Effect of Naturally Acquired Antibody, and Demonstration Serum Antibody

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Journal of Clinical Investigation
Vol. 43, No. 1, 1964
Studies with Rhinoviruses in Volunteers: Production of Illness,
Effect of Naturally Acquired Antibody, and Demonstration
of a Protective Effect Not Associated with
Serum Antibody *
THOMAS R. CATE, ROBERT B. COUCH, AND KARL M. JOHNSON t
(From the U. S. Public Health Service;National Institute of Allergy and Infectious Diseases,
Laboratory of Clinical Investigations and Laboratory of Infectious Diseases,
Bethesda, Md.)
The term "rhinovirus" (1-3) is now used to Johnson and associates (16), our studies were
designate a group of immunologically distinct re- undertaken in volunteers to determine the viruses'
spiratory viruses, possessing many properties of properties as infectious agents of man. The re-
enteroviruses but differing by lability in acid media sults were in agreement with the findings of previ-
(4-6) and almost exclusive multiplication in the ous observers but in addition revealed the exist-
nasopharynx. The first agents isolated that fit ence of a substantial resistance to homologous re-
this description were the JH and 2060 strains of challenge that is distinct from the more limited
ECHO 28 isolated by Price (7) and Pelon, Mo- protective effect of naturally acquired antibody.
gabgab, Phillips, and Pierce (8) in 1956 and clas-
sified by Pelon (9). Modifications in tissue cul- Materials and Methods
ture technique for isolating these and similar
Volunteers and clinical procedures. Subjects were
agents, i.e., incubation on a rotating drum and re-
fifty-seven adult male volunteers from several federal
duction in pH to 7.0 to 7.3, were introduced by correctional institutions who had no medical contraindi-
Pelon and Mogabgab (10) and extended by Tyr- cations to the inoculations. Twenty-eight were inocu-
rell and associates (4) and Tyrrell and Parsons lated at the Clinical Center, National Institutes of
(5) to include an incubation temperature of 330 Health, and the remainder were inoculated at the Fed-
eral Prison Camp, Greenville, South Carolina. The in-
C and use of human tissues. Subsequently, many
oculum used, the dose, the location, the date, and the
rhinoviruses, reported under a variety of names, identifying numbers of participants are shown in Table I.
have been isolated in association with mild respira- At the Clinical Center, men were isolated, three per
tory illnesses (11-16). Some of the strains grow room, for 4 days before and for 10 days after inocula-
tion. The volunteers were examined daily before and
only in tissues of human origin (H strains) and
after inoculation by physicians who did not know which
some as well in monkey kidney tissue culture (M
of several viruses (including agents other than rhino-
strains) (3, 5). It has been estimated that rhino- viruses) had been given.
viruses account for 35 to 40%o of the common At Greenville, S. C., the men were assigned to two
colds in adults (17). large, adjoining rooms without isolation; they continued
Tyrrell, Bynoe, and co-workers (4, 18-20) and their usual work assignments around the camp. Physi-
cians who made daily examinations for 5 days were
Jackson, Dowling, and Mogabgab (21), who have
aware of which agent had been administered. The
administered rhinoviruses to volunteers, have dem- Greenville, S. C., studies were made in August and Sep-
onstrated that the viruses can infect and produce tember, 1962. Respiratory illness was infrequent in the
common colds and that protection is associated camp in the early part of the period, but a mild outbreak
with the presence of specific neutralizing antibody of common cold occurred in late September. Some ex-
traneous rhinoviruses were isolated at this time and on
in the serum. After the isolation of other strains
two other occasions. These are described under Results.
of rhinovirus, NIH 353, 1734, and 11757, by Inocula. The three virus strains used, NIH 353, NIH
1734, and NIH 11757, were isolated from throat swab
*Submitted for publication June 24, 1963; accepted specimens from two Marine recruits and a 2-year-old
September 26, 1963. female (16). Johnson and Rosen have described the
tPresent address: Middle America Research Unit, characteristics of these three strains, which grow only
Balboa Heights, Canal Zone. in tissue of human origin (22). All were isolated in a
56
RHINOVIRUSES IN VOLUNTEERS 57
TABLE I centrifuged at 3,000 to 8,000 rpm for 30 minutes and the
Scheduleofinoculation ofvolunteerswithrhinoviruses supernatant fluid stored at -20° C until tested.
