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An infectious unit can comprise either a single virus particle or a virus

aggregate. Therefore aggregation by antibody will reduce the total number

Neutralization by aggregation does not necessarily involve an intrinsic

loss of virus infectivity. each virus particle nay retain full infectivity

but the infectivity of the population will be reduced.

Aggregation appears to be a major mechanism of neutralization for

poliovirus. Using a panel of polyclonal and monoclonal antibodies, Thomas

et a l . (1965; 1966) found that the neutralization of poliovirus correlated

with the loss of single virus particles and an increase in aggregation. The

poliovirus aggregates were separated using sedimentation velocity gradients

and occurred even when low virus concentrations « 1 0 0 0 pfu/ml) were used.

These workers failed to demonstrate the shift in isoelectric point (pi)

that Mandel (1976) had previously found and which will be discussed later.

Thomas et a l . (1985) treated poliovirus aggregates with papain which

disaggregated the virus and restored the infectivity. demonstrating that

aggregation had not intrinsically damaged virus infectivity. Addition of an

anti-Ig antibody to the resulting poliovirus-Fab‘ complexes caused a return

of both aggregation and neutralization. Interestingly. Thomas et al ■ (1986)

found that the amount of aggregation fell when the concentration of

antibody was in excess of that required for saturation of the virus but

there was no return of infectivity. indicating that aggregation was not the

sole mechanism of poliovirus neutralization. Aggregated poliovirus was

stable and could be separated using sedimentation velocity gradients

(Icenogle et a l .. 1963). Wetz et a l . (1986) also observed a rise in

aggregation as the concentration of neutralizing anti-VPl antibody was

increased. However single virions, separated frcm the aggregated

neutralized virus preparations, had lost 22% infectivity. These had an

average of one antibody molecule bound per five virus particles, indicating

that a single antibody molecule could mediate neutralization in the

78

7 9

absence of aggregation. Wetz et a l . suggested that two mechanisms of

neutralization operated (i) an antibody-mediated conformational change in

the viral capsid proteins, similar to that described by Mandel (1976), and

(ii) aggregation as described by Thomas et a l . (1985; 1966). Neutralization

of adenovirus type 2 by polyclonal anti-fibre antibodies correlated with

the amount of aggregation (Wohlfart et a l .. 1985; Wohlfart. 1968).

Neutralizing anti-hexon and anti-pent on antibodies did not aggregate

adenovirus, except when used in high concentrations, suggesting that the

protein to which the antibody bound was an important determinant of

aggregation.

The relevance of aggregation to influenza virus neutralization has not been

extensively studied. Taylor (1986) found aggregation of influenza virus.

A/Fowl Plague Virus/Host ock/34 (H7N1). was negligible when saturating

amounts of antibody were used for neutralization. When sub-saturating

amounts of antibody (either IgG, IgM or slgA) were used, aggregation did

occur but the effects of aggregation were not investigated. Taylor et al ■

(1987) used the same influenza virus strain and quantified the amount of

aggregation at various monoclonal IgG concentrations. At high IgG

concentrations t hey found no aggregation but as the antibody was diluted

the majority of v irus became aggregated. Surprisingly aggregation of virus

was observed when the antibody was diluted to sub-neutralizing

concentrations, a nd they proposed that aggregation was occurring on the EX

grid. The rise and fall of influenza virus aggregation dependent on

antibody concentration is very similar to that discussed above for

s o

3,2-

In h ib itio n of

virus

attachment to c e lls .

The steric inhibition of virus attachment to cells resulting from the

binding of antibody is conceptually the simplest mechanism of

neutralization.

Lee et al ■ (1961) demonstrated that antibodies against the sigma 1 capsid

protein of reovirus inhibited virus attachment to L cells by up to 99*. For

Epstein Barr virus (EEV). monoclonal neutralizing antibodies against the

gp220 and gp350 glycoproteins inhibited virus attachment to lymphoblastoid

cell lines by more than 96* (Miller & Hutt-Fletcher. 1968). In contrast,

they found neutralizing antibodies against the g p 8 5 glycoprotein failed to

reduce EBV attachment. The inhibition of attachment to cells for other

viruses appears to be a less important as a mechanism of neutralization.

Anti-gD antibodies only partially inhibited (by u p to 30*) the attachment

of herpes simplex virus type 1 (HSV-1) to Vfero cells (Highlander et a l ..

