By Eric Toner, M.D., February 21, 2007

In an article published in the online journal PLoS Medicine on
February 13, 2007, Matthew Sandbulte and colleagues report results
of their study of cross protection between the N1 antigen in
seasonal influenza vaccines and H5N1. In their study, mice were
vaccinated with the neuraminidase antigen, N1, from the human H1N1
strain (A/New Caledonia/20/ 99), which is used in seasonal flu
vaccine. These immunized mice then had significantly reduced
mortality when challenged with H5N1. In addition, they found that
some people carry antibodies that react to the N1 in the avian H5N1
virus [1].

Background
The experience with the 1968 influenza pandemic gave rise to the
hypothesis that cross-immunity to similar neuraminidase antigens of
different influenza strains may reduce the severity of illness. In
1968, a novel virus emerged containing an H3 antigen of avian origin
and the N2 antigen that had been circulating in humans since the
H2N2 pandemic of 1957. Although widespread immunity to N2 in the
human population did not prevent a pandemic, it may have contributed
to the mildness of the 1968 pandemic. Studies demonstrated an
inverse correlation between anti-N2 titers and severity of illness,
suggesting that the neuraminidase antibodies ameliorated the disease
[2]. Neuraminidase, one of the two principle surface proteins of
influenza viruses, is an enzyme that is essential for the release of
newly formed viruses from infected cells. Blocking neuraminidase,
either with drugs such as oseltamivir or antibodies to
neuraminidase, may reduce illness and viral replication.

Methods
Sandbulte's study, which was conducted at the St. Jude's Children's
Research Hospital in Memphis, Tennessee, consisted of 3 parts:

1. Mice were immunized with a DNA vaccine encoding the N1 antigen
used in the current seasonal influenza vaccine and were then
challenged with supra-lethal doses (both 10 and 100 times the mouse
LD50) of one of 3 viruses:
A reassortant virus produced by reverse genetics containing N1 from
a human H1N1 strain
A reassortant virus containing N1 derived from avian H5N1
A wild H5N1 virus recovered in 2004 from a human victim in Vietnam.

2. Serum from immunized mice (including both mice that had been
immunized but not challenged and immunized mice that had been
subsequently challenged with H5N1 and survived) was injected into
other mice to test whether immunity can be transferred passively.
3. Human volunteers were tested for titers of antibodies that react
with avian N1.

Results
As expected, all unimmunized control mice died when exposed to any
of the 3 viruses, and all immunized mice survived challenge with the
virus containing human N1 at both 10 and 100 times the LD50.
Significantly, 50% of immunized mice also survived infection with 10
times the LD50 of the wild type H5N1. A similar result occurred when
the immunized mice where infected with the reassortant virus
containing avian N1, demonstrating that it was immunity to N1 that
protected the mice. However, the protection was dose-dependant.
Immunization did not protect the mice from challenge with doses 100
times the LD50.

In the passive immunity portion of the study, serum obtained from
mice that were immunized with human N1 and survived infection with
wild type H5N1 was injected into other mice and completely protected
them from exposure to 10 times the LD50 of wild type H5N1. This
result demonstrates that humoral immunity is, at least partially,
protective. Serum from mice that had been vaccinated, but not
infected with H5N1, provided a lesser degree of protection: Only 6
of 13 of those mice survived subsequent challenge. Thus, as
expected, subsequent infection boosted the humoral immunity induced
by the N1 vaccination.

In the third part of the study, serum from 38 human volunteers was
tested using a neuraminidase inhibition assay. As expected, most
(31/38) were found to have antibodies to the human H1N1 strain used
in the current seasonal flu vaccine. A minority (~20%) also had low
inhibitory activity (titers 20-80) against avian H5N1.

Discussion
In this mouse model, antibodies to the N1 of human H1N1 cross-react
with the N1 of avian H5N1 resulting in a significant reduction in
mortality. Furthermore, some people have a low degree of anti-avian
N1 activity in their serum, presumably from prior exposure to human
H1N1, which suggests that humans with such antibodies may be more
likely to survive infection with H5N1. If this is true, then the
results of a pandemic with H5N1 may not be quite as catastrophic as
feared, and universal vaccination with seasonal flu vaccine may to
some extent mitigate the consequences of a pandemic. More research
is needed to confirm these results in other animal models and to
ascertain the prevalence and degree of anti-avian N1 antibodies in
the wider population.

References
[1] Sandbulte MR, Jimenez GS, Boon ACM, et al. Cross-Reactive
Neuraminidase Antibodies Afford Partial Protection against H5N1 in
Mice and Are Present in Unexposed Humans. PLoS Med 2007; 4(2): e59.
Available at: http://medicine. plosjournals. org/perlserv/ ?request=
get-document& doi=10.1371/ journal.pmed. 0040059. Accessed February
15, 2007.

[2] Gillim-Ross L, Subbarao K. Can Immunity Induced by the Human
Influenza Virus N1 Neuraminidase Provide Some Protection from Avian
Influenza H5N1 Viruses? PLoS Med 2007; 4(2): e91. Available at:
http://medicine. plosjournals. org/perlserv/ ?request= get-document&
doi=10.1371% 2Fjournal. pmed.0040091. Accessed February 15, 2007