To test for the presence of virus, 0.4 ml of each speci-
men was inoculated into one WI-26 tube. Maintenance
Dateof Volunteeridenti-
Inoculum Dose* Location inoculation ficationno. medium consisted of equal parts of medium no. 199 and
Eagle's medium no. 2 containing 2%o inactivated (560 C,
NIH353 105.2 ClinicalCenter 11/28/61 1-3 30 minutes) calf serum, 0.4% saturated solution of so-
1048 Clinical Center 12/26/61 10-12
dium bicarbonate, and supplemental antibiotics. The
IR, 2R,3Rt
NIH 11757 105° Clinical Center 11/28/61 4-6 cultures were incubated at 340 C on a rotating drum and
105.8 Clinical Center 12/26/61 13, 14,4R,5R,6R observed, for cytopathic effect (CPE) for 13 to 14 days.
NIH 1734 103.5 ClinicalCenter 11/28/61 7-9 Positive cultures were harvested when the CPE involved
105.5 Clinicalcenter 12/26/61 15-17,7R,9R
102.8 Clinical Center 3/12/62 18-28 all of the culture. Positive cultures in which CPE failed
102.8 Greenville, S. C. 8/31/62 29-41 to progress were occasionally seen and were verified on
102.8 Greenville,S.C. 9/21/62 42-57 subpassage. Identity of the first and last isolates from
each subject was established in WI-26 tissue culture by
*Contained in2 mlgivenby nasopharyngeal spray and instillation. neutralization of 32 or more TCID5O 1 with not more than
BasedontitrationsattimeofinoculationsandexpressedasTCIDso.
tRindicates volunteerswhoreceived homologousrechallenge. 20 antibody units of specific hyperimmune guinea pig
serum.
human embryonic fibroblast cell culture (WI-26) (23), Antibody determinhations. Neutralization tests were
and the harvest of a second WI-26 passage of each strain performed in WI-26 with a test dose of virus calculated
was pooled for a volunteer inoculum. Each volunteer to give 3.2 to 10 TCIDOO. Equal volumes of virus and
inoculum was centrifuged for 20 minutes at 2,000 rpm, fourfold dilutions of heat inactivated serum (560 C, 30
filtered through an 800-mA gradocol membrane, and minutes) were incubated for 1 hour at room tempera-
stored in 2-ml amounts in heat-sealed ampules at -600 ture, and then 0.2 ml of this mixture was inoculated
C. Inocula were safety tested in a manner previously into each of two culture tubes. Simultaneous tenfold
described (24). dilutions of the test dose of virus were inoculated into
each of four culture tubes. Five or more foci of CPE in
Volunteers were given 1 ml of virus suspension in-
a culture tube were required to consider that tube posi-
tranasally with a coarse hand spray and another 1 ml
tive. When the presence of 3.2 (not more than 16)
by intranasal instillation with a pipette.
TCID2o was first noted in the simultaneous virus titra-
Nasal secretions. The daily amount of nasal dis-
tion, a definitive reading of the test was made. The
charge in grams was determined by collecting from each
antibody titer, calculated according to the method of Reed
subject used paper handkerchiefs in plastic bags and
and Muench (25), was defined as that initial dilution of
weighing these tissues. From this value was subtracted
serum inhibiting this dose of virus. The test has given
the weight of a like number of unused tissues. To
consistently reproducible results.
correct for any pre-existing rhinorrhea, the weight of
secretion in the 48 hours before inoculation was sub-
Results
tracted from that of the 48 hours after inoculation.
Collection of specimens. Swabs from subjects were Illness. Common cold syndromes occurred in
agitated in vials with 2 ml of veal infusion broth con-
38 of 56 volunteers. Volunteer 21 had a prein-
taining 0.5%o bovine albumin and antibiotics. The swabs
were then discarded. Nasopharyngeal washings were oculation respiratory illness and is not included in
performed by instilling 5 ml of the above medium into subsequent parts of this report except in Table
each nostril with the subject's head tilted back and then, VI. Forty-five of the 56 volunteers were inocu-
with his head tilted forward, collecting the fluid in dis- lated with strain 1734 (including 7 men from
posable paper cups. After being mixed, the wash speci-
whom extraneous rhinoviruses were isolated); of
men was dispensed in vials. Pharyngeal and nasal swab-
bings were always performed before nasopharyngeal these, 29 became ill. Six received NIH 353; four
washes. The isolation results reported are derived from became ill. Five received NIH 11757 and all be-
the nasopharyngeal wash specimen alone unless otherwise came ill. Despite strain, inoculum size, antibody
specified. Essentially identical isolation results were status, and geographic and seasonal differences,
obtained whether specimens were cultured for virus when
the characteristics of the individual illnesses were
fresh or when recultured after storage at -200 C for 1
to 10 weeks. At Greenville, S. C., specimens were stored very much alike, and illness among groups was in-
at -40 C for several days before shipment to the Clini- distinguishable by clinical observation. For that
cal Center. reason the composite description of illness in
Stool specimens were collected and stored at -200 C.
