HA DNA Vaccine Research Articles

Plasmid-encoded CCL27 co-delivery enhances heterologous protection from respiratory virus challenge

Abstract Many pathogens enter the host at mucosal sites. Peripheral vaccination supports robust antigen-specific cellular and humoral immunity but it requires active infection to pull antigen-specific memory cells to the mucosa and relies on passive transudation of antibodies to mucosal surfaces. We reported that co-delivery of the mucosal homing chemokine CCL27 (CTACK) with a DNA plasmid encoding parental SARS-CoV-2 spike (pS) generates antigen-specific IgA in the lung, antigen-specific CD8+ T cells at mucosal surfaces, and was 100% protective in a heterologous Delta VOC challenge model. Here, we extend this work and demonstrate that co-delivery of plasmid-encoded CTACK (+pCTACK) with pS supports protection from heterologous challenge with SARS-CoV-Omicron variants. pCTACK co-delivery significantly lowered viral loads when mice were challenged with Omicron variants BA.2 and XBB.1.5. In the context of Influenza HA DNA vaccination, co-delivery of pCTACK increased HA-specific antibodies in bronchoalveolar lavage, increased antigen-specific CD8+T cells in the lung mucosa, and altered challenge outcomes in vivo. These data have broad implications for the generation of mucosal immunity with parenteral vaccination and support the use of CCL27 to augment parenteral vaccine-induced immunity at mucosal surfaces and support improved outcomes following respiratory virus infection.

Self-destructing Salmonella via temperature induced gene E of phage PhiX174 improves influenza HA DNA vaccine immune protection against H1N1 infection in mice model

The delivery of DNA vaccines is the principle impediment for implementation of DNA vaccination on a mass scale. In this study, we report a temperature induced conditionally expressed phage PhiX174 gene E mediated lysis of Salmonella under in vivo conditions that can increase the immunogenicity of a DNA vaccine delivered via Salmonella carrier system. We electroporated gene E encoding lysis plasmid pJHL187 along with the pcDNA-HA plasmid encoding H1N1 HA into attenuated Salmonella Typhimurium, strain JOL1893. Using C57BL/6 mice as the model, we showed that the mice intragastrically vaccinated with JOL1893 induced significant production of HA-specific humoral and cell mediated immune responses compared to the JOL1837, which carry pcDNA-HA plasmid alone. Furthermore, mice vaccinated with JOL1893 vaccine were fully protected against the lethal H1N1 challenge compared to the JOL1837 strain, which showed 90% protection only. However, none of the animals survived treated with either the PBS or the Salmonella carrying empty vector. Taken together, our results indicate that mucosal immunization with conditional lysis enabled live attenuated S. Typhimurium as a DNA vaccine carrier can induce efficient systemic and mucosal immune responses, and improves immune protection against a highly pathogenic H1N1 infection in mice model.

Humoral and Cellular Immunogenicity Induced by Avian Influenza A (H7N9) DNA Vaccine in Mice.

BackgroundIn March 2013, human infection with avian influenza A (H7N9) virus emerged in China, causing serious public health concerns and raising the possibility of avian-source pandemic influenza. Thus, the development of an effective vaccine for preventing and rapidly controlling avian influenza A (H7N9) virus is needed. In this study, we evaluated the immunogenicity of a synthetic DNA vaccine against H7 HA antigens in mice.Materials and MethodsThe synthetic consensus H7 HA DNA vaccine (25 or 50 µg) was administered to BALB/c mice at 0, 14, and 28 days by intramuscular injection followed by electroporation. Humoral and cellular immune responses were analyzed in a hemagglutination inhibition test and interferon-gamma enzyme-linked immunospot (ELISpot) assay, respectively.ResultsH7 HA-vaccinated mice showed 100% seroprotection and seroconversion rate against H7N9 reassortant influenza virus after both second and third immunizations. The geometric mean titer by the hemagglutination inhibition test increased with an increasing number of immunizations. However, there was no significant difference in geometric titer between the two groups injected with 25 and 50 µg of H7 HA DNA vaccine after two (79.98 vs. 107.65, P = 0.39) and three (159.96 vs. 215.28, P = 0.18) doses. In addition, the ELISpot assay revealed that administration of H7 HA DNA vaccine induced potent interferon-gamma production from mouse splenocytes.ConclusionsThis study demonstrated the humoral and cellular immunogenicity of synthetic consensus H7 HA DNA vaccine in mice. This work demonstrates the potential of the H7 HA DNA vaccine as an efficient tool for the rapid control of emerging influenza A (H7N9) virus.