1967) and HE*>-2 cells (Fuller & Spear. 1965). The small reduction in HSV-1

attachment did not account for the amount of neutralization observed, which

implied that other neutralization mechanisms were also operating. Similar

findings were obtained using rabies virus neutralized by two different

anti-G monoclonal antibodies (Dietzschold et a l .. 1987). They found the

attachment of rabies virus to EH< cells was reduced by 33* and 60* whilst

infectivity was "completely neutralized". Roehrig et a l . (1988) studied the

neutralization of Venezuelan equine encephalomyelitis (VEE) virus by

monoclonal antibodies against the E2 glycoprotein. They showed that the

inhibition of VEE attachment to either Vero or IMF— 90 cells was influenced

8 1 E2c and E2h epitopes inhibited attachment by 80-90%. antibodies against

other E2 epitopes reduced attachment only by 10-30% whilst those against

the E2f epitope actually enhanced attachment by 50-100%. The reduction in

V EX attachment was not due to aggregation because Fab' fragments also

neutralized the virus (although less efficiently) and inhibited virus

attachment, and the addition of an anti-ismunoglobulin failed to increase

either effect. Joklik (1964) found that polyclonal serum reduced the amount

of rabbit pox virus attaching to HeLa cells by 40% but again this did not

account for 92-97% loss of infectivity.

The relevance of inhibition of attachment of poliovirus by antibody to

neutralization is less clear. Handel (1967) found poliovirus neutralized by

polyclonal hyperimmune serum attached to HeLa cells in similar amounts as

infectious virus. In contrast, Efcini et a l , (1963a) found that monoclonal

anti-VP 1 antibodies did reduce poliovirus attachment to HeLa cells by

30-00% but this was not sufficient to account for neutralization (99%).

Inhibition of attachment increased in proportion to antibody concentration

up to saturating amounts. One monoclonal antibody and a polyclonal

hyperimmune serum enhanced attachment to HeLa and L cells by more than

three-fold, which is surprising, because L cells lack poliovirus receptors.

It was not known if the binding of antibody had altered the virus-receptor

specificity as had been found for vesicular stomatitis virus (VSV)

(Schlegel & Wade, 1983), or whether the increased attachment was mediated

by the antibodies themselves. All the neutralizing antibodies induced a pi

shift from pH 7 to pH 3.6-4.1 which had previously been implicated as a

mechanism of neutralization (Handel. 1976). Etoini et a l . (1983a) also used

8 2 by more than 50» depending on the antibody concentration used. This

antibody did not induce a pi shift and the inhibition of virus attachment

by this antibody may be its predominant mechanism of neutralization.

Neutralization of transmissible gastroenteritis virus (TGEV) by polyclonal

IgG or secretary IgA (slgA) antibodies failed to inhibit virus attachment

to swine testis or pig kidney cells although the virus was neutralized by

99.99% (Nguyen et al ■ ■ 1986). Interestingly, increasing the concentration

of slgA resulted in a three-fold enhancement of virus attachment. The

attachment of TGEV to cells did not appear to be mediated by Fc receptors

and they suggested that the enhanced attachment of slgA neutralized virus

was due to an aggregation effect. Skinner et a l . (1988) examined the

effects of neutralizing antibodies against human immunodeficiency virus

(HIV) type 1 on the binding of purified gpl20 to CD4 positive MOLT-4 cells.

Polyclonal antibodies raised against recombinant HIV envelope components

were primarily directed towards a hypervariable region of the gpl20

glycoprotein and failed to inhibit gpl20-CD4 binding. Two monoclonal

antibodies against the same hypervariable region gave similar results. In

contrast, human serum from infected patients did inhibit gpl20-CD4 binding

and reasonable corrrelation (0.92) was found between the titres required

for neutralization and for inhibited gpl20-CD4 binding. These data support

the suggestion that two different mechanisms of HIV neutralization can

operate dependent on the epitope to which the antibody molecules bind.

For influenza virus, some groups have claimed that neutralization results

mainly from the inhibition of virus attachment to cells (Weis et a l ..