They were subsequently thawed and homogenized as a 1TCIDO=the dose of virus that will infect 50%o of
10 to 20% solution in either saline or Hanks balanced tissue cultures, as calculated according to Reed and
salt solution with antibiotics. The homogenate was then Muench (25).
58 THOMAS R. CATE, ROBERT B. COUCH, AND KARL M. JOHNSON
TABLE II
Clinical responsein38volunteers withillnessafterintranasalchallengewithrhinoviruses
Symptom Sign
Constitutional Headache 66% Temperature, elevation, 16%
Malaise 50% > 37.5° C, oral
Chilliness, sweats 39%
Myalgia 24%
Upper respiratory Nasaldischarge 100% Nasal discharge 100%
Nasalobstruction 92% Nasalobstruction 89%
Sneezing 76% Inflamed nasal mucosa 97%
Throat irritation 53% Pharyngeal injection 24%
Eye discomfort 18% Mild cervical adenopathy 26%
Earache 3% Injected palpebralconjuctiva 16%
Dull tympanic membranes 8%
Pulmonary Cough 66%
Chest discomfort 21% Rales 0%
Gastrointestinal Anorexia 21%
Vomiting 5%
Loose bowels 5%
Table II is based on all of the 38 rhinovirus ill- slight elevations over base-line temperatures oc-
nesses that were observed. curred in the majority of volunteers 8 to 12 hours
Characteristically, nasal symptoms began on after inoculation. One other volunteer had a
the day after inoculation. Gradually increasing single oral temperature of 37.60 C on the fifth
complaints followed, with maximal illness on the day after inoculation in association with per-
second or third day. During the acute phase of sistent rhinitis and possible subacute sinusitis.
illness, a typical case was characterized by nasal Generally the volunteers were asymptomatic by
discharge and obstruction, sneezing, coughing, the fifth day after inoculation except for a few
slightly sore throat, headache, and malaise. Tem- with nasal discharge persisting for 2 to 9 more
perature elevations occurred in 6 volunteers dur- days.
ing the acute illness, with maximal oral tempera- On the basis of this description, a classification
tures ranging from 37.6 to 38.50 C. In addition, of severity of illness was prepared as follows: 0 =
W.W.A., 38 Yr.c9 VOLUNTEER * 9
DayAfter Inoculation -2 -I 0 2 3 4 5 6 7 8 9 1013 21
20_
DAILY
NASAL
SECRETION
(GMS) 10 _
Illness Rhinitis, Malaise, Throat Irritation
NIH 1734
Nasopharynx 0 + + + + + + + + +
Neut. Antib. <1:2 1:128
FIG. 1. COMMON COLD WITH RHINOVIRUS NIH 1734 IN A NORMAL VOLUNTEER.
Daily nasal secretions increased to 20 g on the second day after inoculation in
association with a syndrome of common cold. Virus was isolated from naso-
pharyngeal wash specimens taken days 1 through 13.
RHINOVIRUSES IN VOLUNTEERS 59
no illness (but some nasal discharge may be pres- 10-
ent); + = rhinitis with nasal congestion and dis-
9-_ 0
charge; ++ = nasal congestion, discharge, and
systemic reaction; ±++ = nasal and systemic f(0 8-- <'01 <.01
findings and fever (. 37.5° C, oral). This clas- x 7
TotalWBC
sification is used in subsequent parts of the report. (I) +
J 6
ureNa1sails rseepcrreetsieonntsa.tiveThoef ctahseecdoensccurrirbeendceinofFiigll-- LCiaJil 5F _ 0 <01~~<~.01,~,~~
ness and the period of increased nasal secretion in 0 .05
4
men who became ill among the 27 on whom such bi Neutrophiles
data were available. Analyses of data on nasal m 3 0 4
0- --O-_ .05 10o 0
secretion have been limited to the first 48 hours z 2 Lymphocytes .-0
after inoculation, when the quantity of nasal se-
cretion was maximal. The average weight of
excess of secretions (see Methods) for the 27
Pre-Chollenge 2 3 7-10
men in this 2-day period was 8.5 g, with a range
DAYS FOLLOWING CHALLENGE
from 0.0 to 85.5 g. There was a close correlation
FIG. 2. AVERAGE AND INDIVIDUAL CHANGES IN LEUKO-
between the amount of secretion and the severity
CYTE, NEUTROPHILE, AND LYMPHOCYTE COUNTS AFTER
of illness [Kendall's tau = +.543, p <.01 (26)]. CHALLENGE WITH RHINOVIRUSES. The average counts
The quantity of nasal secretion did not correlate are plotted, and the results of analysis of the individual
with the duration of virus shedding nor with the series of counts by the paired sample t test are indicated
titer of postchallenge antibody. The number of by arrows along with respective p values. 0 =no sig-
nificant change.
tissues used correlated with the weight of col-
lected secretions (Kendall's tau = +.688, p <.01).
