Covid human volunteer challenge study

 The first study was just reported on viral kinetics and other outcomes in volunteers inoculated with SARS-CoV-2 virus (see covid human challenge UK NatMed2022 in dropbox, or doi.org/10.1038/s41591-022-01780-9)

Details:

--36 volunteers aged 18 to 29, without evidence of prior infection or vaccination, were inoculated with 10TCID₅₀ of pre-alpha wild type (i.e. the original) of SARS-CoV-2 intranasally, in an open-label nonrandomized study

    -- TCID₅₀ is the Median Tissue Culture Infectious Dose; and, 10TCID₅₀ is a low starting inoculum dose

-- these individuals had none of the following risk factors for severe covid: low or high BMI; abnormal labs including CBC, renal or liver function, clotting, or viral serology to exclude other viruses; spirometry; echocardiography; chest x-ray

-- mean age 22, 75% male, 92% white, BMI 23

-- after inoculation, the volunteers were  housed in a high-containment quarantine unit with individual negative pressure rooms, and with 24-hour medical monitoring and full access to higher-level clinical care

-- two volunteers were subsequently excluded from the per-protocol analysis because of SARS-CoV-2 seroconversion between screening and inoculation

-- per protocol, the first 10 participants inoculated were assigned to receive preemptive remdesivir 100 mg IV for five days after two consecutive nose or throat swabs showed quantifiable SARS-CoV-2 by qPCR (quantitative PCR), in order to mitigate unexpected progression to more severe disease

    -- however, the data and safety monitoring board deemed this was unnecessary and recruited an additional 30 individuals without remdesivir 

    -- and, those who developed indicators of increased clinical severity (persistent fever, persistent tachycardia, severe cough, greater than mild chest CT imaging changes, or oxygen saturation <94%), would get monoclonal antibodies

        -- BUT, no such treatment was ultimately required for anyone

-- patients were quarantined for 14 days after inoculation or until he they met virologic discharge criteria (quarantine a minimum of 14 days after inoculation until the following: 2 consecutive daily nose and/or throat swabs with no viral detection or qPCR Ct>33.5 and no viable virus on culture), with planned follow-up for one year to assess for prolonged symptoms, including smell disturbance and neurologic dysfunction (eg long covid)

-- prespecified infection rate after the inoculum was at least 50%, and, if not achieved, there would be inoculum dose escalation

-- primary objective: to identify an inoculum dose that induced well-tolerated infection in >50% of the volunteers

-- secondary objective to assess viral and symptom kinetics during the infection

Results:

--18 of the 30 volunteers Inoculated became infected (greater than the prespecified 50% minimum, so no inoculum escalation was actually ever done)

--Antibody titers at 28 days after inoculation (all infected individuals developed serum spike specific IgG and neutralizing antibodies):

    -- neutralizing antibody titer, median: 863.5 (interquartile range 403)

    -- spike-specific IgG titer, median: 1549 (IQR 1865)

    -- there was no increase in neutralizing antibodies or IgG titers in those who were deemed uninfected (<2 positive consecutive qPCRs); however, one person who was deemed uninfected did develop natural Covid after discharge from quarantine, suggesting that they were susceptible to the virus

--The viral load rose steeply and peaked about 5 days after inoculation

    -- virus was first detected by quantitative PCR (qPCR):

        -- throat: at median of 40 hours (about 1.67 days after inoculation)

        -- nose: at 58 hours (about 2.4 days)

        -- initial qPCR closely paralleled detection of culturable/ viable virus measured by focus-forming assay (FFA); FFA was positive earlier in the throat than the nose, p=0.0058

-- viral loads increased rapidly, with qPCR peaking in the throat at 112 hours (about 4.7 days) after inoculation and in the nose at 148 hours (about 6.2 days)

-- viral load and viable virus:

    -- nasal: qPCR viral load 8.87 log₁₀ copies/ml (8.41-9.53), FFA viable virus 3.9 log₁₀ copies/ml (3.34- 4.42)

    -- throat: qPCR 7.65 log₁₀ copies/ml (7.39-8.24), FFA 2.92 log₁₀ copies/ml (2.68- 3.56)

        -- highly significant differences between the sites: qPCR difference p<0.0001, and FFA p=0.0024 (ie, throat peaked first but nose got higher viral loads)

    -- culturable/viable virus was recoverable from the nose up to 12 days after inoculation, though swabs for qPCR became undetectable during the resolution phase after about 14 1/2 days

-- above shows number of days of culturable virus (graph g) and detectable by qPCR (graph h, notably longer: 14-15 days vs 9-10 days)

-- and there was no quantitative correlation between viral load and symptoms, with high viral loads in asymptomatic people as well.

