Specific Antibody to Challenge Virus in Serum

BCS

Blood samples for determination of specific antiviral immunoglobulin A (IgA) and Immunoglogulin G (IgG) were collected 1 to 2 days prior to viral challenge and 28-days post-challenge.  Pre-challenge samples were collected by study staff.  Post-challenge samples were collected by subjects’ own physicians and shipped back to the CCU.  Serum from both samples was separated from whole blood, stored at -20°C, and then heated at 56°C before use.

Serum Antibody Titers

Viral specific Immunoglobulin A (IgA) and Immunoglobulin G (IgG) levels for rhinoviruses, coronaviruses (CV), and respiratory syncytial virus (RSV) were determined by enzyme-linked immunosorbant assays (ELISA). 

Specific Antibody to Rhinoviruses¹

Antihuman IgG produced in goats and conjugated to alkaline phosphatase (Miles Laboratories) was used at a dilution of 1:1000 in phosphate buffered saline containing 0.05% Tween-20 (PBS-T; Sigma Chemical Co.) and 0.1% bovine serum albumin (BSA).  Similarly, antihuman IgA produced in goats and conjugated to alkaline phosphatase (Sigma) was diluted 1:1000 in the same diluent, and antirabbit IgG produced in goats and conjugated to alkaline phosphatase (Miles) was used at a dilution of 1:3000 also in PBS-T containing 0.1% BSA.

Polystyrene round-bottomed micro ELISA plates (Nunc Immunoplates U type II, Gibco Inc.) were incubated overnight at 4°C with 100 µl per well of a 1:4000 dilution of rabbit hyperimmune antirhinovirus serum in carbonate/bicarbonate buffer (pH 9.6).  The wells were drained and uncoated sites at which nonspecific binding might occur, were blocked with 1% BSA in PBS for 2 hours at 37°C.  Plates were washed 3 times with PBS-T.  One hundred µl of a 1:10 dilution of crude virus antigen, which had an infectivity of about 107 TCID₅₀ per ml, or control antigen in PBS-T containing 0.1% BSA and 5% uninfected tissue culture fluid was added and plates were incubated overnight at 4°C.  After washing as before, test samples at various serial dilutions in the same diluent were added and incubated for 2 hours at 37°C.  Plates were then washed and the appropriate conjugate added and incubated for 2 hours at 37°C.  Finally, plates were washed 5 times with PBS-T, and 100 µl of p-nitrophenol phosphate (Sigma) dissolved in 10% diethanolamine buffer (1mg/ml) added to each well.  Plates were left uncovered at room temperature and the optical density at 405nm was read at various time intervals in a Titertek Multiskan ELISA reader (Flow Laboratories).

Sera were tested in duplicate 10-fold serial dilutions.  The means from the duplicates were corrected for each dilution by subtracting the mean optical density (OD) in the absence of sample.  From this value, the OD with control antigen coated wells (corrected in the same way) was then subtracted.  The corrected OD was plotted against the log of the sample dilution and this generated a sigmoid curve.  An assay line was chosen at an OD level which intersected this curve in the linear portion for the majority of samples.  The ELISA titer was taken as the reciprocal of the sample dilution corresponding to that OD.

Specific Antibody to Coronavirus and Respiratory Syncytial Virus²

Coronavirus (CV229E) were grown in the C-16 strain of continuous human fibroblast cells, and respiratory syncytial virus (RSV) was grown in HEp-2 cells.  In all other respects, the assay procedure used to determine serum antibody levels was the same for both virus types.

Infected tissue cultures were disrupted by freezing and thawing, clarified by low-speed centrifugation, and stored at -70°C.

Inactivated sera were diluted in phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) and 5% of an extract of uninfected cells (control antigen).

Anti-human IgG produced in swine and conjugated to alkaline phosphatase (Northumbria Biologicals, Ltd., Hertfordshire, U.K.) was used at dilution of 1:400.  Goat anti-human IgA (α chain) and goat anti-human IgM (µ chain) conjugates (Sigma Chemical Co., U.K.) were both used at dilutions of 1:1000. 

Polystyrene round-bottomed micro ELISA plates (Nunc Immunoplates U type II, Gibco Inc.) were coated with virus or control antigen at about 8 µg protein/ml in carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at room temperature.  Plates were then washed three times with PBS-T.  After addition of diluted specimens, the plates were left at room temperature for 4 hours.  Plates were washed as before, diluted conjugates were added, and the plates were left at room temperature overnight.  On the next day, plates were washed, p-nitrophenol phosphate (Sigma) dissolved in 10% diethanolamine buffer (1 mg/ml) was added, and the plates left at room temperature for at least 30 minutes, permitting the color to develop. 

Optical density (OD) was read with a TiterTek Multiskan ELISA reader (Flow Laboratories, High Wycomb, U.K.) at 405 nm.  The low ODs obtained in wells without samples were subtracted from the ODs in wells with samples and then the adjusted ODs obtained in control antigen-coated wells were subtracted from those obtained in virus-coated wells.

Microtiter Neutralization Assay

A microtiter neutralization assay also was used to determine circulating levels of antibody to the challenge virus³.  Neutralization antibodies were determined for the 3 rhinoviruses (RV2, RV9, and RV14) only because suitable neutralizing tests were not available for RSV or CV229E.  Results were recorded as the highest dilution showing neutralization, and a fourfold rise was graded as significant. 