Changes in leukocyte counts and sedimentation ing virus was determined for the nasopharyngeal
rates. Total leukocyte and differential counts wash, nasal swab, and pharyngeal swab specimens
were available on 15 subjects (Volunteers 1 to 9, from 13 antibody-free (< 1: 2) volunteers during
18 to 22, and 24) in the period before challenge the 7-day interval following challenge. As shown
and on days 1, 2, 3, and 7 to 10 after challenge. in Figure 3, the nasopharyngeal wash specimen
Thirteenof thesemendevelopedillness. The aver- was consistently superior to the other two speci-
agecounts for leukocytes,neutrophiles, and lymph- mens in yielding virus. A similar pattern was
ocytes shown in Figure 2 revealed a moderate but noted for all three virus strains. Cumulative
significant increase in total leukocytes because of corresponding rates of virus recovery were as
an increase in the absolute number of circulating follows: nasopharyngeal wash, 62 positive of 82
neutrophiles during the acute illness. Analysis of specimens tested (76%); nasal swab, 36%;
the individual series of counts by the paired sam- pharyngeal swab, 35%; combination of nasal and
ple ttest (27) (Figure 2) showed that the changes pharyngeal swabs, 50%. This superiority of the
in average counts were due to uniform changes in nasopharyngeal wash specimens also continued
counts from each volunteer. Of interest was the into the second and third weeks following chal-
slight decrease in lymphocytes during the acute lenge. Subsequent studies were performed using
illness. only this latter specimen for virus detection, and
Erythrocyte sedimentation rates (ESR) were with one exception all of the virus recovery data
determined at the same times as the leukocyte reported are based on this specimen alone. Since
counts. Five of the men (Volunteers 2, 3, 4, 7, Volunteer 16 could not tolerate nasopharyngeal
and 9) developed ESR elevations in the range of washing, his isolation results are based on nasal
27 to 50 (Westergren) by 7 to 10 days after chal- and pharyngeal swab specimens.
lenge. None of the other volunteers tested had Challenge of antibody-free (< 1: 2) volunteers.
ESR elevations greater than 16 at this time. Table III shows the results of challenge of 21
Comparison of virus yield according to source antibody-free volunteers with the 3 virus strains.
of specimen. The proportion of specimens yield- Seventeen illnesses were observed. No significant
60 THOMAS R. CATE, ROBERT B. COUCH, AND KARL M. JOHNSON
e)
z
w
Q
w
0.
LU
Cf)
w
0-
0
C0.)
z
w
w
a.
1 2 3 4 5 6 7
DAY POST-INOCULATION
FIG. 3. COMPARISON OF RHINOVIRUS YIELD BY SOURCE OF SPECIMEN.
TABLE III
Response ofantibody-free (<1:2) volunteers afterintranasal challengewithrhinoviruses
Volunteer Virusshedding,nasopharynx Postchallenge
Virusstrain no. days 1-7 days8-14 antibodytiter Ilinesst
1 5/7 1/3 1:4 ++
10 5/7 1/2 1:8 +++
353 2 7/7 3/3 1:16 ++
11 4/7 2/2 1:32 +
3 7/7 1/3 1:64 0
Totals 28/35 (80%) 8/13 (62%) 5/5 > 4-fold 4/5
rises
4 3/7 1/3 <1:2 ++
11757 5 6/7 1/3 1:4 +++
6 7/7 1/3 1:8 +
Totals 16/21 (76%) 3/9 (33%) 2/3 > 4-fold 313
rises
7 7/7 2/3 1:4 +
18 1/7 0/3 1:4 0
42 0/5* 0/3 1:4 +
15 3/7 0/2 1:4 ++
8 4/7 1/3 1:8 ++
29 0/4* 3/5 1:16 0
1734 43 1/5* 0/3 1:32 0
30 3/4* 1/4 1:64 +++
16 7/7 0/2 1:64 +++
19 6/7 1/3 1:64 +
20 4/7 2/3 1:64 ++
9 7/7 313 1:128 ++
44 2/5* 0/4 >1:128 ++
Totals 45/79 (57%) 13/41 (32%) 13/13 > 4-fold 10/13
rises
Inoculated atthe Greenville, S. C., Federal Prison Camp.
t0 = noillness; + = rhinitiswithnasalcongestionanddischarge; ++ = nasalcongestion,discharge,andsystemic
reaction; +++ - nasaland systemic findings and fever.
RHINOVIRUSES IN VOLUNTEERS 61
difference could be detected among the strains in These 3 instances involved all 3 viruses and in
the quality or quantity of illness produced. each case began fronm titers of 1: 16 to 1: 32 in
Virus was recovered from the nasopharynges of the prechallenge serum.
