Edwards., Matthew J. (1999) Interactions of monoclonal antibodies with an influenza A virus. PhD thesis, University of Warwick.

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Abstract

This investigation examines the efficiency and mechanism of neutralization of influenza A/PR/8/34 virus by three haemagglutinin (HA)-specific monoclonal IgGs (H36, H37 and H9) and their Fabs. The efficiency of neutralization by the Fabs was lower than that of their IgGs. This was concentration-dependent: 50% neutralization (N50) required 104- to 375-fold more Fab than IgG, and at N$>o required 15- to 208- fold more Fab than IgG. Affinities of the IgGs and Fabs were very similar and thus did not contribute to the difference in their neutralization efficiencies. The mechanism of neutralization of influenza virus by the IgGs was complex. At N90, the majority of the loss of infectivity was attributable to inhibition of virus attachment to cells, whilst at N50 inhibition of attachment accounted for only a minority of the infectivity loss, with the majority attributed to inhibition of fusion. In between N50 and N90 both mechanisms operate simultaneously. In contrast, inhibition of attachment by the H36 and H37 Fabs was the dominant mechanism of neutralization throughout the range of concentrations studied. The H9 Fab did not inhibit virus- attachment, but inhibited virus-cell fusion. Therefore, it was concluded that the difference between the IgGs and their Fabs was the ability of the IgGs to display simultaneous neutralization mechanisms.

Post-attachment neutralization (PAN) provides compelling evidence of the ability of antibodies to neutralize by mechanisms other than inhibition of attachment. H36, H37 and H9 IgGs and their Fabs gave PAN at 4°C and 37°C. Efficiency of PAN by the IgGs was lower than the standard neutralization of free virus (STAN). The efficiency of PAN and STAN by the Fabs was similar, except for H9 Fab whose PAN activity was significantly greater than its STAN activity. The mechanism of PAN by both IgGs and their Fabs was inhibition of fusion.

The HA of influenza A/PR/8/34 virus goes through a series of conformational intermediates in the fusion process. Examination of the pre-fusion intermediate (created by treating at pH 5 and 4°C) using conformation-specific MAbs detected changes in the viral HA, but these were less extensive than in virus treated at pH 5 and 37°C (post-fusion conformation). The pre-fusion intermediate does not cause fusion, but is a fusion-committed state, as it can subsequently undergo fusion at high efficiency at pH 7.5 when the temperature is raised to 37°C. IgGs and Fabs did not inhibit the haemolysis of CRBCs by the pre-fusion intermediate, nor did they inhibit the conformational changes of the HA that trigger the fusion process. It was concluded that these antibodies interfere sterically with events that occur prior to the formation of the pre-fusion intermediate.

The point at which neutralization becomes irreversible was investigated using the three IgGs and a monoclonal IgA. Neutralization of virus in solution was shown to be reversible by removing antibody from the virus with ammonium hydroxide (pH 11.5) (AmOH). The reversibility of neutralization was also examined under conditions designed to mimic the early steps in the virus infectious cycle, (1) virus attachment to cells and (2) low pH-induced virus-cell fusion. Neutralization could be reversed by removal of antibody with AmOH, after neutralization of (1) virus attached to cells and (2) after low pH treatment of virus attached to cells. This suggested that these MAbs do not trigger a permanent loss of virus infectivity, but are required to be bound to the virus for the duration of the neutralization process.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology > QR355 Virology
Library of Congress Subject Headings (LCSH):	Influenza A virus -- Infections, Monoclonal antibodies, Immunoglobulins
Official Date:	1999
Dates:	Date Event 1999 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dimmock, N.J.
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain)
Extent:	xxii, 287 leaves : charts, illustrations
Language:	eng

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