Optimal designs of an HA-based DNA vaccine against H7 subtype influenza viruses

The outbreak of a novel H7N9 influenza virus in 2013 has raised serious concerns for the potential of another avian-source pandemic influenza. Effective vaccines against H7N9 virus are important in the prevention and control of any major outbreak. Novel vaccination technologies are useful additions to existing approaches. In the current report, DNA vaccine studies were conducted to identify the optimal design of an H7 HA antigen using the HA gene from a previously reported H7N7 virus that is lethal in humans as the model antigen. New Zealand White rabbits were immunized with DNA vaccines expressing 1 of 3 forms of H7 HA antigen inserts encoding the HA gene from the same H7N7 virus. High-level H7 HA-specific IgG was detected by ELISA, and functional antibodies were confirmed by hemagglutination inhibition assay and pseudotyped virus-based neutralization assay against viruses expressing HA antigens from either the previous H7N7 virus or the novel H7N9 virus. HA antigen design under the tissue plasminogen activator leader (tPA) was the most immunogenic. The data presented in the current report confirm the immunogenicity of the H7 HA antigen and provide useful guidance to prepare for an optimized H7 HA DNA vaccine to help to control the emerging H7N9 virus if and when it is needed.

Cross reactivity of serum antibody responses elicited by DNA vaccines expressing HA antigens from H1N1 subtype influenza vaccines in the past 30 years

In the past three decades, ten H1 subtype influenza vaccines have been recommended for global seasonal flu vaccination. Some of them were used only for one year before being replaced by another H1 flu vaccine while others may be used for up to seven years. While the selection of a new seasonal flu vaccine was based on the escape of a new emerging virus that was not effectively protected by the existing flu formulation, there is limited information on the magnitude and breadth of cross reactivity among H1 subtype virus circulation over a long period. In the current study, HA-expressing DNA vaccines were constructed to express individual HA antigens from H1 subtype vaccines used in the past 30 y. Rabbits naïve to HA antibody responses were immunized with these HA DNA vaccines and the cross reactivity of these sera against HA antigen and related H1 viruses in the same period was studied. Our data indicate that the level of cross reactivity was different for different viral isolates and the key mutations responsible for the cross reactivity may involve only a limited number of residues. Our results provide useful information for the development of improved seasonal vaccines than can achieve broad protection against viruses within the same H1 subtype.

A polyvalent influenza A DNA vaccine induces heterologous immunity and protects pigs against pandemic A(H1N1)pdm09 virus infection

The composition of current influenza protein vaccines has to be reconsidered every season to match the circulating influenza viruses, continuously changing antigenicity. Thus, influenza vaccines inducing a broad cross-reactive immune response would be a great advantage for protection against both seasonal and emerging influenza viruses. We have developed an alternative influenza vaccine based on DNA expressing selected influenza proteins of pandemic and seasonal origin. In the current study, we investigated the protection of a polyvalent influenza DNA vaccine approach in pigs. We immunised pigs intradermally with a combination of influenza DNA vaccine components based on the pandemic 1918 H1N1 (M and NP genes), pandemic 2009 H1N1pdm09 (HA and NA genes) and seasonal 2005 H3N2 genes (HA and NA genes) and investigated the protection against infection with virus both homologous and heterologous to the DNA vaccine components.We found that pigs challenged with a virus homologous to the HA and NA DNA vaccine components were well protected from infection. In addition, heterologous challenge virus was cleared rapidly compared to the unvaccinated control pigs. Immunisation by electroporation induced HI antibodies >40 HAU/ml seven days after second vaccination. Heterologous virus challenge as long as ten weeks after last immunisation was able to trigger a vaccine antibody HI response 26 times higher than in the control pigs. The H3N2 DNA vaccine HA and NA genes also triggered an effective vaccine response with protective antibody titres towards heterologous H3N2 virus.The described influenza DNA vaccine is able to induce broadly protective immune responses even in a larger animal, like the pig, against both heterologous and homologous virus challenges despite relatively low HI titres after vaccination. The ability of this DNA vaccine to limit virus shedding may have an impact on virus spread among pigs which could possibly extend to humans as well, thereby diminishing the risk for epidemics and pandemics to evolve.