20 volunteers. In general, there was profuse vi- Homologous rechallenge. Four weeks after
rus shedding from the nasopharynx during the primary challenge, 8 of the initially antibody-
first 2 or 3 days after challenge. Thereafter, fre- free volunteers were rechallenged with the same
quency of virus recovery progressively decreased. virus strain. Eight additional volunteers were
Nine volunteers shed virus through days 11 to 14, available for primary challenge at the same time
and 4 of 12 tested continued to shed virus through to verify continued infectivity and illness-produc-
days 17 to 20. For reasons possibly related to ing capacity of the inocula. The results recorded
inadequate refrigeration, less virus was recovered in Table IV demonstrate the nearly complete ab-
from volunteers in Greenville, S. C., than from sence of virus shedding and total absence of ill-
similar men at the Clinical Center. If the volun- ness in the homologously rechallenged volunteers.
teers studied in Greenville are excluded, the aver- Nasal secretions were negligible or absent. Three
age percentage of virus-positive specimens is simi- of the 8 developed a further rise in antibody titer
lar for all 3 virus strains. Except for isolation of after rechallenge. In contrast were the frequent
herpes simplex from one volunteer before and virus isolations, uniform rise in antibody titer,
after inoculation, preinoculation cultures were and 6 illnesses in the 8 volunteers who had pri-
negative, and no agents except the strain adminis- mary challenge at this time. Two groups of
tered were recovered. physicians examined these 16 volunteers to check
One hundred twenty rectal swab specimens and the accuracy of clinical diagnosis, and identical
75 stool specimens obtained from Volunteers 1 to results were recorded. Both the continued po-
9 were tested for the presence of virus. Virus tency of the inocula and a uniform potent protec-
was recovered from one, a stool specimen obtained tive effect in the homologous rechallenge group
on day 2 from a volunteer challenged with NIH were thus demonstrated.
353, and was subsequently identified as the agent Challenge of volunteers having naturally ac-
administered. quired neutralizing antibody to NIH 1734. More
Twenty of the 21 volunteers had a fourfold extensive studies to assess the effectiveness of
or greater rise in homologous neutralizing anti- naturally acquired antibody were undertaken with
body titer 3 weeks following challenge. Sera one virus strain, NIH 1734. Volunteers were
from 9 volunteers with no prechallenge antibody, chosen on the basis of approximations of antibody
3 volunteers with each strain, were tested for neu- titer in sera taken 1 to 3 months before challenge.
tralizing antibody at weekly intervals for 4 weeks With consideration for the minimal time needed to
following challenge. One developed a fourfold develop antibody response, the volunteers with
rise in antibody titer after 1 week, 2 after 2 weeks, naturally acquired antibody must have had ini-
and 5 after 3 weeks. One volunteer did not de- tial contact with 1734 virus a minimum of 6 weeks
velop antibody in this period. None of the vol- before challenge. The distribution of antibody
unteers exhibited further increase in titer between titers in the volunteers chosen for study is a fair
the third and fourth weeks. Most of the antibody representation of the antibody levels found in the
titers were low (12 of 21 < 1: 16) and would correctional institution population as a whole,
have gone undetected if larger amounts of virus with 20 to 40% being free of detectable antibody.
had been used in the test. All of the sera taken from volunteers immediately
Paired sera from Volunteers 1 to 17 were also before challenge were tested in a single neutrali-
tested for antibody against the two of these virus zation test to achieve a valid ranking according
strains with which the individual had not been to prechallenge antibody titer. Paired sera were
inoculated. Antibody to one or both heterologous tested for rise in titer in one or more separate neu-
strains was present in the preinoculation sera of tralization tests. Volunteers who had no detec-
all of these volunteers. Three of the 34 tests of table antibody were included in each study, and
paired sera against heterologous virus demon- results from their challenge are tabulated in Table
strated a bare fourfold rise in antibody titer. III.
