Humoral immunity plays an important role in the host defense against influenza virus infection. One of the key functions of antibodies is to neutralize the virus, rendering it noninfective, and these antibodies can be measured by MN assay, which detects antibodies that neutralize viruses, or indirectly by hemagglutination inhibition (HI) assay, which detects antibodies targeting the sialic acid binding sites of the surface HA protein. Antibody titers determined by MN and HI assays usually have good correlation with recent A(H1N1)pdm09 infection (15
). MN or HI titers of 40 or more are associated with protection from influenza virus infection and are often used as markers of successful immunization in influenza vaccine trials (23
). During the first few days of an infection, the neutralizing antibody titers are often low (34
). However, the magnitude of nonneutralizing antibodies during this period has not been studied. We hypothesized that the nonneutralizing antibodies in the early stage of infection may play a role in the outcome of an infection. To detect nonneutralizing antibodies, we used an ELISA plate coated with a split-virion inactivated A(H1N1)pdm09 virus vaccine as antigen, which could detect both neutralizing and nonneutralizing antibodies against the vaccine proteins. We showed that within 2 to 4 days after symptom onset, significantly higher ELISA titers were found for patients with severe disease than for those with mild disease, but there was no significant difference in their MN titers. The ELISA/MN titer ratio, which is a surrogate marker for the amount of nonneutralizing antibody, was higher for patients with severe disease than for those with mild disease. Patients requiring PPV, representing those who had the most severe respiratory disease, had higher ELISA titers than those who did not require PPV. The results from our study suggested that an exaggerated nonneutralizing antibody response during the early stage of infection was associated with severe disease.
The nonneutralizing antibody present in patients during the early stage of infection was likely to be preexisting or was the result of a secondary heterotypic antibody response against conserved epitopes, which may be found outside the receptor binding pocket of HA or in the highly conserved nucleoprotein, matrix proteins, and polymerase proteins, or even the less conserved NS proteins (11
). This early IgG response can happen within a few days after infection because of immune priming by previous exposure to similar viral epitopes. In our ELISA, we used a split-virion inactivated A(H1N1)pdm09 vaccine as the coating antigen, and it contained mainly HA but also other proteins of the A(H1N1)pdm09 virus, including the neuraminidase, the matrix protein, and the nucleoprotein (4
). The matrix proteins and nucleoprotein have conserved amino acid sequences, and therefore antibodies against these proteins from prior seasonal influenza virus infection or vaccination could be induced. Upon infection with the A(H1N1)pdm09 virus, memory B cells can proliferate rapidly and generate a large amount of these high-avidity nonneutralizing antibodies, especially in patients with severe disease. This is consistent with the observation that the number of peripheral blood B cells is higher in patients with severe disease than in those with mild disease during the early stage of infection (14
Nonneutralizing antibodies against influenza virus can have either protective, neutral nonprotective, or detrimental effects. Antibodies against the M2 ectodomain and the nucleoprotein have been shown to be protective in animal models (2
). On the other hand, antibodies against NS1 have been shown to delay viral clearance (21
). This is consistent with the previous finding that a delay in viral clearance rather than a high initial viral load in the respiratory tract is associated with severe disease (37
). Antibodies against PB1-F2 and M1 can be detected in patients with influenza virus infection, but the functional significance of these antibodies is unknown (20
). Other potential effects of nonneutralizing antibody include the activation of the complement cascade and antibody-dependent cellular cytotoxicity, which can be protective or detrimental if excessively proinflammatory (1
). Besides the uncertainty of these effects on the clinical outcome, the avidity of such nonneutralizing antibodies for complement activation and antibody-dependent cytotoxicity is also unknown. If these effects turn out to be detrimental, it is conceivable that such nonneutralizing antibodies with high avidity would produce more damage to patients than antibodies with low avidity. Though the notion that prior seasonal influenza vaccination is associated with worse outcomes for patients with subsequent A(H1N1)pdm09 virus infection has been disputed, nonneutralizing antibodies may play a role if this is indeed the case (33
). Detrimental effects of nonneutralizing antibody have been speculated to be associated with poor outcomes of dengue virus, respiratory syncytial virus, and measles virus infections via antibody-dependent enhancement (42
). Though the laboratory phenomenon of antibody-dependent enhancement has also been shown for influenza virus, no clinical study has ever been performed to ascertain the link (35
). The pro- or anti-inflammatory effects of nonneutralizing antibody in the pathogenesis and outcome of influenza should be investigated further (3
