DISCUSSION
This analysis compared the infectivity and virulence of two SMV inocula
used in two different human challenge trials by examining infection
rates, illness rates, severity of illness, viral shedding, and IgG
seroconversion among subjects challenged with a single dose of one of
these inocula. Inoculum 1 was prepared at NIH sometime between 1977 and
1979 from the stool filtrate of a subject infected in the original Snow
Mountain Virus outbreak in Colorado in December 1976. This safety-tested
inoculum was first used in a human challenge study with 12 subjects
conducted at NIH by Dolin et al. [12] and then again in a human
challenge study with 15 subjects conducted between 2000-2002 at the
University of North Carolina-Chapel Hill [11]. Inoculum 2 was
prepared from the stool filtrate of an infected subject in the 2000-2002
human challenge trial and was then used in a human challenge study with
44 subjects at Emory University from 2016-2018 [9]. The goal of this
analysis is to examine whether the infectivity, pathogenicity, and
virulence of the SMV changed due to passage in the human host by
assessing differences in the outcome measures of the two more recent
challenge trials.
Both inocula were associated with similar overall infection rates as
measured by the proportion of challenged subjects who developed
infection and illness after challenge in each trial. However, the median
infectious dose (ID50) of Inoculum 2 was about 100-fold
higher than that estimated for Inoculum 1 [13]. Comparing the
severity scores of infected subjects with clinical symptoms indicated
that subjects infected with SMV Inoculum 2 had less severe acute
gastroenteritis and more delayed viral shedding even though these
subjects were challenged with a much higher dose than the subjects who
were infected with Inoculum 1. These results are consistent with what we
recently reported [15] that subjects challenged with a
first-generation NV inoculum (8FIIa, prepared in 1971) had significantly
higher severity scores of acute gastroenteritis but shorter duration of
viral shedding compared with those challenged with a second-generation
NV inoculum (8FIIb, prepared in 1997 from the stool filtrate of a
subject infected with the first NV inoculum).
Viral shedding and clinical illness are two important outcomes evaluated
in NoV challenge trials. Based on outbreak investigations and human
challenge studies, most infected subjects experience clinical symptoms
along with viral shedding in their feces during the first several days
of infection [16]. Symptoms usually resolve after 48-72 hours,
however viral shedding can continue for up to three weeks [16] and
even longer in immunocompromised subjects [17, 18]. In this study,
all subjects infected with SMV Inoculum 1 shed virus for up to six days,
but 3% of subjects infected with Inoculum 2 had viral shedding between
days 15 to 45 days post challenge. We hypothesize that the less severe
clinical illness, but possibly longer SMV shedding, observed in subjects
infected with Inoculum 2 may be associated with viral mutations that
reduced the virulence of the inoculum, rather than host factors or
laboratory assessment methods. However, it is possible that the RT-qPCR
assay used to measure viral shedding in the more recent challenge trial
with Inoculum 2 is more sensitive and better able to detect prolonged
shedding compared to the conventional RT-PCR assay used in the trial
with Inoculum 1 [19].
As the prototype of NoV GII.2, SMV is associated with a small percentage
of NoV outbreaks, and little is known about the mutation of this virus.
Swanstrom et al., [20] reported that the sequence of the P2 domain
of SMV strains collected between 1976 and 2010 has evolved over time but
less extensively than has been reported for GII.4 NoV strains. Mutations
in the surface protein could significantly alter the antigenicity of
representative strains, which could also change viral function,
pathogenesis, transmission, and infectivity. Studies of other RNA
viruses indicate that viral mutations can be deleterious or favorable to
the pathogens in terms of infectivity and virulence. Both host and viral
factors may explain the change in infectivity and virulence that we
observed between the two SMV inocula. Some host factors, including
acquired and innate immunity prior to challenge and secretor status, can
affect the likelihood of NoV infection and illness. Other host factors,
including age, sex, and race/ethnicity may or may not impact the risk of
NoV infection and illness. Rouphael et al [9] reported that
prechallenge anti-SMV serum IgG concentration, carbohydrate-binding
blockade antibody, and salivary IgA were not associated with infection
with Inoculum 2. Given the evidence from viral evolution of other RNA
viruses such as influenza and SARS-CoV-2 [21-23], we hypothesize
that the less severe symptoms associated with the second generation SMV
inoculum in this study may be due to intra-host SMV mutations during the
course of infection in the subject who was the original source of
Inoculum 2 and possibly further inter-host mutations in the subjects who
were infected with Inoculum 2 after challenge.
This analysis is the first to compare the clinical outcomes associated
with two different SMV inocula used in two human challenge trials
[9,11]. We examined detailed clinical and laboratory data collected
from well-controlled human challenge studies with nearly identical
protocols that included follow-up sample collection and analyses to
carefully characterize infection, illness, and immune response. The
limitations of this analysis include small sample size in both studies
that may not provide enough power to detect a significant difference in
some outcomes, retrospective analysis of two studies that span over 20
years and had different study populations, and slightly different
laboratory methods for measuring viral shedding and serum antibodies.
Finally, we were not able to assess how differences between inocula
preparation in the late 1970’s (Inoculum 1) vs. 1997 (Inoculum 2) and
storage may have affected the infectivity, pathogenicity, and virulence
of these two inocula. The time between inoculum preparation and the
challenge trials that provided the data we analyzed was approximately 20
years for both inocula: Inoculum 1 (1979-2000) and Inoculum 2
(1997-2016).
Understanding the difference in the severity of illness associated with
these two SMV inocula is critical for those who plan to use second
generation NoV inoculum to evaluate new NoV vaccine candidates in
vaccination-challenge trials. As we observed for the NV inocula, the
second generation SMV Inoculum 2 was associated with a higher
ID50 and less severe clinical illness when compared with
the first generation SMV inoculum. If second generation NoV inoculum
does not elicit expected symptomatic illness rates in challenged
subjects, it will become more complex and costly to conduct
vaccination-challenge trials because they may require larger numbers of
study subjects and challenges with higher doses of inoculum in order to
assess the efficacy of a candidate vaccine to reduce illness.
Funding: This study was supported by the National Center for
Advancing Translational Sciences of the National Institutes of Health
(UL1TR002378); the National Institute of Allergy and Infectious Disease
(R01 AI148260); and the Division of Microbiology and Infectious Diseases
to the Emory Vaccine and Treatment Evaluation Units (VTEU):
HHSN272200800005C, HHSN272201300018I, HHSN27200003, and HHSN27200018
Table 1: Modified Vesikari score (18-point scale) [14] for
SMV-infected subjects
with clinical symptoms