62 THOMAS R. CATE, ROBERT B. COUCH, AND KARL M. JOHNSON
TABLE IV
Homologous rechallenge (4weeks) andsimultaneousprimary challenge ofvolunteers withrhinoviruses
Virusshedding,
Volunteer Prechallenge nasopharynx Postchallenge
no. Virusstrain antibodytiter days 1-7 antibodytiter Illness
Homologous rechallenge
4R 11757 <1:2 1/7 1:128 0
7R 1734 1:4 1/7 1:8 0
IR 353 1:4 1/7 1:128 0
5R 11757 1:4 0/7 1:16 0
6R 11757 1:8 0/7 1:16 0
2R 353 1:16 0/7 1:32 0
3R 353 1:64 0/7 1:32 0
9R 1734 1:128 0/7 1:128 0
Totals 3/56 (5%) 3/8> 4-fold 0/8
rises
Simultaneous primary challenge
11 353 <1:2 4/7 1:32 +
10 353 <1:2 5/7 1:8 +++
15 1734 <1:2 3/7 1:4 ++
16 1734 <1:2 7/7 1:64 +++
13 11757 1:2 5/7 1:64 ++
14 11757 1:4 6/7 1:16 ++
12 353 1:32 5/7 1:128 0
17 1734 1:128 1/7 >1:512 0
Totals 36/56 (64%) 8/8 > 4-fold 6/8
rises
Results of challenge of 25 volunteers with vary- agents for the latter illness, and for Volunteer 45,
ing levels of naturally acquired antibody to NIH were unsuccessful.2
1734 are described in Table V. Sixteen of these According to the data in Tables III and V from
men shed virus from the nasopharynx, 6 for volunteers challenged with NIH 1734, there is a
longer than 10 days. Paired sera from 18 of 24 significant association of decreasing frequency
demonstrated a fourfold or greater rise in anti- and severity of illness with increasing antibody
body titer. The majority of the paired sera have level [Greenville volunteers, Kendall's tau =-
been tested on more than one occasion to confirm .244; Clinical Center volunteers, tau = -.260;
the result, most notably those from Volunteers p =.03 (combined taus)]. There was no statis-
26 and 45, in whom a fourfold rise in antibody tically significant relationship between duration of
titer was detected in the absence of virus shedding. virus shedding and increasing antibody titer when
Of the 7 volunteers who did not meet one of these the Greenville and Clinical Center data were simi-
two criteria of infection, 4 had a prechallenge larly analyzed.
antibody titer of 1: 1,024 or greater. Extraneous viruses were isolated from 8 vol-
Thirteen of 25 men developed an illness that, in unteers during the course of these studies (Table
all but one instance, was indistinguishable from VI). Each of these volunteers had been inocu-
the illnesses occurring in antibody-free volunteers. lated with NIH 1734, but nasopharyngeal wash
One volunteer with prechallenge antibody titer of specimens contained a virus that was not neutral-
1: 128 (No. 27) had an afebrile upper respiratory ized by guinea pig serum specific for NIH 1734.
illness with nausea and vomiting on days 1 and 2 These extraneous virus strains all produced en-
following challenge; then along with continued
virus shedding, he had a recurrence of his acute 2Paired sera from Volunteer 57, as well as 45, failed
symptoms on days 14 through 16 with oral tem- to fix complement when tested against adenovirus, para-
influenza Types 1, 2, and 3, influenza Types A and B,
perature elevation to 37.7° C. The typical, mod-
respiratory syncytial virus, and Eaton agent. These
erately severe illness experienced by Volunteer 57 studies were performed by Mr. Horace C. Turner, Lab-
is unexplained. Attempts to detect other etiologic oratory of Infectious Diseases, NIH.
RHINOVIRUSES IN VOLUNTEERS 63
TABLE V
Challenge ofvolunteers having naturallyacquired neutralizingantibodyto NIH1734
Volunteer Prechallenge Virusshedding, nasopharynx Riseinantibody
no. antibodytiter days 1-7 days8-14 titer,>4-fold Illness
45 1:4* 0/5 0/3 Yes ++
31 1:8* 5/5 1/4 Yes +
32 1:8* 0/5 0/4 No specimen 0
22 1:16 7/7 1/3 Yes +
33 1:16* 5/5 1/3 Yes +++
23 1:16 6/7 0/4 Yes 0
24 1:16 3/7 0/3 Yes ++
47 1:16* 3/5 0/3 Yes ++
48 1:32* 5/5 0/3 Yes ++
50 1:64* 2/5 0/3 Yes +
25 1:64 0/7 013 No 0
52 1:64* 2/5 1/3 Yes ++
35 1:128* 4/5 2/4 Yes 0
53 1:128* 4/5 0/3 Yes 0
26 1:128 0/7 0/3 Yes 0
27 1:128 7/7 2/3 Yes +++
17 1:128 1/7 0/2 Yes 0
37 1:256* 5/5 2/4 Yes +
38 1:256* 0/5 0/4 No 0
39 1:256* 4/5 4/4 Yes +
56 1:512* 1/5 1/3 Yes 0
40 1:1024* 0/5 0/4 No 0
41 1:1024* 0/5 0/4 No 0
57 1:2048* 0/5 0/4 No ++
28 1:2048 0/7 0/3 No 0
Geometric
Totals mean 1:86 64/141 (45%) 15/84 (18%) 18/24 13/25
* Inoculated in Greenville, S. C., Federal Prison Camp.
terovirus CPE and were acid labile (3); they are Eleven other volunteers in this group (Volunteers
hereafter termed "rhinoviruses." Identification 42 to 57) were exposed during this outbreak of
of the extraneous rhinovirus from Volunteer 21, HGP infection, but in paired sera from only one,
to which only 2 volunteers, 22 and 24, were ex- Volunteer 50, could a fourfold or greater rise in
posed, has not been completed. antibody titer be detected.