1988). This suggestion is based solely on the fact that the antigenic sites

8 3 No data were presented to demonstrate a reduced attachment of influenza

virus to cells. Contrary to the suggestion of Weis et a l .. others have

found the ability of antibody to inhibit attachment to cells depends on

class of antibody and the cell type used. Poesee et a l . (1962) demonstrated

that polyclonal anti-HA antibodies from hyperimmune rabbit serum against

A/Fowl Plague Virus/Rostock/34 (H7N1) (A/FPV/R) did not prevent virus

attachment to CEF cells although the virus was neutralized by 99.999%. This

finding was confirmed using B-K cells and A/FPV/R neutralized by saturating

amounts of monoclonal IgG antibodies directed against different antigenic

sites on the H7 haemagglutinin (Taylor, 1986; Taylor & Diamock, 1985a).

When polyclonal IgM anti-HA antibodies were used to neutralize A/FPV/R then

a reduction in virus attachment to EH< cells was found (Taylor & Dimmock.

1985b). The reduction in attachment was temperature dependent; at 4*C the

amount of virus attaching was negligible, but as the temperature was raised

more virus attached and at 37*C the amount of IgM-neutra 1 ized virus

attaching had increased to approximately 50% of non-neutral ized virus

levels. These workers suggested that the larger molecular size of IgM

antibodies sterically hindered virus attachment and that thermal agitation

was responsible for bringing the cell receptor and haemagglutinin into the

correct juxtaposition. Taylor & Dimmock (1985a) found that neutralizing

polyclonal slgA antibody also inhibited 4/FPV/R attachment to EHK cells in

a manner very similar to that found using IgM. Monomeric IgA. produced by

differential reduction of the slgA, did not prevent A/FPV/R attachment but

continued to neutralize the virus. Therefore the molecular size of the

antibody influences its ability to inhibit the attachment of influenza

virus to cells; large polymeric antibodies (IgM and slgA) reduce

attachment, whilst smaller monomeric antibodies (IgG and monomeric IgA) do

8 4

Although neutralizing anti-HA IgG antibodies at saturating concentrations

do not reduce the attachment of A/ITV/R to CEF or EH< cells (as discussed

above), they can inhibit virus attachment to chicken erythrocytes (Poesee.

1961). Dinmock (1967) proposed that the difference was due to the length of

the cell receptors. The erythrocyte receptor for influenza virus is

probably glycophorin (Jackson et a l .. 1973) which only extends 5nm from the

erythrocyte surface (Viitala & Jamefelt, 1965). Dimmock argues that the

bound IgG molecule is sufficiently large enough to prevent the virus

haemagglutinin making contact with the glycophorin. Although the receptor

for influenza virus on cultured cells has not been identified, Dimmock

suggests that the receptor is long enough to penetrate the fringe of IgG

molecules bound to the virus allowing the haemagglutinin to make contact.

It is possible that glycophorin (or a similar size of molecule) is the

influenza virus cell receptor on all haemopoietic cells, because Eisenlohr

et a l . (1967) found that a neutralizing monoclonal IgG2a anti-HA antibody

inhibited the attachment of A/PR/8/34 (H1N1) to A20 murine B-lymphoma cells

by more than 90* and (Fab' )2 fragments were equally effective.

The attachment of influenza virus to chicken erythrocytes does not result

in a productive infection (Cook et a l .. 1979). The attachment of virus

particles cross-links erythrocytes and is visualized as haemagglutination.

The inhibiton of attachment of influenza virus to erythrocytes probably

accounts for the inhibition of haemaggluti nation (HI) but this does not

necessarily reflect neutralization. Polyclonal (Yoden et a l .. 1962) and

monoclonal (Kida et a l .. 1962) anti-HA antibodies can exert a neutralizing

effect on influenza virus in the absence of HI activity. These workers

8 5 determine whether antibodies had HI activity or not. Gitelman et a l . (1966)

produced monoclonal antibody escape mutants which were not neutralized by

the generating antibody but the antibody still had HI activity against the

variant. They suggested that this may result from altered specificity

and/or avidity of the virus variant for cell receptors because the pattern

of haemaggluti nation titres using erythrocytes from different animal

species was also altered. Casino et al ■ (1906) reported that a monoclonal

IgM antibody had HI activity but did not neutralize. They suggested that

"either the epitope for neutralization overlaps with, but is smaller than,

the epitope for hemagglutination, or that it only partially overlaps with

the latter epitope" but this interpretation is open to question (section

10.3.5.). The fact that neutralization and haemagglutlnatlon-inhibitlon are

not closely linked suggests that the erythrocyte is a poor model on which

to study neutralization of influenza virus by antibody. Escape mutants of

poliovirus have been produced to which the generating antibody continues to

b i n d (Thomas et a l .. 1966).

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