COMPARATIVE ASPECTS OF REGIONAL IMMNITY TO INFLUENZA VIRUS
INFECTION AND DNA VACCINATION
Dr. D. Paul Lunn and Dr. Chris W. Olsen, School of Veterinary Medicine,
University of Wisconsin-Madison.
Equine influenza remains one of the most important infectious diseases of the
horse throughout the world and is enzootic in North America. Strenuous
attempts are made to control equine influenza virus infection by vaccination, but
this approach is of limited value because currently available commercial
vaccines provide extremely short lived protection,1 and in the field these
vaccines have little ability to control infection.2
In pigs influenza can occur as either an enzootic problem in a herd or, more
commonly, as explosive outbreaks of acute respiratory disease. Although rarely
fatal, swine influenza can be of substantial economic impact and may also play
an important in the global ecology of influenza A viruses in humans. The major
pandemics of human influenza this century were caused by viruses that were
reassortants between pre-existing human and avian strains.3 Pigs have been
implicated as the intermediate species for adaptation of avian viruses to
mammals and as the “mixing vessels” in which human-avian virus reassortment
occurs. As in horses, the inactivated whole-virus vaccine that is commercially-
available in the United States does not consistently provide complete protection
from challenge infection.
Immunity to influenza virus infection
Protection from influenza virus infection is thought to depend primarily on
secretory IgA and transudated IgG antibodies at mucosal surfaces, with serum
IgG and cytotoxic T lymphocytes (CTL) preventing progression and eliminating
virus during the recovery phase.4-6 In murine models it has been demonstrated
that influenza-specific IgA in respiratory secretions protects against influenza
virus infection.7 Differences in IgG sub-isotype responses to influenza virus are
likely to be important in determining protective immunity. In murine models,
vaccines that induce IgG sub-isotype responses most like those resulting from
natural infection produce the most complete and long lasting immunity.8
Natural equine influenza virus infection confers complete clinical immunity for
over six months and partial immunity for over one year.9 Long-term protective
immunity against infection is therefore obviously possible and a successful
equine influenza vaccine should stimulate the same type of immune responses
as infection does. An experiment was performed to compare local and circulating
antibody responses and protection resulting from a conventional commercial
vaccine or natural influenza virus infection.1 The horse possess a particularly
complex array of immunoglobulin isotypes, with four sub-isotypes of IgG: IgGa,
IgGb, IgGc, and IgG(T), in addition to the other immunoglobulin isotypes. It was
found that 3 months after administration of two dose of a conventional vaccine,
ponies were left unprotected when subjected to a challenge infection. In
contrast, 3 months after being given an initial influenza virus infection, ponies
were completely immune to a repeat challenge infection. A critical difference
between natural infection and vaccination was that infection induced high levels
of IgA in nasal mucosal secretions whereas vaccination induced no IgA
antibodies. In addition there were marked differences in the isotypes of IgG
induced by infection compared to vaccination, with natural infection inducing
IgGa and IgGb responses and conventional vaccines inducing IgG(T)
responses. In the horse the IgGa and IgGb sub-isotypes are capable of
mediating important anti-viral activities such as complement fixation and
antibody-dependent cellular cytotoxicity, while IgG(T) responses can actually
inhibit complement fixation. Overall there is clearly a need for vaccines better
able to induce protective components of the immune response.
Current active vaccination strategies can be broadly divided into the
administration of “live”, “dead” and DNA vaccines, and these approaches have
been recently and extensively reviewed.10-12 Live vaccines include attenuated
microbes and recombinant vaccines that utilize a living vector, while dead
vaccines include killed whole pathogens, soluble pathogen subunits or protein
subunits. Immunization based on administration of plasmid DNA (variously
termed “genetic, “nucleic acid” or “DNA” immunization) is a radically different
form of vaccination that enjoys many of the immunological and safety
advantages of both live and dead vaccines. DNA vaccination results in the in
vivo synthesis of antigenic proteins in a manner identical to that occurring in
natural infection.13 This endogenous production results in presentation of
antigens by MHC I and presentation to CD8+ T-cytotoxic lymphocytes, and
uptake and presentation of soluble proteins by MHC II to CD4+ T-helper
lymphocytes. As a result DNA vaccination has been shown to induce both potent
CTL and antibody responses. To date no undesirable side effects have been
associated with the use of DNA vaccination. Overall DNA vaccines can deliver
the immunogenic advantages of live vaccines but at a low cost and with minimal
safety concerns. The intense scientific interest in DNA vaccination can be
gauged by visiting the World Wide Web site maintained at
Influenza virus has been used extensively as a model pathogen in DNA vaccine
studies in mice, chickens, ferrets, pigs, horses and non-human primates, and
clinical trials of DNA-based influenza virus vaccines are underway in humans.
Our studies have focused on gene gun delivery of DNA vaccines against equine
and swine influenza viruses in mice, ponies and pigs, including studies
employing co-administration of interleukin-6 DNA or cholera toxin as an
approach for modulating and adjuvanting influenza virus hemagglutinin-specific
immune responses. The results indicate that gene gun administration of
plasmids encoding hemagglutinin genes from influenza viruses is an effective
method for priming and/or inducing virus-specific immune responses, and for
providing partial to complete protection from challenge infection in mice, ponies
and pigs.14-17 In addition, studies of interleukin-6 DNA co-administration in mice
clearly demonstrate the potential for this approach to enhance vaccine efficacy
The results of these DNA vaccination experiments demonstrate the capacity of
immune responses directed solely against the hemagglutinin protein to protect
against influenza virus infection in a number of species. In addition, DNA
vaccination also provides a powerful tool for identifying the role of specific
immune responses in this protection and studying the regulation of these
responses. For example, in horses, DNA vaccination responses are primarily
restricted to the IgGa and IgGb sub-isotypes and demosntrate the importance of
this phenotype of immune response and its ability to confer protection even in
the absence of a primary mucosal IgA response. The regulation of immunity to
influenza virus infection can also be characterized in terms of the type of T-
helper cell associated with specific immune responses. To date studies in horses
using analysis of mRNA production by PCR analysis, and in pigs using
ELISPOTS, indicate that systemic IFN-g production characteristic of a T-helper 1
response are sequels to both DNA vaccination or influenza virus infection. These
studies and others involving modulation of responses to DNA vaccination by co-
administration of cytokine genes or mucosal adjuvants such as cholera toxin are
helping to build our understanding of regional respiratory immunity to this
important viral pathogen.
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Vaccination Strategies for use in Horses. In: Wernery U, Wade JF, Mumford JA,
Kaaden O-R, eds. Equine infectious diseases VIII. Proceedings of the Eighth
International Conference, Dubai 23rd-26th March, 1998. Newmarket: R & W
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vaccination of horses using the influenza virus hemagglutinin gene. Vaccine
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efficacy of baculovirus-expressed and DNA-based equine influenza virus
hemagglutinin vaccines in mice. Vaccine 1997;15:1149-1156.
17. Macklin MD, McCabe DE, McGregor MW, et al. Immunization of pigs with a
particle-mediated DNA vaccine to influenza A protects agianst challenge with
homologous virus. J.Virol. 1998;72:1491-1496.
18. Larsen DL, Dybdahl-Sissoko N, McGregor MW, et al. Co-administration of
DNA encoding interleukin 6 and hemagglutinin confers protection from influenza
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