Antibody avidity was assessed in the current study by comparing the ELISA OD values with and without urea, which is a dissociating agent that can disrupt the interaction between the antibody and the coating antigen. If an antibody binds to the antigen weakly, then the addition of urea will disrupt the binding, resulting in a lower OD value. We have shown that higher antibody avidities are independently associated with severe disease. This is consistent with our finding that patients with severe disease have higher levels of secondary antibody production, presumably from memory B cells. Our finding appears to contradict a study by Monsalvo et al., who showed that patients with severe A(H1N1)pdm09 virus infection had lower antibody avidity than those with mild disease (28
). Several groups have also examined the relationship between age and antibody avidity in patients with natural infection, and they have shown that antibody avidity is higher in older than in younger age groups (28
). For antibody responses after influenza vaccination, one study showed a higher antibody avidity in the elderly than in younger vaccine recipients, while another study did not find any differences (18
). In our study, we did not find any correlation between age and antibody avidity. There are several important differences between other studies and ours which may explain the discrepancy of the results. First, we examined the serum antibodies that were present shortly after a natural infection, which are probably different from the antibodies arising in the convalescent phase after natural infection or vaccination. Second, we used a split-virion influenza vaccine, which contains HA and other viral proteins, while other studies used pure recombinant HA without other viral proteins. There may be differences in avidity for antibodies directed against HA but not for those against other viral proteins. Finally, a sampling bias may have occurred in the severe cases, because for those patients who died early on, no sera could be collected in the convalescent phase.
In this study, we employed a split-virion inactivated vaccine. This approach has several advantages over using recombinant antigens. First, by using the vaccine as the coating antigen, we could detect the sum of a wide range of IgGs against different viral components instead of a limited IgG response specific to a particular protein. Since it has been demonstrated that antibodies may have synergistic effects, our study is deemed more relevant by examining the antibody response against a range of different viral proteins instead of just the antibody response against a single viral protein (43
). Second, recombinant proteins may assume an altered conformation, and conformation-dependent antibodies may not recognize the altered antigen structure (49
). The use of a vaccine also differs from the use of inactivated virus, because zonal centrifugation will remove nonviral host materials which may lead to nonspecific reactions (13
There were several limitations to our study. We did not test the antibody titers in serum samples collected on day 0 and day 1 after symptom onset because most patients did not attend the hospital during this early stage of illness. Except for HA, the exact quantity of each protein present in our batch of the vaccine was not known. However, we used the same batch of vaccine across all experiments to ensure consistency of the antigen content. Another limitation of our assay was that the high ELISA titers may have been related to antibodies that could neutralize the virus in vivo
but could not be detected with the current MN assay (25
). Finally, although the high ELISA titers were likely related to the rapid production of nonneutralizing antibody targeting conserved epitopes, we do not know the quantity of preexisting antibody, as preinfection serum samples were not available.
Our study is the first to look at the early antibody response (both neutralizing and nonneutralizing antibodies) against current vaccine components and the association between these antibodies and patient outcomes. The excessive nonneutralizing antibody response during early infection may have contributed to the dysregulated inflammation in severe infection (38
). It is important that even an HI titer of 40, which is considered to be a protective titer reflecting mainly a neutralizing antibody response in vaccine studies (12
), was associated with a protective efficacy of only 70% in an exposed population (30
). Moreover, some vaccinated patients with very high HI titers (up to 2,048) developed disease after exposure to influenza virus (29
). This protection is therefore not absolute and can be overcome by a larger dose of virus (3
). Once the virus enters cells and the viral life cycle is started, every infected cell will produce millions of infectious virions, which can easily overcome the neutralizing antibody in the interstitial fluid to infect adjacent cells as far away as where the balance occurs between the dilution of virus and the neutralization effect of antibody. Thus, at the anatomical site where the viral load cannot be controlled by the initial innate immune response, the concomitant innate inflammatory response and the subsequently mounted adaptive immune response with nonneutralizing cross-reactive antibody may just fuel inflammatory damage. This is a significant issue in the elderly and those with severe comorbidities, whose respiratory reserve is marginal and tissue regenerative power is poor. In addition, inflammatory cytokines spilled over into the circulation may precipitate acute catastrophes such as stroke or heart attack (46
). Further studies should be conducted to ascertain the role of nonneutralizing antibody in the pathogenesis of severe influenza.