Hemagglutinin Antigen Research Articles

Proteotyping to establish the lineage of type A H1N1 and type B human influenza virus

The ability to establish the lineage of type A H1N1 and type B human influenza virus strains using a new proteotyping approach is demonstrated. Lineage-specific signature peptides have been determined for the hemagglutinin antigen of type A H1N1 and type B influenza viruses. The detection of these peptides alone within the high resolution mass spectra of whole antigen digests enables the lineage of the strain to be rapidly and unequivocally assigned. This proteotyping approach complements conventional PCR approaches and should aid in the monitoring of the evolution of the influenza virus in both humans and animals.

Improved haemagglutinin antigen content in H5N1 candidate vaccine viruses with chimeric haemagglutinin molecules

The candidate vaccine virus NIBRG-14 was derived by reverse genetics and comprises the haemagglutinin (HA) and neuraminidase (NA) genes derived from the clade 1 virus A/Viet Nam/1194/2004 on an A/Puerto Rico/8/34 (PR8) backbone. The HA gene was modified to remove the multibasic cleavage site motif associated with high pathogenicity. Reports from manufacturers, confirmed by data generated in this laboratory, have shown that this virus yields a low amount of HA antigen. We have generated a panel of new viruses using reverse genetics in which each virus consists of the PR8 backbone, the NA gene from A/Viet Nam/1194/2004 and a chimeric HA gene with sequences from both PR8 and A/Viet Nam/1194/2004. Here we show that a number of these viruses have improved HA antigen content and yield and are therefore better candidate vaccine viruses for use in production of H5N1 vaccine.

AS03A‐Adjuvanted Influenza A (H1N1) 2009 Vaccine for Adults up to 85 Years of Age

Vaccination of high-risk groups was started shortly after the emergence of the influenza A (H1N1)2009 pandemic virus. Healthy adults were enrolled into 2 age strata: 18-60 years and 160 years, and received monovalent influenza vaccine containing 3.75 microg of A/California/2009 (H1N1) hemagglutinin antigen, adjuvanted with AS03A. Hemagglutination inhibition assay-based antibody titers against H1N1 vaccine were assessed after 1 vaccine dose(primary endpoint), after which subjects were randomized 1:1 to receive no further vaccination or a second dose.Immunogenicity endpoints were European licensure criteria for influenza vaccines. Exploratory analyses assessed the effect of previous seasonal influenza vaccination on responses to the H1N1 vaccine. Licensure criteria for immunogenicity were fulfilled after 1 dose of H1N1 vaccine (N=240). For subjects 18-60 years of age, previous vaccination against seasonal influenza within the preceding 2 seasons resulted in significantly lower geometric mean titers (adjusted for baseline antibody titer) after 1 or 2 doses of H1N1 vaccine (P <.001 and P=.003, respectively). Transient mild or moderate injection-site pain was reported by 87.5%and 65.0% of subjects 18-60 years of age and >60 years of age, respectively, after the first dose, and in 63% of subjects overall after the second dose. A single dose of 3.75 microg hemagglutinin antigen, AS03A-adjuvanted H1N1 2009 vaccine was immunogenic and well tolerated in adults. In exploratory analyses (of subjects 18-60 years of age), postvaccination antibody titers were lower in subjects who had previously received seasonal influenza vaccination, compared with those who had not. This phenomenon warrants further investigation. NCT00968526.

Standardization of the Peste des Petits Ruminants (PPR) Haemagglutinin antigen for Haemagglutination-Inhibition Test

Ezeibe, M.C.O., Eze, J.I., Ijabo, O., Ngene, A.A., Okoroafor, O.N., Chukwu, O.S., Ukomadu, N.M., Sanda, E.M., Eze, I.C. and Ugonabo, J.A.C. 2010. Standardization of the peste des petits ruminants (PPR) haemagglutinin antigen for haemagglutination-inhibition test. J. Appl. Anim. Res., 38: 113–115. Samples of tisssue culture Peste des Petits Ruminants (PPR) monospecific vaccine were incubated with equal volume of aluminium-magnesium silicate. The cultured PPR virus which were haemagglutination negative became haemagglutination positive following the incubation with AMS. The haemagglutination was in each case inhibited by a known PPR antiserum. It has been concluded that incubation with AMS can be adopted to convert cultured PPR virus to standard PPR haemagglutinin antigin for use in haemagglutination inhibition test to confirm diagnosis of PPR.

Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein

BackgroundRecent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA) protein were studied.ResultsSerological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI), and recognition of linear epitopes by peptide scanning (PepScan). Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes.ConclusionsInfection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

Immune response of single dose vaccination against 2009 pandemic influenza A (H1N1) in the Taiwanese elderly

We conducted a multi-center, randomized and laboratory-blinded clinical trial with subgroup analyses, involving adults aged greater than 60 years old (range 61–86 years old), to investigate the immunogenicity and the potential factors affecting the immune response of a monovalent, unadjuvanted, inactivated, split-virus vaccine. A total of 107 subjects were randomized to receive 15 and 30 μg of hemagglutinin antigen in a 1:1 ratio. The immunogenicity was detected through hemagglutination inhibition (HAI) test of serum obtained before and 3 weeks after vaccination. By 3 weeks after vaccination, HAI titer ≧1:40 was observed in 75.5% and 81.1% of participants receiving 15 and 30 μg of hemagglutinin antigen, respectively. Positive seroconversion was observed in 71.7% and 81.1% of recipients of the 15 and the 30 μg, respectively. The GMTs increased by a factor of 10.7 and 17.4 in the groups of 15 and 30 μg, respectively. This study indicated that one dose of 15 μg hemagglutinin antigen without adjuvant induced protective immune response in the majority of elderly. Multivariate logistic regression analyses showed that gender, age and diabetes were statistically significant factors affecting the seroprotection rate ( p = 0.04, 0.01 and 0.01, respectively) and seroconversion rate ( p = 0.01, 0.01 and 0.01, respectively).

Age-related changes in magnitude and diversity of cross-reactive CD4 + T-cell responses to the novel pandemic H1N1 influenza hemagglutinin

Accumulating evidence suggests that T-cell responses play a significant role in controlling influenza disease. Although humoral responses to the major antigenic component hemagglutinin (HA) have been studied in considerable detail, our understanding of the cellular responses against this antigen is limited. Here, we systematically characterized the magnitude and diversity of immunity to pandemic H1N1 HA and its relationship to seasonal H1N1 HA responses in a cohort of healthy nonimmunized adults. We observed considerable diversity in the magnitude of crossreactive pandemic CD4 + T-cell responses among the subjects, with a subset of the individuals demonstrating higher magnitude T-cell responses against the pandemic antigen compared with the seasonal antigen. Importantly, the data suggest that age-related changes in CD4 + T-cell responses preferentially segregate to the antigenically drifting globular region of HA, more so than the conserved region involved in membrane fusion. These results have important ramifications for our understanding of influenza immunity in humans and development of vaccine strategies against this important pathogen.

Immunogenicity of a monovalent pandemic influenza A H1N1 vaccine in health‐care workers of a university hospital in Japan

A phase III observational study evaluating a single-dose of an inactivated, split-virus, unadjuvanted AH1pdm vaccine in HCW was conducted. A safe and effective vaccine was needed after the emergence of AH1pdm in April 2009. We analyzed the immunogenicity and safety of the vaccine. A total of 409 subjects were enrolled and given 15 μg hemagglutinin antigen by s.c. injection. Antibody titers were measured using hemagglutination-inhibition antibody assays before vaccination and 28 days after. The co-primary immunogenicity end-points were the proportion of subjects with antibody titers of 1:40 or more, the proportion of subjects with either seroconversion or a significant increase in antibody titer, and the factor increase in geometric mean titer. We collected 389 pair samples. Antibody titers of 1:40 or more were observed in 148 of 389 subjects (38.0%, 95% CI: 33.2-42.9). The immunogenicity was also confirmed in other end-points, but was not sufficient and was lower than in previous reports. A total of 96 of adverse events was reported: 51 local events and 57 systemic events. There were 12 subjects with both local and systemic events. Nearly all events were mild to moderate except in four subjects. A single 15-μg dose of AH1pdm vaccine did not induce sufficient immunogenicity in HCW, with mild-to-moderate vaccine-associated adverse events. We need to consider further improvement of the AH1pdm vaccine program in HCW for the prevention of nosocomial infection, as well as for the benefit of HCW.