The extraneous rhinovirus from 5 volunteers The extraneous rhinovirus isolated from Vol-
(Table VI) was identified as HGP virus (4, 5) unteer 34 ("No. 34 virus") did not produce CPE
by neutralization with specific guinea pig serum. in monkey kidney cells. Specific guinea pig sera
TABLE VI
Volunteers challenged with NIH1734who shedanextraneous rhinovirusfromthenasopharynx
NIH 1734 Extraneousvirus
Risein Riseinanti-
antibody bodytiter,
Volunteer Prechallenge Virus titer, >4-fold
no. antibodytiter isolated > 4-fold Illness Identity
21 1:8 Yes Yes +* Not tested See text
34 1:32 No No 0 No
36 1:128 Yes Yes + Yes See text
46 1:16 Yes Yes ++ Yes
49 1:32 Yes Yes + No
51 1:64 Yes Yes ++ Yes HGP
54 1:128 No Yes ++ Yes
55 1:256 Yes No + Yes
*This volunteer had a minor upper respiratory illness before inoculation that persisted after inoculation.
64 THOMAS R. CATE, ROBERT B. COUCH, AND KARL M. JOHNSON
prepared against No. 34 virus did not neutralize nificant reduction in frequency and severity of
any of the following rhinoviruses: NIH 1734, illness as the titer of naturally acquired prechal-
353, 11757, 33342, and 1059 (22), HGP (4, 5), or lenge antibody increased (p =..03). Sixteen ad-
2060 (8, 9). However, hyperimmune guinea pig ditional volunteers with prechallenge antibody ti-
serum prepared against No. 34 virus did neutralize ters of < 1: 2 or 1: 2 have been challenged with
the extraneous rhinovirus from Volunteer 36. NIH 1734 in a dose of 102-8 TCID50 as part of a
Eleven other volunteers in this group (Volunteers separate study (30). They all developed colds
29 to 41) were exposed to No. 34 virus, but in with an average illness identical to that of the 13
none of their paired sera could a fourfold rise in antibody-free volunteers included in the present
antibody titer be detected (no late serum was ob- report. This enhances the confidence in the as-
tained from Volunteer 32). sociation of decreasing illness with increasing pre-
The illnesses observed in the volunteers listed challenge antibody. Evidence that neither the in-
in Table VI were similar to those in other volun- oculation procedure itself, nor some unrecog-
teers in these studies. Because of the presence of nized agent in the NIH 1734 inoculum produced
an extraneous virus, however, illness data from illness has been obtained by giving 6 volunteers
these volunteers cannot be interpreted in relation- this inoculum, which had been inactivated with
ship to prechallenge antibody versus NIH 1734, specific hyperimmune guinea pig serum prepared
and their data have been omitted from Table V. against a strain grown in different cells. There
Even if these men are included with the other vol- was no illness or virus shedding in this group
unteers challenged with NIH 1734, the significant (30).
relationship of decreasing illness with increasing The superiority of the nasopharyngeal washing
prechallenge antibody titer is not affected. method over nose and pharyngeal swabs for re-
covery of virus in the present study confirms a
Discussion
similar finding with the HGP agent by Bynoe and
These studies in volunteers have shown a con- associates (19). With specimens obtained by this
sistent association of challenge with NIH 353, method, virus was recovered from 47 of 57 vol-
NIH 1734, and NIH 11757, and the appearance of unteers. Frequent occurrence of virus shedding
an illness resembling experimentally induced and more than 10 days after challenge and at least 5
natural disease caused by similar agents (21, 28). days after acute illness is over has not been gen-
Illness usually began within 24 hours of inocula- erally appreciated, but the very rare recovery of
tion, and there was considerable uniformity of these strains from stool or rectal swab specimens
the syndrome regardless of the virus strain used is in keeping with the experience of others (1).