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.

Characterization of immune responses induced by immunization with the HA DNA vaccines of two antigenically distinctive H5N1 HPAIV isolates.

The evolution of the H5N1 highly pathogenic avian influenza virus (HPAIV) has resulted in high sequence variations and diverse antigenic properties in circulating viral isolates. We investigated immune responses induced by HA DNA vaccines of two contemporary H5N1 HPAIV isolates, A/bar-headed goose/Qinghai/3/2005 (QH) and A/chicken/Shanxi/2/2006 (SX) respectively, against the homologous as well as the heterologous virus isolate for comparison. Characterization of antibody responses induced by immunization with QH-HA and SX-HA DNA vaccines showed that the two isolates are antigenically distinctive. Interestingly, after immunization with the QH-HA DNA vaccine, subsequent boosting with the SX-HA DNA vaccine significantly augmented antibody responses against the QH isolate but only induced low levels of antibody responses against the SX isolate. Conversely, after immunization with the SX-HA DNA vaccine, subsequent boosting with the QH-HA DNA vaccine significantly augmented antibody responses against the SX isolate but only induced low levels of antibody responses against the QH isolate. In contrast to the antibody responses, cross-reactive T cell responses are readily detected between these two isolates at similar levels. These results indicate the existence of original antigenic sin (OAS) between concurrently circulating H5N1 HPAIV strains, which may need to be taken into consideration in vaccine development against the potential H5N1 HPAIV pandemic.

Broader Neutralizing Antibodies against H5N1 Viruses Using Prime-Boost Immunization of Hyperglycosylated Hemagglutinin DNA and Virus-Like Particles

BackgroundHighly pathogenic avian influenza (HPAI) H5N1 viruses and their transmission capability from birds to humans have raised global concerns about a potential human pandemic. The inherent nature of antigenic changes in influenza viruses has not been sufficiently taken into account in immunogen designs for broadly protective HPAI H5N1 vaccines.MethodsWe designed a hyperglycosylated HA vaccine using N-linked glycan masking on highly variable sequences in the HA1 globular head. Immunization of these hyperglycosylated HA DNA vaccines followed by a flagellin-containing virus-like particle booster in mice was conducted to evaluate neutralizing antibody responses against various clades of HPAI H5N1 viruses.ResultsWe introduced nine N-X-S/T motifs in five HA1 regions: 83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 156NTT, 161NRS, 182NDT, and 252NAT according to sequence alignment analyses from 163 HPAI H5N1 human isolates. Although no significant differences of anti-HA total IgG titers were found with these hyperglycosyalted HA compared to the wild-type control, the 83NNT and 127NSS mutants elicited significantly potent cross-clade neutralizing antibodies against HPAI H5N1 viruses.ConclusionsThis finding may have value in terms of novel immunogen design for developing cross-protective H5N1 vaccines.

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.

Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection

Achieving broad-spectrum immunity against emerging zoonotic viruses such as avian influenza H5N1 and other possible pandemic viruses will require generation of cross-protective immune responses. Strong antibody responses generated against the H5HA protein are protective, however, antigenic variation between diverging isolates can interfere with virus neutralization. The current study investigates co-administration of an H5 HA DNA vaccine with other variable and conserved influenza antigens (NA, NP, and M2). All antigens were derived from the A/Hanoi/30408/2005 (H5N1) virus and the contribution towards overall protection and immune activation was assessed against lethal homologous and heterologous challenges. An (HA+NA) combination afforded the best protection against homologous challenge and (HA+NP) was comparable to HA alone against heterologous A/Hong Kong/483/1997 challenge. Interestingly, combining all four H5 antigens at a single site did not improve protection against matched challenge and unexpectedly reduced survival by 30% against a heterologous challenge. Survival was also significantly decreased against heterologous challenge following combination of (HA+NP) with an unrelated antigen. Although there were no significant changes in antibody titres, significantly lower T-cell responses were detected against all antigens except HA in each combination. Co-administration of the vaccines at different injection sites restored T-cell responses but did not improve overall protection. Similar observations were also recorded following combination of HA and NP antigens using two different adenovirus-based backbones. Overall, the data suggest that co-administering certain H5N1 antigens offer better or comparable protection to HA alone, however, combining extra antigens may be unnecessary and lead to unfavourable immune responses.