High-dose influenza vaccination in the elderly

On December 23, 2009, the Food and Drug Administration (FDA) approved a new high-dose seasonal influenza vaccine designed for adults 65 years or older. Fluzone High-Dose (sanofi pasteur), approved through the accelerated-review pathway, contains 60 mcg hemagglutinin (HA) antigen from each of the three influenza strains, which is four times the amount of each antigen in traditional injectable seasonal influenza vaccines.

Efficacy, immunogenicity and stability of a novel intranasal Nanoemulsion-adjuvanted influenza vaccine in a murine model

Mutations of influenza virus increase concerns of worldwide epidemics resulting from the newly emergent strains. Current influenza vaccines are inefficient and require annual vaccinations. W805EC adjuvant is an oil-in-water emulsion composed of nanodroplets with an average diameter of approximately 400 nm. The nanoemulsion adjuvant has been used successfully to stimulate the immune response when mixed with several other antigens in animal models. In this study, W805EC nanoemulsion adjuvant activity was evaluated using nasal influenza vaccination in a murine model. Five to twenty percent W805EC adjuvant was used to inactivate influenza A/Puerto Rico/8/34/05 (H1N1). Mice immunized with the nanoemulsion adjuvanted influenza virus intranasally showed a robust specific humoral immune response as demonstrated using ELISA and HAI assays. Serum HAI titers were more than 104 following two vaccinations. Vaccinated mice were also protected against challenge with an LD80 of live influenza virus. Splenocytes from vaccinated mice were assayed for cytokine production following virus stimulation. The cytokine profile demonstrated a robust cellular immune response with enhanced Th1 and Th17 immunity that provided balanced immunity against both intracellular and extracellular forms of the virus. Additionally, the vaccine preparations showed minimal protein degradation but remained potent when stored at 4ºC for up to three months. This work demonstrates that W805EC nanoemulsion adjuvant can effectively enhance the immunogenicity of influenza hemagglutinin antigen. The nanoemulsion adjuvant can result in antigen sparing and cross-protection. The potential exists for a nasally administered influenza vaccine that may require little or no refrigerated storage.

Safety and immunogenicity of an inactivated split-virus influenza A/H1N1 vaccine in healthy children from 6 months to

This study was conducted to determine the immunogenicity and safety of an inactivated split-virus influenza A/H1N1 vaccine in healthy Korean children from 6 months to <18 years of age. The immunization schedule consisted of two vaccinations, 21 days apart. The unadjuvanted vaccine contained 7.5 μg (subjects 6 months to <3 years of age) or 15 μg (subjects 3 to <18 years of age) of hemagglutinin antigen per dose. A total of 251 subjects were enrolled and 248 and 242 subjects, respectively, were included in the post-first dose and post-second dose immunogenicity evaluations conducted on a per protocol basis. By day 21, after the first dose, hemagglutination-inhibition titers of 1:40 or more were observed in 5.9% of subjects 6 months to <3 years of age, 34.9% of subjects 3 to <9 years of age and 81.4% of subjects 9–18 years of age. By day 21 after the second dose, the titer had been achieved 55.9%, 69.5% and 90.5%, respectively. No vaccination-related serious adverse events were observed. A single 15-μg dose of vaccine was highly immunogenic in subjects equal to or more than 9 years of age. However, a two-dose regimen is needed to produce potentially protective antibody titers in younger children.

