Harriet L. Robinson

Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA

Direct DNA inoculations have been used to demonstrate that in vivo transfections can be used to elicit protective immune responses. The direct inoculation of an H7 haemagglutinin-expressing DNA protected chickens against lethal challenge with an H7N7 influenza virus. Three-week-old chickens were vaccinated by inoculating 100 micrograms of plasma DNA by each of three routes (intravenous, intraperitoneal and subcutaneous). One month later, chickens were boosted with 100 micrograms of DNA by each of the three routes. At 1-2 weeks postboost, chickens were challenged via the nares with 100 lethal doses of an H7N7 virus. Low to undetectable levels of H7-specific antibodies were present postvaccination and boost. High titres of H7-specific antibodies appeared within 1 week of challenge. In a series of four experiments, 50% (28/56) of the DNA-vaccinated and < 2% (1/67) of the control chickens survived the challenge. This exceptionally simple method of immunization holds high promise for the development of subunit vaccines.

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin

Immunization of ferrets with a plasmid DNA expressing influenza virus haemagglutinin (pCMV/H1 DNA) provided complete protection from challenge with the homologous A/PR/8/34 (H1N1) influenza virus. Delivery of DNA-coated gold beads by gene gun to the epidermis was much more efficient than intramuscular delivery of DNA in aqueous solution. The antibody response induced by DNA delivered by gene gun was more cross-reactive than DNA delivered in aqueous solution or after natural infection. This novel approach to vaccination against influenza may afford broader protection against antigenic drift than that provided by natural infection.

DNA vaccines: A novel approach to immunization

Direct DNA inoculations are being developed as a method of subunit vaccination. Plasmid DNAs encoding influenza virus hemagglutinin glycoproteins have been tested for the ability to provide protection against lethal influenza challenges. In immunization trials using inoculations of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens were protected against the lethal challenge. Good protection was achieved by intramuscular, intravenous and intradermal injections. In mice, 95% protection was achieved by gene gun delivery of 250-2500 times less DNA than the saline inoculations. Successful DNA vaccination by multiple routes of inoculation and the high efficiency of gene-gun delivery highlight the potential of this promising new approach to immunization.

Protective immunity induced by rotavirus DNA vaccines.

It is estimated that Group A rotavirus diarrhea causes as many as one million deaths per year in children worldwide, and effective vaccines will be essential for their control. Plasmid DNA vaccines encoding murine rotaviral proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and provide protection against rotavirus challenge. The vaccines were administered by inoculation into cells of the epidermis with an Accell gene gun. (Auragen, Inc., Middleton, WI, USA). Each vaccine elicited rotavirus-specific serum antibodies as measured by ELISA. Virus neutralizing antibodies were detected in mice receiving plasmid DNAs encoding for outer capsid proteins VP4 and VP7, but not for VP6, an inner capsid protein, and all of the vaccines generated virus-specific CTL responses. Each vaccine was effective in protecting mice against infection after homotypic rotavirus (100 ID50) challenge, showing reductions (P < 0.0002) in viral excretion measured over a 9 day period. Increased rotavirus-specific intestinal IgA antibodies were seen in vaccinated mice after rotavirus challenge, particularly in mice that received the VP6 DNA vaccine. This suggests that intracellular IgA-mediated neutralization may be involved in protective immunity induced by the VP6 DNA vaccine, and may represent a new mechanism for protection by DNA vaccines.

Early studies on DNA-based immunizations for measles virus.

DNA-mediated immunizations have been used to raise neutralizing antibodies for measles virus. Single inoculations of plasmids expressing measles hemagglutinin or fusion glycoproteins raised neutralizing antibody in BALB/c mice. Plasmids expressing the hemagglutinin glycoprotein (both normal and secreted) raised neutralizing responses that persisted for 1 year. For both forms of hemagglutinin, the effectiveness of the raised antibody (ratio of neutralizing activity to ELISA activity) was similar. High titers of neutralizing antibody were also raised by inoculation of rabbits with the hemagglutinin and fusion glycoprotein-expressing plasmids.

Influence of avian leukosis viral sequences on transmission to the egg and embryo.

Rous associated virus type-0 (RAV-0), a subgroup E replication-competent endogenous virus of chickens, is associated with a low efficiency of virus shedding into the egg albumen and failure to establish congenital transmission. In contrast, RAV-1, a subgroup A virus of exogenous origin, is efficiently shed into the albumen and readily infects the embryo. Among a series of in vitro constructed recombinants between RAV-0 and RAV-1, we have identified subgroup E recombinants that efficiently shed virus into the egg albumen but do not undergo efficient congenital transmission. The LTR region, subgroup-determining sequences in env, and sequences within a 375 bp Sacl-Xhol fragment at the 5 end of the genome each influenced the efficiency of virus shedding into the albumen. Egg inoculations with viruses differing only in env were used to confirm the low rate of congenital transmission of subgroup E viruses. These studies revealed that subgroup A envelope antigens are at least 100-fold more effective for the establishment of embryonic infection than subgroup E.