Influenza H5 Hemagglutinin DNA Primes the Antibody Response Elicited by the Live Attenuated Influenza A/Vietnam/1203/2004 Vaccine in Ferrets

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness.

GM-CSF Increases Mucosal and Systemic Immunogenicity of an H1N1 Influenza DNA Vaccine Administered into the Epidermis of Non-Human Primates

BackgroundThe recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a world-wide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques.Methodology/Principal FindingsMacaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particle-mediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1–3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine.Conclusions/SignificanceThese results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skin-delivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract.

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Heterologous HA DNA vaccine prime—inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses

The trivalent inactivated vaccine (TIV) is used to prevent seasonal influenza virus infection in humans, however, the immunogenicity of this vaccine may be influenced by the priming effect of previous influenza vaccinations or exposure to antigenically related influenza viruses. The current study examines the immunogenicity of a clinically licensed TIV in rabbits naïve to influenza antigens. Animals were immunized with either the licensed TIV, a bivalent (H1 and H3) HA DNA vaccine or the combination of both. Temporal and peak level serum anti-influenza virus IgG responses were determined by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were measured by hemagglutination inhibition and microneutralization against either A/NewCaledonia//20/99 (H1N1) or A/Panama/2007/99 (H3N2) influenza viruses. Our results demonstrate that the immunogenicity of the TIV is low in sero-negative animals. More significantly, the heterologous DNA prime-TIV boost regimen was more immunogenic than the homologous prime-boost using either TIV or DNA vaccines alone. This finding justifies further investigation of HA DNA vaccines as a priming immunogen for the next generation of vaccines against seasonal or pandemic influenza virus infections.

Vaccination with hemagglutinin or neuraminidase DNA protects BALB/c mice against influenza virus infection in presence of maternal antibody

BackgroundMaternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.ResultsThe results showed that: i) when the offspring were immunized with the same vaccine as their mothers, whether inactivated or DNA vaccine, the presence of maternal antibody inhibited offspring immune response and the offspring could not be protected from a lethal influenza virus infection; ii) when the offspring, born to mothers immunized with inactivated vaccine, were immunized with NA DNA vaccine, the interference of maternal antibody were overcome and the offspring could survive a lethal virus challenge; iii) when the offspring were immunized with different DNA vaccine from that for their mothers, the interference of maternal antibody were also overcome. In addition, high-dose inactivated vaccine in maternal immunization caused partial inhibition in offspring when the offspring were immunized with HA DNA vaccine, while lower dose caused no significant immunosuppression.ConclusionTo avoid the interference of maternal antibody in influenza vaccination of offspring, mothers and their offspring shall not be immunized with the same vaccine. If mothers are immunized with inactivated vaccine, NA DNA vaccine for the offspring shall be effective; and if mothers are immunized with HA (NA) DNA, NA (HA) DNA for the offspring shall be effective.

Distinct Contributions of Vaccine-Induced Immunoglobulin G1 (IgG1) and IgG2a Antibodies to Protective Immunity against Influenza

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.

Influenza HA DNA induces Th1 cells and protection despite limited antibody responses

The relative ability of influenza HA DNA vaccines to induce cellular and humoral immunity after one or more doses and the persistence of this response have not been fully elucidated. HA DNA induces a potent Th1 response. Although this response wanes 12 weeks after a single immunisation, Th1 cells persist in the spleen for at least 6 months after two booster immunisations. In contrast, influenza specific ELISA IgG titres reached significant levels only after booster immunisations and HI antibodies were generally weak or undetectable. Nevertheless, two doses of HA DNA confer almost complete protection against respiratory challenge with live virus.

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.