Evaluation of a Subunit H5 Vaccine and an Inactivated H5N2 Avian Influenza Marker Vaccine in Ducks Challenged with Vietnamese H5N1 Highly Pathogenic Avian Influenza Virus

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

Immunogenicity and Safety of H5N1 A/Vietnam/1194/2004 (Clade 1) AS03-Adjuvanted Prepandemic Candidate Influenza Vaccines in Children Aged 3 to 9 Years

The development of vaccines against pandemic influenza viruses for use in children is a public health priority. In this phase II, randomized, open study, the immunogenicity and reactogenicity of H5N1 A/Vietnam/1194/2004 (NIBRG-14) (clade 1) prepandemic influenza vaccine were assessed in children aged 3 to 5 and 6 to 9 years. Children were randomized to receive 2 doses, given 21 days apart, of A/Vietnam/1194/2004 vaccine containing 1.9 microg or 3.75 microg hemagglutinin antigen (HA), adjuvanted with a tocopherol-based oil-in-water emulsion (AS03) containing 11.86 mg (AS03(A)) or 5.93 mg (AS03(B)) tocopherol. Control groups received 2 doses of trivalent influenza vaccine (TIV). Humoral immune responses, reactogenicity, and safety were the primary outcome measures; cross-reactivity and cell-mediated responses were also assessed (NCT00502593). Between 49 and 51 children in each age stratum (aged 3-5 and 6-9 years) received H5N1 vaccine, and between 17 and 18 children in each age stratum received TIV. After the second dose, recipients of H5N1 vaccine (1.9 microg HA/AS03(B), 3.75 microg HA/AS03(B), and 3.75 microg HA/AS03(A)) achieved humoral antibody titers against the vaccine-homologous strain, which fulfilled the United States influenza vaccines licensure criteria for immunogenicity. With the exception of 1 child, there were no H5N1 immune responses in children who received TIV. The most frequent injection-site event was pain in all groups, and the H5N1 vaccine had a clinically acceptable reactogenicity and safety profile. Exploratory analyses in children aged 3 to 5 years indicated that the induction of CD4 T-cell responses polarized in favor of a T-helper 1 profile. The results showed that 2 doses of AS03-adjuvanted H5N1 influenza vaccine at antigen-sparing doses of 1.9 microg or 3.75 microg HA elicited broad and persistent immune responses with acceptable reactogenicity, and without safety concerns, in children aged 3 to 9 years.

Safety and Cross‐Reactive Immunogenicity of Candidate AS03‐Adjuvanted Prepandemic H5N1 Influenza Vaccines: A Randomized Controlled Phase 1/2 Trial in Adults

The tocopherol-based oil-in-water emulsion adjuvant system family (AS03) improves antigen sparing with split-virion H5N1 influenza vaccines, representing an important development for pandemic preparedness. In this phase 1/2 randomized, controlled, observer-blinded study in 680 adults, we assessed the immunogenicity and safety of A/Indonesia/5/05 H5N1 (IBCDC-RG2, clade 2.1) prepandemic candidate vaccines produced at 2 separate manufacturing sites. Two doses, each of which contained 3.75 microg of hemagglutinin antigen, were given 21 days apart either without adjuvant or with an adjuvant system containing 11.86 mg or 5.93 mg of tocopherol (AS03). The AS03-adjuvanted A/Indonesia/05/2005 (NIBRG-14) vaccines were significantly more immunogenic than nonadjuvanted vaccine in homologous assays. Neutralizing cross-clade immunogenicity against clades 1, 2.2, and 2.3 was demonstrated at day 42 with all vaccines; at 6 months, seroconversion rates were highest for clade 2.2 (60.7%) and for clade 1 (38.3%). Adjuvantation was associated with increased short-term injection-site reactions (pain) in 80% of participants, with such reactions assessed as being of grade 3 severity for 4.0% of doses. No other safety or reactogenicity concerns were identified over 6 months of follow-up. Humoral responses against the adjuvanted 3.75-microg hemagglutinin antigen vaccines from both manufacturing sites fulfilled European and US licensure criteria for immunogenicity for influenza vaccines.

Erythrocyte binding preference of 16 subtypes of low pathogenic avian influenza and 2009 pandemic influenza A (H1N1) viruses

All 16 subtypes of avian influenza viruses of low pathogenicity (LPAIV) as well as their hemagglutinin (H) antigens, and four 2009 pandemic influenza A (H1N1) virus isolates were assayed for hemagglutinating activity against 5 erythrocyte species: goose, guinea pig, human group O, chicken and horse. Of all viruses and antigens assayed, the highest hemagglutination (HA) titers were obtained with goose and guinea pig erythrocytes. Hemagglutinating activity of replicating LPAIV and LPAIV antigens decreased, in order, with chicken and human group O; meanwhile, horse erythrocytes yielded lowest or no HA titer. Moreover, the 2009 pandemic viruses did not agglutinate both horse and chicken erythrocytes. Our study concluded that goose and guinea pig erythrocytes are the best in HA assay for all subtypes of influenza viruses.

