Kenneth; Kelsey, Douglas K.; Meschievitz, Carlton K.
title: Prevention of Experimental Coronavirus Colds with Intranasal α-2b Interferon
date: 1986-09-03
journal: J Infect Dis
DOI: 10.1093/infdis/154.3.443
sha: db342c89d2ebda647a1aa46751b190e27248c871
doc_id: 683487
cord_uid: dwkjwst1

Fifty-five volunteers treated with either intranasal recombinant interferon (rIFN; 2 × 10(6) IU/day) or placebo for 15 days were exposed to coronavirus by direct intranasal inoculation on the eighth day of treatment. Symptom scores were recorded, and cultures of virus were taken daily for all volunteers for seven days after inoculation. Nineteen (73%) of the 26 placebo recipients met symptom-score criteria for a cold, compared with 12 (41%) of the IFN recipients (P = .02). The mean nasal symptom scores in the placebo and IFN groups were 9.2 and 5.4, respectively (P = .03), and the mean total symptom scores in the two groups were 23.2 and 9.4, respectively (P = .003). The mean number of days with a total symptom score >4 was 1.6 in the placebo recipients and 0.5 in the rIFN recipients (P = .02). Prophylactic intranasal rIFN effectively shortened the duration and reduced the severity of coronavirus cold symptoms.

Common colds occcur an average of two to four times per year per adult and six to 10 times per year per child [1, 2] . Recent studies have examined the efficacy of interferon prophylaxis for prevention of colds in the family setting [3, 4] . These studies indicate that rhinovirus colds can be effectively prevented. The effect on total respiratory illness, however, is less impressive and suggests that a major impact on the incidence of the common cold will require preventing infections caused by other viral pathogens.

Coronaviruses are believed to be responsible for at least 10070 of upper-respiratory-tract illnesses [5] , although information about coronavirus epidemiology is limited by the lack of reliable methods of isolating virus from clinical specimens. A recent report [6] suggests that several immunologically distinct strains of coronavirus exist that are capable of causing human respiratory tract infections. Infection confers protection to the homologous strain of virus; however, the duration of this immunity is not known. The existence of several viral strains and the uncertainty about the possibility of reinfection suggest that prevention of coronavirus infection by active immunization will be difficult. A previous study [7] and the observation that coronavirus 229E is sensitive to recombinant human a-2b interferon (rIFN) in vitro (authors' unpublished observations) suggest that intranasal interferon may be useful for treating or preventing coronavirus colds. The purpose of this randomized, double-blind study was to determine the efficacy of rIFN in preventing coronavirus colds in human volunteers.

Volunteers. Healthy young adult volunteers were recruited from the University of Utah. Individuals who reported upper-respiratory-tract illness in the preceding week or who were taking oral or intranasal medications that would interfere with infection or assessment of symptoms were excluded. Each volunteer was documented to have a normal, complete blood cell count, blood chemistry, and urinalysis before participating in the study. No attempt was made to select antibody-free volunteers for the study. All but two volunteers had prechallenge titers of antibody >1 :4.

Interferon. rIFN (Schering, Kenilworth, NJ) was provided in lyophilized form and reconstituted to a final concentration of 5 x 10 6 IU/m!. A placebo that was identical to the rIFN in protein content, pH, tonicity, and appearance was also provided. The in-terferon or placebo treatment was administered as a nasal spray by using a metered pump device that delivered 0.05 ml per spray. Each nostril was sprayed with 0.1 ml of medication twice each day.

Virus inoculum pool. The virus inoculum used in this study was a coronavirus 229E strain provided by Dr. D. A. J. Tyrrell (MRC Common Cold Unit, Salisbury, England). The inoculum pool consisted of nasal wash from a human volunteer with a coronavirus cold. This material was tested for the presence of pathogenic bacteria and then diluted in Earle's balanced salt solution to a titer of rv200 TCIDso/ml before inoculation into volunteers. Each volunteer was challenged by intranasal inoculation with 0.25 ml/nostril for a total inoculum of rv 100 TCID so.

Symptom scoring. Symptom scoring was done by a modification of a previously published method [8] . Each volunteer was asked to record symptom scores daily during the study. Symptoms such as fever, chills, headache, muscle ache, sneezing, sore throat, hoarseness, and cough and the nasal symptoms of rhinorrhea and nasal obstruction were judged as absent to severe by assigning a score of 0-3. Symptom criteria for a cold were a total symptom score of at least 5, in addition to either the presence of rhinorrhea on at least three of the six study days after coronavirus inoculation or the subjective impression of the volunteer that he had a cold.