-- average time after inoculation to clearance of viable (culturable) virus:

    -- nose: 244 hours (208-256), for average of 10.2 days

    -- throat: 208 hours (172-244), for average of 8.7 days

-- accuracy of the rapid antigen testing by lateral flow assay, LFA (see http://gmodestmedblogs.blogspot.com/2021/04/covid-uk-variant-likely-responds-to.html for more information about this assay):

    -- LFA was done using the same swab to assess viral load

    -- none of the uninfected volunteers had positive LFA at any time, whereas all of the infected ones did for at least two days

    -- median time to first positive LFA was four days after inoculation in both the nose and throat, within 24-48 hours after the first positive qPCR, and within 24 hours of positive FFA

        -- in 9 of the 18 infected participants, viable virus was detectable by FFA one or more days before the first positive LFA (i.e. there was a lag from culturable virus to the positive LFA antigen test)

        -- after about four days after inoculation, LFA had high-sensitivity as a surrogate for qPCR or FFA positivity, with a few false positives in relation to FFA but none to qPCR (and clinically/public-health-wise, having a few false positives is better than having false negatives)

--Clinical infection:

    --symptoms, by self-reported diary:

        -- all who developed symptoms, 16 (89%) of the infected participants, had mild-to-moderate ones, beginning 2 to 4 days after inoculation; two participants (8%) remained asymptomatic

        -- major symptoms were from the upper respiratory tract including nasal stuffiness, rhinitis, sneezing, and sore throat. Symptoms of headache, myalgias, joint pain, malaise, and feverishness were also recorded

        -- no serious adverse effects

        -- no evidence of pulmonary disease (which was more common with this initial SARS-CoV-2 virus) by either clinical or radiological assessments

    -- peak symptoms were at 112 hours after inoculation (closely aligning with peak viral load in the nose), but there was no correlation between the amount of viral shedding by qPCR or FFA and symptom score

        -- no difference in symptoms between those treated with remdesivir or not

            -- of interest, there was also no difference in viral load or viral culture in the 6 of the 10 participants who became infected and were treated with remdesivir

        -- anosmia developed more slowly in 15 (83%) of volunteers, either by self-report or by the University of Pennsylvania smell identification tests

            -- complete smell loss occurred in nine participants (50%), most improved noticeably before day 28, though five still reported symptoms by day 180 (only one had measurable smell impairment at that time)

       -- fever > 37.8°C: 7 people (39%)

       -- CRP>5 mg: 5 people (28%)

-- no quantitative correlation was noted between viral load and symptoms (with high viral loads even in asymptomatic volunteers)

-- mathematical modeling suggested that twice-weekly rapid antigen tests with LFA would diagnose infection before 70 to 80% of viable virus had been generated

Commentary:

--This is the first study where there is clear evidence of a specific time of inoculation (much more precise than using contact data) with a specific standardized viral inoculum in controlled study conditions

    -- we have certainly developed a lot of insight into this virus through some of the very well-researched viral contact studies, though those involved unknown quantities of virus in the index individual, with spread in different households with different levels of ventilation, different people with different amounts of time at different distances from that index person, and with the potential for the contacts to have been exposed to others with SARS-CoV-2 in the community (aside from the index person). And some of the observational studies may have missed sampling at the time of the peak viral loads, leading to lower loads recorded than the true peak. the detailed sampling in this group of volunteers provides more robust data

-- several prior studies had suggested that symptoms from natural infection followed exposure by about 5 days; this study with its detailed design found viral shedding within 2-4 days, peaking at 4-5 days, and suggested that up to 44% of transmissions occur before symptoms are noted

    -- and, as an important perspective, the best guess is that about 40-50% of those with confirmed SARS-CoV-2 infection are asymptomatic (for a recent large meta-analysis, see covid asx percent meta-anal jama2021 in dropbox, or doi:10.1001/jamanetworkopen.2021.37257.

-- it was especially notable that the qPCR was positive about 5 days longer than culturable virus by FFA (ie, using repeat PCR to assess patients being able to return to work, though not advocated by CDC or public health folks, is sometimes required by workplaces, and is pretty likely useless...)

