Having effective vaccines against influenza virus A (H5N1) is a public health priority. However, interlaboratory assay variation limits comparison of vaccine strategies without direct comparative studies. We compared the reproducibility of hHI and neutralization against a candidate standard. Overall, both assays were consistent, although neutralization displayed more intralaboratory variability than did hHI; 3 of 15 laboratories reported >2-fold differences in >25% of identical replicates.
Titers determined by neutralization were higher and had a greater range than those determined by hHI, which suggests that neutralization may be more sensitive, particularly with low-titered serum. However, for some prevaccination serum, e.g., sample N, 6 (40%) laboratories reported neutralization titers of 20–160 but negative hHI titers, which suggests nonspecific reactivity or that neutralization detects functionally different antibodies than HI. This finding is consistent with findings of seroprevalence surveys in which titers to influenza virus subtype H5N1 may be detected by neutralization but not HI or Western blot among some persons with no exposure to subtype H5N1 (7
Sample K was from a person who had no known exposure but had detectable antibodies against H5. Most (93%) laboratories detected anti-H5 reactivity to NIBRG-14 by neutralization in this sample, but fewer (21%) detected antibodies to IBCDC-RG5. Studies suggest that antibodies against subtypes H1N1 and H3N2 detected by neutralization may be more strain specific than those detected by HI (10
); however, we did not observe this difference.
Consistent with previous serologic comparisons (10,11
interlaboratory variation was noted when absolute titers for the same serum samples were compared. Neutralization displayed more variability and had differences of 35-fold (%GCV 68%–232%) compared with differences of 16-fold (%GCV 22%–163%) for hHI. Although difficulty of measuring hHI values due to fragility of erythrocytes has been noted, the intralaboratory reproducibility of hHI appears better than that of seasonal HI (10
). Both assays for subtype H5N1 compared favorably with those for subtype H3N2 evaluated previously, which found 128-fold (%GCV 138%–261%) and 724-fold differences (%GCV 256%–359%) with HI and neutralization, respectively (10
and up to 32-fold differences (%GCV 90%–128%) with HI to human influenza subtypes H1N1, H3N2, and B viruses (11
Although HI is straightforward, most laboratories preferred their own assays. Variable parameters that may influence hHI include pretest serum hemabsorption (lowers titers) and erythrocyte suspension (higher concentration lowers titers). Because no common neutralization protocols exist, laboratories have developed their own protocols, which creates potential for variability. Because operator inexperience may have influenced reproducibility of assays for subtype H3N2 (10
), laboratories were selected for expertise in serologic testing for H5. Although most used microneutralization based on an assay described by the World Health Organization (18
), protocols differed by starting dilution of serum; preparation of cells; and virus inoculation, incubation, and endpoint estimation. Laboratories that performed assays with virus infection of cell monolayers generally reported lower titers than those that used suspensions. Assays with long incubation times and non-ELISA endpoints (e.g., cytopathic activity) were associated with greater interlaboratory variation than ELISAs with shorter incubation times. A biostandard should reduce variation associated with assay differences because standardization of protocols may be limited by local availability of reagents.
Expression of the initial serum dilution, which clearly influences absolute titers, should be standardized. Although HI titers are typically expressed as the serum starting dilution before any addition of virus, calculation of starting dilutions for neutralization varies among laboratories. We propose that the calculated starting dilution for seasonal and avian influenza neutralization be expressed as serum dilution before the addition of virus (e.g., 5 µL serum in 45µL diluent plus 50 µL virus solution is considered as 1:10) as it is with HI.
Because the correlation between serum antibodies detected by hHI and protective efficacy against influenza subtype H5N1 is unclear, by default, immunogenicity criteria established for seasonal vaccines (3
) are used for subtype H5N1 vaccines despite the lack of established immune correlates for neutralizing antibodies. Although hHI and neutralization titers correlate closely (9
), this and other studies (10
) find that the relationship depends on individual laboratory-antigen-serum combinations and cannot be generalized.
A potential limitation to this study is that 07/150 was derived from recipients of adjuvanted whole-virus vaccine but test serum samples were obtained from persons who received plain or adjuvanted split-product vaccines. Interlaboratory agreement improved when NIBRG-14, but not heterologous antigens, was used, which suggests that 07/150 is clade specific. Although no association between vaccine formulation and %GCV was noted in test serum, the quality and cross-reactivity of antibodies induced by whole-virus vaccine may differ from quality and cross-reactivity induced by alternative formulations including adjuvanted, subunit, or recombinant vaccines. To reduce potential variation in antibody isotypes, we obtained day-42 postvaccination samples when possible; however, the avidity of antibody to hemagglutinin or presence of antibody against denatured viral proteins after whole-virus vaccination (20
) could influence the effectiveness of 07/150 against test serum. Differences among vaccine formulations should be examined, if possible, during evaluation of clade 2 standards; however, because production requires substantial donations of plasma, providing separate standards for specific vaccine formulations is impractical.
The overall reproducibility of sheep antiserum raised against clade 1 H5 hemagglutinin was poor; reported titers ranged widely. Because some laboratories failed to detect antibodies in sheep antiserum, the expression of relative titers did not reduce %GCV. When these laboratories were excluded from analysis, sheep serum improved interlaboratory agreement to NIBRG-14 by hHI but not by neutralization or for clade 2 antigens. This finding suggests that if assays can detect antibodies, sheep antiserum is a useful internal control; however, its role as an international standard is limited if some hHI assays appear unable to detect antibody titers. The reason for this discrepancy is unexplained because no clear association with assay method has been found. The antibody repertoire induced by cleaved hemagglutinin in Freund adjuvant in sheep antiserum will differ from that induced in humans by purified antigens. An alternative animal source and/or production method may be more reliable.
The World Health Organization Expert Committee on Biologic Standards has accepted 07/150 as an antibody standard for clade 1 H5 hemagglutinin and has assigned an arbitrary value of 1,000 IU. The assigned value of 1,000 IU is equivalent to an hHI titer of 140 (i.e., GMT to NIBRG-14 found across study laboratories), giving a seroprotective titer for 07/150 of ≈285 IU. For neutralization, a standard value of 1,000 IU for 07/150 would be equivalent to a neutralization GMT of 518. Because the relationship between hHI and neutralization is inconsistent and immune correlates are lacking, assigning a seroprotective level to neutralization is not possible. Useful information may be obtained by retesting serum from completed trials of clade 1 subtype H5N1 vaccine candidates against 07/150. Regulators will be required to discuss the interpretation of a standard before vaccine licensure for clinical use.
For standardizing serologic assays that use different influenza (H5N1) clades, a reliable animal serum source would be most convenient, but failure of some laboratories to detect antibody in sheep antiserum limits their use. The production of clade-specific standards for subtype H5 viruses will require human plasma donations, which can only be produced after initial clinical trials have been conducted. This requirement must be considered in future vaccine studies.