Using human volunteers to understand influenza virus behaviour and its impact....

This will be the first in a long term project to catalogue the literature's record of volunteer infection studies using influenza virus (IFV). Much like the one I started on rhinovirus transmission back in Nov-2013...see those posts here and here, so far.

I don't guarantee it will be done before I'm too old to type, but I'll be adding to it as time allows. ;)

So first up is a paper.

Author: Frederick Hayden et al
Journal: J Clin Invest 101(3):643-9
Year: 1998
IFV strain used: A/Texas/36/91 (H1N1)
Inoculation route/amount/viral titre: intranasal/0.25ml/10⁵ TCID₅₀
Volunteers included: 19 healthy susceptible males (26%) and females, median age 21-year
Antibody levels: =1:8, haemagglutinin-inhibition (IH)

Some key results...

• Volunteers were kept in isolation for 1-day prior to inoculation.
• Most (74%) volunteers shed virus on day 1 (1 day after inoculation) as determined by viral growth from recovered nasal washings
• All volunteers developed symptoms with scores peaking at day-2 and returned to normal by day-8
• 12/19 (63%) developed fever, peaking at day-2
• Upper respiratory tract signs of illness (runny nose, sore throat occurred in 15/19 (79%), peaking at day-2
• Lower respiratory tract symptoms (cough, hoarseness) peaked on day-5
• Systemic symptoms (muscle aches, fatigue) also peaked on day-2
• Proinflammatory cytokines in nasal washes revealed several patterns:
1. Interleukin (IL)-6 and interferon (IFN)-a increased (day-2), decreased, then increased again (day-5) - a biphasic pattern
2.  IL-8 tumour necrosis factor (TNF)-a began rising at day-2, peaking at day-4; IL-8 (peaking when nasal mucous turbidity increased) not decreasing as rapidly as TNF-a
3. IL-1�, IL-2 and tissue growth factor (TGF)-� did not rise
• Cytokines in plasma and serum
• IL-6 and TNF-a peaked weakly but at the same times as in the nose 
• IFN-a and IL-8 were not detectable
• IL-1�, IL-2 and TGF-� did not rise
• Nasal lavage virus levels (titres) correlated with fever, total symptoms, systemic symptoms and upper respiratory symptoms, TNF-a, IFN-a and IL-6 on day-2. Total symptoms, viral titre, IL-6 and TNF-a all correlated on day-5
• Lower respiratory tract symptom scores peaked in correlation with IFN-a and IL-6 on day-5 & day-6 (also when IL-8 peaked)

As you'd expect given the inoculation route, signs and symptoms of disease occurred first in the upper respiratory tract and then progressed to the lower respiratory tract over time. This is obviously not a "natural" infection as we do not get 0.25mL of flu-containing liquid flying up our noses when someone sneezes on/near us. So the value of this study lies other than in studying natural transmission.

The authors note a few things in the discussion...

• The response to infection at the site likely leads to the local (runny nose,cough) and the systemic (fever, myalgia) signs and symptoms. 
• Cytokines are produced by a variety of cells; monocytes/macrophages, bronchial epithelial cells, CD4+ and CD8+ T cells and the infected epithelial cells themselves.
• Mouse models seem to have more severe lower respiratory tract disease after influenza virus infection than do human volunteers
• Other cytokines may be involved during more severe natural influenza infections of humans
• TNF-a, although rising earlier, peaked later perhaps acting to control inflammation after virus has been contained (in the upper respiratory tract at least; what's happening in the lower respiratory tract?)
• TNF-a and IL-8 were part of the 2nd wave of cytokines and may be better markers of more severe lower respiratory tract influenza disease

Cytokine background information...

• IL-6
• A proinflammatory cytokine
• Secreted by T cells and macrophages
• IL-8
• Also known as neutrophil chemotactic factor
• A chemokine secreted by macrophages and epithelial cells and anything with tool-like receptors
• Attracts neutrophils and other granulocytes and indices phagocytosis in them
• TNF-a
• Also called cachexin or cachectin
• Mostly secreted by macrophages,but also CD4+ lymphocytes and natural killer (NK) cells
• It is a pyrogen, inducing fever, apoptotic cell death, cachexia (weight loss) and inflammation
• IFN-a
• A Type I interferon secreted by leukocytes, macrophages, epithelial cells, endothelial cells etc
• Involved in the innate immune response against viral infection by triggering many other antiviral proteins and processes
• 
  

Read More

Rhinovirus transmission by aerosol and lower respiratory tract disease after inoculation

In the next instalment to answer the question posed in last week's post, we also find that rhinovirus can a lower respiratory tract infection (LRTI), if it is delivered directly to the site; several issues around this topic are contentious in current age of PCR diagnosis of lower respiratory tract disease using specimens from the upper respiratory tract (URT).

From Thomas R. Cate et al, Am J Epidemiol.

Author: Thomas R Cate et al
Journal:  Am J Epidemiol 81(1):95-105
Year: 1965
RV type used: NIH 1734 (RV-A15¹)
RV receptor type: major group; ICAM-I

This study set out to investigate the impact of RV on the lower respiratory tract.

Key features of the study layout..

