Selection and Optimisation of a Mucosal Sampling
Method for Application in Phase 1 Clinical Trials
Bergin
1
P,
Langat
2
R,
Omosa-Manyonyi
2
G,
Farah
2
B,
3
J,
3
G,
3
J,
2
G,
Bizimana
Umvirigihozo
Nyombayire
Ouattara
Ingabire
4
5
4
1
3
2
Cox J , Stevens G , Fast P , Gilmour J , Karita E , Anzala O
1 International
3
R,
Park
4
H,
Chetty
5
P,
Verlinde
4
C,
Laufer
4
D,
2Kenya
AIDS Vaccine Initiative, Human Immunology Laboratory, Imperial College London, UK,
AIDS Vaccine Initiative, University of Nairobi, Nairobi, Kenya,
3Project San Francisco, Kigali, Rwanda, 4International AIDS Vaccine Initiative, New York, USA and 5International AIDS Vaccine Initiative, Johannesburg, South Africa
BACKGROUND
A)
In sub-Saharan Africa the main route of transmission of HIV-1 remains across the genital mucosa during
RESULTS
B)
Mid turbinate
sexual intercourse. A successful HIV preventative vaccine will likely require the induction of an anti-HIV
Nasopharynx
immune response at the mucosal surface. There are several approaches that may be used in order to
induce a longer lasting immune response at the mucosal surface. The International AIDS Vaccine
Anti-HIV antibodies were detected in nasal secretions of 100% of HIV-seropositive samples with IgG expressed
Detection antibody
anti-human IgG/IgA
and 93.4% of nasal samples, respectively. IgA anti-gp140 and anti-p24 IgA were detected in 94% and 88.5% of
Sample
containing Ab
Initiative (IAVI) has generated a panel of potential vaccine candidates; replicating vectors containing a
vaccine insert intended to induce a stronger and longer lasting immune response. Administered nasally,
these vectors may target the vaccine induced immune response to the genital mucosa and/or gastric
Parotid papilla, marks the entry to
the parotid duct
associated lymphoid tissue. A pilot study was performed to determine if anti-HIV antibodies could be
Figure 1. A) Nasopharyngeal tract with midturbinate (NC) and nasopharynx (NP) indicated and B) parotid gland
detected in nasal secretions collected from the mid-turbinate region of the nasal cavity (NC), nasopharynx (NP) and oral secretions (Figure 1). To determine the optimal location for sampling that would
Substrate /
colour development
UG37 Ag Clade A
gp-140/ LAI p24
Clade B
Figure 2. ELISA method
allow for the most compliancy, the collection device/material that would give the highest yield of sample,
and whether anti-HIV-1 antibodies could be detected in the nasal compartment. Volunteers were
All volunteers provided all nasal and salivary samples although volunteers indicated that the NC collection was
recruited at Clinical Research Centers in Nairobi, Kenya (Kenya AIDS Vaccine Initiative; KAVI) and
preferable to the deeper NP sample.
A)
B)
At KAVI 15 HIV-1 seropositive and 15 HIV seronegative volunteers were recruited into the study. KAVI
staff transferred the sampling and processing methodologies to PSF, where 20 HIV-1 seropositive and 20
HIV-1 seronegative volunteers were recruited. Nasal samples were collected using FloQ swabs
C)
(transudate) was collected into Falcon tubes. In addition, volunteers at KAVI provided a mid-turbinate
secretion collected using a Synthetic Absorptive Matrix (SAM) strip. Eluted samples were aliquoted and
stored at -80C until they were tested for gp140 Env (Clade A UG37) and Gag p24 (Clade B LAI) IgG and
D)
Figure 3. Anti-HIV-1 IgG and IgA against
p24 (Clade B LAI) and envelope gp140
(Clade A UG37) were present in HIV-1
seropositive samples from KAVI and PSF.
KAVI samples from A) HIV seronegative
volunteers and B) HIV seropositive
volunteers and PSF samples from C)
HIV-1 seronegative volunteers and D)
HIV-1
seropositive
volunteers.