and the antibody status of the inoculated volun- Neutralizing antibody titers were determined
teer. The illness resembled what has classically against small doses of virus, which gave a sensi-
been described as "common cold," with nasal dis- tive, reproducible test. Fourfold or greater rises
charge and obstruction, rapid recovery, and few in titer were detected in sera from 47 of 56 (84%)
sequels. A slight but significant leukocytosis due volunteers 3 weeks after challenge. No increases
to neutrophiles occurred on the first and second in titer occurred in sera from 9 volunteers between
days following challenge with a simultaneous the third and fourth weeks, and only 3 of 8 demon-
slight decrease in lymphocytes. Similar changes strated a further . fourfold rise at 2 months de-
in neutrophile counts have also been noted in this spite intervening homologous rechallenge. Jack-
laboratory in studies with Coxsackie A-21 (29). son and co-workers (31) have presented data to
The independent finding of a positive correlation the effect that following challenge with an in-
between increasing nasal secretions and severity fectious nasal secretion containing an unknown
of illness offers confirmation of the accuracy of "common cold" virus, the capacity of convales-
the clinical observations on the volunteers. cent serum to inactivate the same or antigenically
Establishing that the illnesses were related to similar infectious nasal secretions continues to in-
the virus strain given was important, and such crease in titer for 1 year. This finding, despite
was done with the inoculum most extensively em- differences in methodology, does not appear to be
ployed, NIH 1734; there was shown to be a sig- in keeping with our studies,
RHINOVIRUSES IN VOLUNTEERS 65
The 8 volunteers who received homologous re- feron is the responsible factor in resistance to
challenge at 1 month had relatively low titers of rechallenge.
antibody (mean titer, 1: 10) and yet exhibited The appearance of antibody in the nasal secre-
substantial resistance to the second challenge. tions in response to infection is thought to be im-
They developed no illness following reinoculation portant in reduction of virus spreading and pre-
and almost no virus shedding (57% of specimens vention of illness (20, 37). However, iil volun-
positive for virus in the first week). In contrast, teers with naturally acquired antibody, there was
illnesses occurred in 13 of 25 volunteers with relatively poor protection against illness and
naturally acquired antibody (mean titer, 1: 86), frequently prolonged virus shedding. This sug-
and there was a 45% incidence of specimens posi- gests that specific antibody in the nasal secretions
tive for virus the first week following inocula- does not account for the potent protection ob-
tion. These results suggest a mechanism of re- served on rechallenge. Thus, resistance to rein-
sistance in the rechallenge group other than that fection with rhinoviruses not dependent on cir-
afforded by circulating antibody as usually meas- culating neutralizing antibody as usually meas-
ured. Of possible significance in explaining this ured has not been satisfactorily explained and
phenomenon is the 1-month time interval between remains an important subject for study.
the challenges, as opposed to an estimated interval One of the difficulties of performing virological
of at least 6 weeks and probably longer between studies in human volunteers, the appearance of
natural infection and inoculation of the naturally extraneous viruses, was amply demonstrated in
acquired antibody group, i.e., some relatively these studies. It was possible, however, to pre-
short-lived mechanism of resistance. vent these events from significant interference
A similar phenomenon, undoubtedly involving with interpretation of the results. Dual infection
more than one virus strain, has been described by with two rhinoviruses was detected in at least 6
Dochez, Shibley, and Mills (32) in chimpanzees. volunteers (21, 36, 46, 49, 51, and 55, Table VI).
They found that these animals rarely developed Assuming that the volunteers were not exposed to
colds spontaneously or after inoculations within the two infecting rhinoviruses simultaneously,
3 months of a previous infection. Sabin (33, 34) then there was limited resistance to superinfection
has described a possibly related situation in the early in the course, as compared to the potent pro-
resistance to reinfection of the gastrointestinal tection observed on homologous rechallenge at 1
tract, lasting more than 1 year after oral attenu- month. If a nonspecific mechanism of resistance
ated poliovirus vaccine. This resistance appears is involved, then it is apparently not induced in the
to be independent of serum antibody and is not first few hours or days of the initial infection.
conferred by 2 doses of parenteral Salk vaccine. The rare occurrence of heterologous antibody
A possible mechanism of local resistance to re- responses in the present volunteers is evidence for
infection suggested by Stuart-Harris and Francis the antigenic distinctiveness of the strains studied,
(35) may have relevance to the present results. as had been previously demonstrated in animals by
They observed in ferrets, following challenge with Johnson and Rosen (22). With the apparent
influenza, a change from the normal columnar epi- multiplicity of serologically distinct strains of
thelium of the respiratory tract to a transitional rhinovirus (6) and the relatively poor protection
reparative form resistant even to chemical injury. afforded by specific antibody in these studies, con-
The resistance lasted 3 to 4 weeks. Another trol of the common cold by means of parenteral
mechanism of local resistance to be considered is vaccines will apparently be a formidable task.
an effect of interferon. Resistance to rechallenge
with rhinoviruses was present 1 month after first
Summary
challenge and for an average period of 13 days
after cessation of virus shedding. This would Inoculation of adult male volunteers with three
require the local persistence of interferon in the strains of rhinovirus, NIH353, 1734, and 11757,
absence of evident viral mniultiplication for a pe- was followed by a high frequency of common
riod longer than has been experimentally recorded colds. The illnesses usually began the day after
(36). On this basis it seems unlikely that inter- inoculation and lasted 2 to 3 days. Rhinorrhea