• 16 healthy adult male inmate volunteers
• Safety-tested preparation of RV-15
• 6 volunteers given 1ml nasopharyngeal serum-inactivated virus via a hand atomizer (coarse droplets expected to mainly deposit in the upper respiratory tract), and 1ml instilled intransally by pipette with subject lying on back
• 8, RV-15-antibody-free volunteers were exposed to 10l of air (16, 20 or 66 TCID₅₀ RV15), via a mask, containing 15-second-old 0.2-3.0um particles generated from a Collison atomizer (see Figure)
• A number of re-inoculations were also performed on each virus-delivery group
• Aerosols was also sampled using a Shipe impinger (this device contained cell culture medium onto which some aerosol was impacted) for virus isolation, after storage at -70�C. These data determined the dose that had been used
• Prior (2-days) to inoculation, nasal, pharyngeal and anal swab specimens and 10ml of nasopharyngeal wash (NPW) were collected, frozen at -20�C for testing to identify pre-existing viruses or bacteria (all culture based). The same specimen types were collected after inoculation (minus the anal swab). RV culture was conducted on human embryonic fibroblast cultures, with rotation at 33�C)

Key results included...

• Only 1 other virus, apart form RV-15, was found in the subjects. Culture may have missed fastidious or unculturable respiratory viruses (like the RV-Cs) however.
• During the 1st week after inoculation, usually starting from day-2..
• NPWs contained culturable virus in at least 1 specimen from 8/8 subjects
• During the 2nd week after inoculation..
• 7/8 subjects gave virus-positive NPWs
• During the 3rd week after inoculation..
• 5/8 subjects gave intermittent virus-positive NPWs
• Maximal virus titre aligned in time with most severe illness
• Nasal and pharyngeal swabs specimens did not yield virus as often as NPWs
• All subjects had a rise in antibody titre of 4-fold or greater, indicating infection, by 3-weeks with a further bump after 4-5-weeks
• Tracheobronchitis was diagnosed in 6/8 antibody-free aerosol-inoculated volunteers. This is a lower respiratory tract disease.
• Signs and symptoms included cough (sometimes in fits), substernal chest pain,, wheezing, tender trachea.
• 3 had a primary diagnosis of tracheobronchitis , the other 3 also had a prominent coryzal illness (nasal obstruction/discharge, sneezing, sore throat, swollen neck lymph nodes). 
• Fever was determined in 5/8, within the 1st 1-2-days.
• Signs and symptoms lasted for 1-4 days, a little longer for a rhinitis-alone
• No tracheobronchitis developed among 31 antibody-free volunteers inoculated through a course spray/drop method into the nasopharynx
• No infection (no suitable rise in antibody) or illness was detected among 6 volunteers inoculated with a preparation of virus that had first been inactivated by incubation with an antibody-positive serum. This identified that there were no other viruses/bacteria in the preparation that could have caused the disease. This had been, infrequently, found in other preparations by the authors so this step was important part of their comprehensive approach.
• 4-weeks later, 2 volunteers from the aerosol infection group, 2 from the inactivated virus group, and 2 new volunteers, were (re-)inoculated
• No infection, illness or virus shedding resulted in the aerosol pair
• No illness but infection and shedding occurred in the pair previously inoculated with inactivated virus
• Infection, illness and shedding were apparent in the new volunteer pair
• Neutrophil counts were significantly raised in aerosol-inoculated volunteers at illness onset and also, but to a lesser extent, in the 6 volunteers given inactivated virus. This explains to me why in those with a predisposition to severe RV outcomes, including those with asthma, a symptomatic RV infection is not necessary to trigger an attack.

The authors concluded...

• The aerosols generated here, which carried relatively small amounts of virus, would likely travel beyond the nasopharynx and tracheobronchial tree and be carried into the lungs, probably with <50% deposited and the remainder exhaled
• No evidence of pneumonia was found
• If RV is suitably aerosolized in sufficiently small particles, inhalation can result in lower respiratory tract disease while site-specific installation into the upper respiratory tract usually results a typical URTI or "common cold"

How do these findings translate to everyday exposures to RV coughs and sneezes and in children? In the general community we are constantly exposed to virus and have a complex, person-specific panoply of antibodies resulting from different infections beginning in childhood. This is probably why we are incapacitated by bad colds and LRTIs all the time! An addendum in the discussion of Cate's paper highlights how symptoms resulting from RV infection are best considered as part of the entire spectrum of possible outcomes. 

Previous symptomatic infection, as shown above, protects from lower respiratory tract disease hence adults are less likely to have LRTIs than children who see these viruses for the first time. Also, there is literature showing that the antibody to some RVs can protect against, or moderate, disease due to infection by other RVs. If you are antibody-free, then disease can potentially be more severe.

Cate's studies are all conducted without knowledge of the 50+ RV-Cs because they could not be grown (detected) using the cells employed by the culture methods of the day. Why is that relevant? Because some consider RV-Cs to be more asthmagenic/pathogenic and because we don't know the receptor or natural tissue tropism/distribution of the RV-Cs in humans. How the RV-Cs perform in human volunteer infections is unknown.

Certainly room remains for some new research building upon excellent studies like this one by Cate et al and highlighting (a) that RV can infect the lungs and cause disease if an aerosol is encountered and (b), that one outcome from RV infection does not fit all.

Further reading and references...

1. First HRV nomenclature assignment publication
   http://www.nature.com/nature/journal/v213/n5078/pdf/213761a0.pdf

Read More