-- would be really useful to know the relationship between some different rapid antigen tests and culturable virus. if one has covid with a positive antigen, is there culturable virus when the antigen turns negative?? if not, that would be an easy way to figure out if the infected person were able to resume normal activities (perhaps after 2 negative antigen tests, perhaps 12 hours apart? or 24 hours?). the contrary issue (remaining antigen positive), also of unknown significance, does raise the possibility that the person is still contagious, as reflected in the opinion in https://khn.org/news/article/rapid-antigen-test-isolation-guidance/ . This volunteer trial did provide important data for the LTA done in the UK, but this test may well have different sensitivity/specificity from the rapid antigen kits in the US (and there may be important differences in the LTA done in this study by the researchers and self-administered ones at home by US test kits)

-- there will be continued monitoring of these volunteers over the next year to assess for long covid symptoms as well as local and systemic immune markers, including cross-reactive antibodies, T cells, and soluble mediators

Limitations:

-- small number of people, all under 30 years old, and all without known risk factors for severe Covid. Hard to know if this applies to other patients as well as those with comorbidities

-- study involved the initial strain of SARS-CoV-2, which has different viral kinetics than the more current variants, also limiting generalizability to our current situation (in particular, several variants with known different viral kinetics, including much increased transmissibility and higher viral loads: eg see https://www.nature.com/articles/s41591-022-01816-0 )

-- and, the intranasal inoculum was on the low range, which might also affect viral kinetics (though the viral loads measured were even higher than what is found in natural infection)

-- we do know that qPCR can remain positive longer than the culturable virus. But does lack of culturable virus (still just a lab test) definitively mean that there would be no actual human transmission of the virus?? in this small study in healthy people, there was a 5-day lag between culturable virus by FFA and positive qPCRs. so, this is an important public health question

    -- one of their findings regarding the qPCR being positive 14 days after inoculation is that that would suggest quarantine should be extended another 5 days on average after our current approach (and, 6 of 18 infected people had continuing qPCR positive at 28 days in the nose and 2 of 18 in the throat; all were negative by day 90, but the prolonged positive qPCR likely represents nonviable viral remnants). And in fact 3 of the volunteers not meeting criteria of being infected after inoculation did have low level qPCR positivity in the nose and 6 in the throat up to 14 days after inoculation (infection was defined as 2 consecutive positive qPCRs, and these people had only 1)

    --and, they used a qPCR Ct cutoff of 33.5 on 2 consecutive samples to determine positivity. is this the appropriate cutoff? why not 35? or even higher (though the higher the cutoff, the more likely the results are not a true positive/higher likelihood of contaminant, for example)

so, several important findings in this human study:

-- viral shedding began within two days of exposure to the initial SARS-CoV-2 virus, reaching high levels with viable virus detectable up to 12 days after inoculation (which was 8 to 10 days after symptoms in those who became symptomatic)

-- no difference in qPCR in those with symptoms or asymptomatic: ie, all infected individuals seemed to be equally contagious

-- in the real world, it is clear from this and other less robust studies, that there are high viral loads/culturable virus prior to symptoms (and just as high in asymptomatic people). But, in the case of someone being exposed to a symptomatic person (and there is a reasonable assumption that that time is the initial exposure time), then it makes sense to think of the time frame of potential infectivity to others to be more on the order of 10-11 days (with the strong caveat that the above data refer to the initial SARS-CoV-2 and not any of the more transmissible subsequent variants). and not just 5 days per the CDC (https://www.cdc.gov/coronavirus/2019-ncov/if-you-are-sick/quarantine-isolation-background.html)

-- and, as noted above, there are so many questions raised by this study, including the precise significance of the measured values (qPCR, FFA) in this laboratory setting with a currently extinct virus vs real-world current viral transmissibility. ie, this study provides important information, but more studies with newer variants need to be done, including for breakthrough infections in those already vaccinated or having had prior infection

-- lateral flow test results for antigen were strongly associated with viable virus. but important to have data on other rapid tests self-administered at home. might be very useful to know that some people can leave quarantine soon (perhaps if 2 negative tests)

--and, this study does suggest that masking that masking (a pretty easy thing to do) should still be considered important, as well as distancing, avoiding high-volume venues..., despite the dramatic current relaxation of these mitigation measures. and especially so for those inadequately vaccinated/boosted (a significant population around the world).

geoff

 

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