Isolation ofvirus. Specimens for isolation of virus were collected daily by nasal wash for seven days after challenge. Each nostril was rinsed with 5 ml of PBS that was then mixed 3:1 (vol/vol) with fourtimes concentrated beef-heart infusion broth containing 4070 fetal calf serum and antibiotics (vancomycin, gentamicin, and amphotericin B). Antibody to IFN sufficient to neutralize 10 4 IV of rIFN was then added to each milliliter of sample. After incubation at room temperature for at least 30 min, 0.2 ml of sample was inoculated into each of two tube cultures of human embryonic lung fibroblast cells (MRC-5) maintained in Eagle's MEM containing 2070 fetal calf serum. One tube from each specimen was placed in a stationary rack and the other in a roller drum; all tubes were incubated at 33 C. The tubes were examined ever other day for the development of CPE. Tubes with apparent CPE were passaged to fresh MRC-5 cells and again observed. Samples with CPE both in the original tube and after passage were considered positive for coronavirus. Isolates of virus were confirmed as coronavirus by ELISA. A nasal wash done before challenge was inoculated into cynomologous monkey kidney, human foreskin fibroblast, HEp-2, and mink lung cells to detect unsuspected viruses.

ELISA for coronavirus antigen. Antiserum to coronavirus 229E was produced in C3H mice and in guinea pigs. The mouse antibody was diluted 1:500 in 0.1 M sodium bicarbonate (pH 9.0) and adsorbed to the wells of polystyrene microtiter plates by incubation overnight at 4 C. V nreacted binding sites were blocked by incubating the plates for an additional hour with Eagle'sMEM containing 10070 fetal bovine serum. After rinsing with tap water, 50 J..lI of uninfected media or cell culture supernatant from a coronavirus isolate was placed into each antibodycoated well and diluted 1:3 with media. The samples were incubated for 1.5-2.0 hr at room temperature, and the plates were then washed three times with tap water. The second antibody (guinea pig antibody to 229E) was then added to each well and incubated for 1 hr at room temperature. After again washing the plate with tap water, we incubated the third antibody, horseradish peroxidase-conjugated rabbit antibody to guinea pig IgG (Cappell Laboratories, West Chester, Pa) in the plate 1'.Jr 1 hr at room temperature. The plate was again washed with tap water, and the substrate, 0.05070 1, 2-phenyldiamine in PBS (pH 7.4) with 0.03070 H 202 , was added to each well and incubated for 20 min at room temperature. The absorbance was measured at a wavelength of 492 nm by using a spectrophotometer (Titertek Multiskan'"; Flow Laboratories, McLean, Va). Specimens were considered positive if the absorbance was >2 SD above the mean absorbance of the negative control wells.

Serology. An assay for neutralizing antibody was done in 96-well microtiter plates. Serial twofold dilutions of serum were incubated with 17 TCID so of coronavirus 229E for five days at 33 C. Wells were examined for CPE, and the titer of neutralizing antibody was calculated. Volunteers were considered infected if there was at least a fourfold increase in titer of neutralizing antibody over the course of the study.

Study design. Assignment to treatment groups was done randomly and was double-blind for volunteers and investigators. All volunteers were treated with intranasal rIFN (10 6 IV twice a day) or placebo for one week before challenge with the study virus, on the day of challenge, and for one week after challenge. Thus, treated volunteers received a total of 30 X 10 6 IV of rIFN over 15 days. Volunteers indicated that each dose was taken by marking a form at the time the medication was administered; these forms were checked by the study staff each time the volunteers were seen. Symptom scores were monitored beginning on the first day of rIFN or placebo treatment and continuing for three weeks. On the eighth day of interferon or placebo treatment, all volunteers were challenged with the study virus. Nasal washes for detecting shedding of virus were collected for seven days beginning on the day after challenge.

Statistical analysis. Proportions were compared by a one-sided Fisher's exact test. Symptom scores in the two groups of recipients were compared by a two-sided Mann-Whitney U test.

Twenty-seven volunteers received placebo and twenty-nine received rIFN. One of the placebo recipients reported a symptom score of 8 on the day of coronavirus inoculation and was excluded from further data analysis.

