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Paradigm Shift Intervention Monitoring
DR. GELLIN: I am always impressed by
what a body of work that is. Two questions. One of them, you had
mentioned about the low reactors and the biological issues at the lab
in London, is that the same issue for the H1 as well as the H3? Because
they showed, I think, that there was a higher percentage of H1 low
reactors in the London lab, as well. The question really is that a lab
problem or does that signal some other evolution, since they have had
more H1 this year?
DR. COX: Yes. What we know for H1N1 viruses is that if you isolate them in a particular cell line, you tend to get more of the variance with changes within that region that I mentioned -- the 153 to 158 region. They had been using the particular cell line that pulls out that particular variant or that selects for that particular variant.
They have subsequently switched to normal MDCK cells. I think what they are seeing is a combination of the two phenomena. Plus, they receive a lot of viruses that have been isolated COT, the special kind of MDCK cells. I think it is a combination of they are receiving viruses with changes that cause that reduction in activity and then having some additional low reactors, but they are scattered throughout the tree.
The above comments from the 2011/2012 vaccine advisory meeting provides additional data supporting a emphasis on using cell lines that reduce the likelihood of identifying low reactors, which explains the failure of the CDC to report H1N1 low reactors in US isolates from the 2009/2010 and 2010/2011 seasons. The above comments do not claim that the “normal” MDCK cells produce isolates that are reflective of the H1N1 in the patient, and in fact the data released in weekly FluViews for the past two seasons strongly suggests extreme bias in the CDC data.
The antigen characterization test can be heavily influenced by the isolation procedures for the test virus, as well as the creation and use of the ferret reference sera. CDC data has been internally and externally inconsistent with data produced by the CDC other WHO regional centers, including Mill Hill, as described above. These inconsistencies involve the antigenic site described above, as well as receptor binding domain changes, including D225G and D225N.
The data bias and inconsistencies for the antigenic reason are best demonstrated by G158E (H3 numbering), which has been demonstrated to be an important change associated with low reactor status via multiple approaches by multiple labs, including the CDC. One of the first H1N1 low reactors identified by the CDC, A/Bayern/69/2009, was an isolate from Germany that had G158E. This result was supported by Mill Hill, who characterized another G158E isolate from Germany, A/Bayern/62/2009, as a low rector. This designation was supported by data from MedImmune who had characterized various clones from California/07/2009 (CA/07), the H1N1 vaccine target. One clone had G158E and grew well in chicken eggs and therefore was a vaccine candidate. However, antisera directed against wild type CA/07 had a dramatically reduced titer against the G158E clone, so the clone was not selected. Similarly, escape mutants that evaded monoclonal antibodies directed agains CA/07 also had G158E, providing another line of evidence supporting the role of G158E in the generation of low reactors.
However, the assay used by the CDC subsequently failed to identify isolates with G158E as low reactors. One dramatic example was an isolate from Ukraine, A/Lviv/N6/2009, which had D225G. Mill Hill designated this isolate as a low reactor. However, the CDC isolated a virus from this patient that had D225G and G158E, yet did not designate the isolate as a low reactor. This isolate subsequently was used as a control in antigen characterization tests and data published by the Health protection Agency in the UK as well as the WHO regional center in Australia used the target and clearly showed a dramatic reduction in titer against a number of ferret anti-sera generated against various targets, including CA/07.
The lack of sensitivity in the CDC is reflected in FluView reports. Although other labs report the frequent detection of G158E, the CDC reported only 6 low reactors out of 1847 tests of 2009/2010 isolates and all six involved changes at position 159. Thus, the CDC found no examples of low reactors in US isolates with G158E. The frequency of low reactors in the 2010/2011 season is even lower, with only one reported example out of 490 tests. Recent sequences from Germany and Japan have high frequencies of changes at positions 156-159, with G158E being the most common change.
This failure of the CDC to identify isolates with G158E, as well as the failure to classify isolates with G158E as low reactors raises serious issues of bias in the nCDC isolation procedures as well as the sensitivity of the antigen characterization test.
This selection bias extends to the recent emergence of the Chihuahua sub-clade with D225N and associated 2011 pandemic alert issued by WHO. D225N is a raely reported receptor binding domain change, yet it was found in 2 of 11 Chihuahua sub-clade sequences from sever or fatal cases in Mexico. These data were generated by direct sequencing of the clinical sample (pharyngeal swab). Similarly, when the CDC released data from direct sequencing of their first two samples from Ukraine, both had D225N, which was also seen at a high frequency in direct sequences generated by Mill Hill of Ukraine autopsy lung samples.
Recently, the CDC has release 8 sequences from 2011 US samples that are closely related to the D225N sequences from Mexico, but none of the CDC sequences have D225N. However, none are direct sequences, raising concerns that this important change is also being selected against by CDC isolation procedures.
These isolation issues and biased antigen characterization tests can have serious consequences. At the vaccine selection meeting, the Air Force provided dramatic evidence of recent vaccine breakthrough in military recruits, consistent with release of Chihuahua sequences from vaccinated military dependents, raising serious concerns about the current utility of an H1N1 vaccine directed against CA/07. However, in spite of this compeling evidence against the efficacy of the current H1N1 target, the committee voted 15-0 with one abstention, to leave the H1N1 target unchanged, largely based on the antigen characterization data presented by the CDC.
The reliance on this heavily biased test for vaccine target selection continues to be hazardous to the world’s health.