50 µl of HeLa maintenance medium with 5% fetal calf serum was added to each well of a microtiter plate, except for the cell control wells to which 100 µl of HeLa maintenance medium was added.  50 µl of heat-inactivated acute and convalescent serum was then added to each of the first two wells, and another 50 µl to the last two wells (serum controls).  Doubling dilutions of the serum from 1:2 to 1:128 were then made in the plate using a multichannel pipette.  50 µl of 50% tissue culture infective dose (TCID₅₀) virus was added to all wells except the serum and cell control wells.  Plates were shaken using a microshaker and incubated in the presence of 5% CO₂ (using a plastic box) for 1 hour at room temperature.  100 µl of freshly stripped Ohio Hela cells at a concentration of 3x10⁵ cells/ml in 5% fetal calf serum and maintenance medium was added to each well.  Plates were then incubated at 33°C in 5% CO₂ for approximately 5 days and then examined microscopically for inhibition of cytopathic effect.  The neutralization titer of the serum was computed as the reciprocal of the highest dilution that completely inhibited the virus cytopathic effect.

PCS1, PCS2, PCS3, & PMBC (Rhinovirus)

All three Pittsburgh Cold Studies and the PMBC rhinovirus trials used a microtiter neutralization assay to assess antibody to the challenge virus³.  Aside from the timing of the pre-challenge sample collections, all procedures for the assay were identical across the four studies.

For all blood draws, 5 ml of whole blood was collected in 7-ml red-topped vacutainer tubes and then placed in a refrigerator to clot.  Serum was collected by centrifugation of clotted blood at 2,000 rpm (900 X g) for 20 minutes.  Using sterile glace Pasteur pipettes, serum was transferred into numbered cryovials, and then stored at -20^oC until shipped to the laboratory for assay.

Enough 96-well plates were prepared for all samples using human embryonic lung fibroblast (MRC-5) cells.  Frozen serum samples were heat-inactivated in a H₂O bath at 56°C for 1 hour.  Trypsin for cell removal was thawed, and growth media comprised of 10% fetal bovine serum in EMEM with L-glutamine, and penicillin-streptomycin was heated, both at 37°C.

After 50 μl of growth media was added to all wells in a labeled 96-well plate, 50 μl of participant serum was added to rows A and E, two wells per sample (four wells per participant), producing a 1:2 dilution of the serum in these rows.  A multi-channel pipetter was used to mix the serum and growth media and then to remove 50 μl of the mixture from row A and add it to row B producing a 1:4 dilution of sera. This procedure was repeated to produce serial 2-fold dilutions (1:8 and 1:16, respectively) in rows C and D.  Contents of each well in row D were mixed again and then 50 μl from each well was discarded.  This procedure repeated for rows E-H.

A virus pool of the desired serotype was diluted to provide a final virus concentration of 10-50 TCID₅₀/50 μl, and 50 μl of the diluted virus was added to all wells using a multi-channel pipetter.  The mixture of participant serum and virus was incubated for 1 hour at 33°C in 5% CO₂.  At the end of one hour, the participant serum/virus mixture was transferred into 96-well plates with cells removing all media from cells first, and then incubated again at 33°C in 5% CO₂.

Positive (virus and MRC-5 cells with no participant serum) and negative (MRC-5 cells only without serum or virus) control wells were included with each batch.  A virus titer of final virus concentration was used for the assay, with serial ten-fold dilutions and a minimum of six replicate wells at each dilution.

Plates were examined every two days for development of typical virus cytopathic effect. The results of the assay were recorded between 3 and 10 days post-inoculation when all positive control wells were clearly positive.

PMBC (Influenza)

Antibody to influenza was determined using a standard hemagglutination inhibition (HI) assay⁴.  Hemagglutination is the process by which the viral surface protein, hemagglutinin (HA), binds to red blood cells and causes them to clump together or agglutinate.  This property of viruses is the basis for the hemagglutination assay, which frequently is used to detect the presence of virus in bodily fluids (see Isolation and Confirmation of the Challenge Virus in Nasal Secretions, PMBC – Influenza).  The HI assay is a modification of the HA assay that is used to detect the presence of antibody to the virus.  Antibodies to virus function by preventing attachment of viral HA to red blood cells and by extension inhibiting hemagglutination.  Accordingly, if a serum sample containing no antibody to influenza is incubated with influenza virus antigen, hemagglutination will be observed.  By comparison, if the serum does contain antibody to influenza, hemagglutination will be inhibited.  The HI titer of the serum is then computed as the highest dilution of serum that prevents hemagglutination.  

References

¹Barklay, W. S. & Al-Nakib, W. (1987). An ELISA for the detection of rhinovirus specific antibody in serum and nasal secretion. Journal of Virological Methods, 15, 53-64.

²Callow, K. A. (1985). Effect of specific humoral immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection. Journal of Hygiene, 95, 173-189.

³ Al-Nakib, W. & Tyrrell, D.A.J. (1988). Picornviridae: Rhinoviruses—common cold viruses. In E.H. Lennett, P. Halnen, & F.A. Murphy (Eds.), Laboratory diagnosis of infectious diseases: Principles and practice (Vol. 2, pp. 723-742). New York: Springer-Verlag.

⁴Dowdle, W.A., Kendal, A.P., & Noble, G.R. (1979). Influenza virus. In E.H. Lennett & N.J. Schmidt (Eds.), Diagnostic procedures for viral, rickettsial, and chlamydial infections (pp. 595-606). Washington, D.C.: American Public Health Association.