All
seronegative samples were below the cutoff, except samples from 4 PSF
seronegative volunteers with low levels of
anti-p24 antibodies (Figure 3 C).
Anti-HIV antibodies were present in both whole oral fluid and parotid saliva from HIV seropositive volunteers,
much more mucus than parotid saliva and was more difficult to process and assess on the ELISA.
IgG antibodies were expressed at higher levels than IgA antibodies.
Midturbinate (NC) samples were similar in expression to naso-pharyngeal (NP).
described (Keefer et al., Cranage et al.; Figure 2). All mucosal samples were diluted 1:20 prior to
analysis, and plasma samples were diluted 1:100. All samples from KAVI were assessed on site at
samples gave a higher yield than the SAM strip.
DISCUSSION
NP sampling appeared to have little benefit over NC sampling, and HIV antibodies were detected in all HIVseropositive individuals. Volunteers indicated that the NC sampling was preferable to the deeper NP collection,
provide a unique and tolerable method to collect antibodies in an HIV vaccine trial following intranasal
Key:
to yield a better sample, likely due to collecting from the entire cavity.
Circles  = Nasal FloQ swab
Squares  = Nasal SAM strip
Triangles  = Nasopharyngeal FloQ swab
Black = Env (gp140) IgA
Blue = Env (gp140) IgG
Red = Gag (p24) IgA
Green = Gag (p24) IgG
CONCLUSIONS
E)
vaccination. The SAM strip has been used in studies where there has been rapid, repeated, sampling (i.e.
minutes between samples). In this case, where a single sample was taken per visit, the FloQ swab appeared
Samples collected from the turbinate nasal cavity demonstrate antibodies against HIV-1
Sample collected from multiple research centres were comparable
The methods described here are likely to be acceptable to volunteers in a Phase 1 Clinical Trial
REFERENCES
1.
Figure 4. Devices used for nasal sample collection. A) COPAN FloQ
swab used to collect samples from the midturbinate region, or up to
10cm into the naso-pharynx. B) The Synthetic Absorptive Matrix (SAM)
strip (Hunt Diagnostics UK) used to collect repeat sample from the
midturbinate region of the nasal cavity.
In all cases FloQ swab
E) A comparison of the samples collected
at KAVI and PSF. PSF had a trend
towards a higher yield of IgG and IgA
antibodies
IgA antibodies using a Enzyme Linked Immunosorbent Assay (ELISA) modified from previously
B)
In all cases the antibodies were present in their plasma samples, suggesting these
suggesting that NC sampling may result in greater compliance with repeated sampling. NC sampling may
saliva was collected from parotid glands (Figure 1) using Salimetrics Oral Swabs and whole oral fluid
A)
salivary samples.
KAVI samples.
Committee and Emory University IRB (PSF), and all volunteers provided informed consent to take part.
test plasma were used to ensure parity between the two testing laboratories.
gp140/p24 except for 4 Rwandan volunteers with low levels of anti-p24 antibodies in nasal secretions and/or
Data from KAVI and PSF were comparable, although PSF samples consistently yielded higher O.D.’s than the
Ethical approval was granted by University of Nairobi Ethics Committee (KAVI), Rwanda National Ethics
KAVI, while PSF samples were assessed at the Human Immunology Laboratory in London. A panel of
magnitude or quantity. All nasal samples from HIV-seronegative volunteers were negative for IgG and IgA anti-
but at much lower levels than that seen in the nasal secretions (data not shown). Whole oral fluid contained
METHODS
(COPAN), either from the mid-turbinate region (NC) or deeper in the naso-pharynx (NP) (Figure 1),
nasal samples, respectively. No significant differences were detected between NC and NP samples in
volunteers may have been exposed to HIV or early in infection.
RESULTS
Kigali, Rwanda (Project San Francisco; PSF) to prepare for future Phase I Clinical Studies.
at a higher level than IgA (by optical density O.D.). Anti-gp140 IgG and anti-p24 IgG were detected in 100%
2.
Keefer et al. PLOS One
2012;7(8):e41936
Cranage et al. Vaccine. 2011
Feb 4;29(7):1421-30
ACKNOWLEDGEMENTS