Effect of rIFN on symptoms. The use of rIFN significantly reduced the symptoms of coronavirus colds (table 1) . Nineteen (73070) of the 26 placebo recipients met symptom-score criteria for a cold, compared with 12 (41070) of the 29 rIFN recipients (P = .02). The mean nasal symptom score for the six days after challenge was 9.2 in the placebo recipients and 5.4 in the rIFN recipients (P = .03). The total symptom scores in the two groups were 23.2 and 9.4, respectively (P = .003). The mean number of days with a total symptom score >4 to .seroconvert than were those with lower initial titers. Four (22010) of 18 volunteers with an initial titer >1 :32 had at least a fourfold increase in antibody titer, compared with 29 (85010) of 34 volunteers with an initial titer <1 :32 (P < .001). Convalescent sera were not available for three volunteers. The presence of antibody in the acute serum specimens had no discernible effect on the rate of isolation of virus or on symptom scores. Side effects ofrIFN. Five placebo and 10 rIFN recepients reported bloody nasal mucus at least once during the 15 days of the study. Four placebo recipients and six rIFN recipients reported bloody nasal mucus in the first week of observation, before inoculation with coronavirus. These differences were not statistically significant. There was also no difference in nasal or total symptom scores in the two groups before challenge. Nasal speculum examinations done on days 1, 8, and 15 revealed no mucosal ulcerations in either of the treatment groups; however, bleeding sites were seen in eight (28010) of the rIFN recipients and in one (4010) of the placebo recipients (P = .05). Abnormalities in blood counts, blood chemistries, or urinalyses that could be attributed to rIFN treatment were not seen. No volunteers withdrew from the study due to side effects of the interferon or placebo treatment.

The results of this study indicate that prophylactic intranasal rIFN at a dose of 2 x 10 6 IU/day effectively reduces both the duration and severity of coronavirus colds. This beneficial effect on symptoms was not, however, associated with a decreased incidence of infection. In a previous study, Higgins et al. [7] gave 12 x 10 6 IU of intranasal interferon/day to volunteers beginning one day before Turner et al. coronavirus challenge. In that study, both infection and illness were reduced in the interferon-treated group. The ability of interferon to modify illness at doses that do not prevent infection has also been reported in studies of rhinovirus infection. Sarno et al. [9] reported that 2.4 and 10 x 10 6 IU of interferon daily modified rhinovirus illness but did not affect the rate of viral infection. At higher daily doses of interferon, however, both illness and infection were significantly reduced. The mechanism by which symptoms are reduced by interferon without an associated reduction in infection is not known. No attempt was made inthis study to quantify the amount of virus shed in the nasal secretions or to determine the effect of interferon on the host response to the virus. Either of these factors may be important in the production of symptoms during respiratory viral infection [10, 11] .

The study of the human coronaviruses has been hampered by difficulties in isolating the virus from clinical specimens. In this study, 13 of the 26 placebotreated volunteers had virus isolated from nasal wash specimens. Unlike many other viruses, the coronaviruses do not produce a distinctive CPE in cell culture and cannot be reliably distinguished from nonspecific cytotoxicity. To assure the specificity of the cell culture isolations, we routinely passaged cultures with apparent coronavirus CPE to fresh cells. Only those specimens that again produced CPE were considered positive for coronavirus when the data were analyzed. The positive cultures were confirmed as coronavirus by an ELISA. This evidence for the specificity of isolation of virus is further supported by the fact that 32 (74010) of the 43 volunteers with virus isolated also had a fourfold increase in titers of neutralizing antibody to the virus. Of the 11 patients who did not have a significant rise in titers of neutralizing antibody, nine had an initial titer 1:32, and the other two did not have convalescent sera available for analysis. An inverse relation between the frequency of seroconversion and the level of preexisting antibody to coronavirus 229E has been previously reported [12] .

An interesting and important feature of coronavirus epidemiology is the ability of this virus to infect and produce symptoms associated with shedding of virus in spite of the presence of preexisting neutralizing antibody [12] . The presence of high titers of antibody had no effect on either illness or infection in this study. Volunteers with high initial titers of antibody were less likely to have a fourfold rise in titer, an effect that has been previously reported. Reed [6] has recently reported that there are strain differences within the serogroup 229E and that infection may provide protective immunity to the homologous strain but not to heterologous strains.

The role that intranasal rIFN therapy can play in the prevention or treatment of coronavirus colds remains to be determined. Regimens that have been studied for preventing colds include prophylaxis during epidemic periods and contact prophylaxis in the home [3, 4, 13] . The lack of information about coronavirus epidemiology makes it difficult to predict if either or both of these regimens will be practical for preventing coronavirus infection. It is also not known whether rIFN will be effective for treating coronavirus colds. Further studies that address the issue of practical dosing regimens for preventing or treating the common cold are needed before rIFN can be useful in a clinical setting.

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