A candidate H1N1 pandemic influenza vaccine elicits protective immunity in mice.

BackgroundIn 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model.MethodsWe generated two adenovirus(Ad5)-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA) from the recently emerged swine influenza isolate A/California/04/2009 (H1N1)pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFNγ Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus.Conclusions/SignificanceA single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunization.

The use of calcium phosphate nanoparticles encapsulating Toll-like receptor ligands and the antigen hemagglutinin to induce dendritic cell maturation and T cell activation

Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells. Antigen presentation by mature (activated) DCs is a prerequisite for the stimulation of antigen-specific T cells, whereas antigen presentation by immature DCs results in the generation of specific tolerance. Our aim was to develop calcium phosphate nanoparticles which can serve as carriers of immunoactive oligonucleotides into dendritic cells for their activation. We analyzed size, surface charge, and morphology of calcium phosphate nanoparticles loaded with the TLR ligands CpG and poly(I:C) and also with the antigen hemagglutinin (HA) by scanning electron microscopy, dynamic light scattering, Brownian motion analysis and ultracentrifugation. The uptake of fluorescence-labeled nanoparticles into dendritic cells was illustrated by confocal laser scanning microscopy. Immunostimulatory effects of these nanoparticles on DCs were studied, i.e., cytokine production and activation of the cells in terms of upregulation of surface molecules. We show that functionalized calcium phosphate nanoparticles are capable to induce both innate and adaptive immunity by activation of DCs.

Generation and characterization of a fusion protein of single-chain fragment variable antibody against hemagglutinin antigen of avian influenza virus and truncated protamine

The hemagglutinin antigen (HA) of avian influenza virus (AIV) is an immunogen abundant on the surfaces of infected cells, and can be used as a target for specific antibodies to clear viral infection. Protamine has been demonstrated to deliver DNA into cells effectively. Accordingly, a fusion protein of anti-HA single-chain fragment variable (scFv) and truncated protamine (tP) may be used as a vehicle for delivering the anti-AIV siRNA into the AIV-infected cells for gene therapy. To test this hypothesis, we constructed a novel recombinant plasmid, pET28-scFv-tP, by connecting the genes for anti-H5N1 AIV HA-specific scFv with synthesized oligonucleotides encoding the 22 amino acids of human tP and a linker. Furthermore, the recombinant scFV-tP was expressed and purified, with a yield of 7–8 mg of scFv-tP and a purity of >92% from 1 L of bacterial culture. Characterization of its bioactivity revealed that scFv-tP recognized HA, similar to its scFv control, in a dose-dependent manner and that the scFv-tP, but not its scFv control, bound to DNA and delivered plasmid and oligonucleotide DNA into the AIV-infected MDCK cells effectively. More importantly, transfection with the mixture of the scFv-tP and plasmid for the NP-specific siRNA significantly inhibited the replication of AIV in MDCK cells, as compared with that transfection with the scFv–plasmid mixture, even with the plasmid in liposome. Our data demonstrated that the recombinant scFv-tP retained the functions of both scFv and tP, and might be potentially used for delivering genetic materials for targeting therapy of AIV infection in vivo.

Immunogenicity of Avian H5N1 Influenza Virus Recombinant Vaccines in Cats

Domesticated cats can be infected with highly pathogenic avian influenza H5N1 virus and potentially can be vector or reservoir hosts in an influenza pandemic. We examined the immunogenicity of the hemagglutinin (HA) of H5N1strain VN/1203/04 in cats. ELISAs showed that a single dose of recombinant H5 HA protein can induce a robust antibody response against both whole inactivated virus and recombinant HA antigen. A marked increase in hemagglutination inhibition (HI) titers was also observed in sera from cats given a single dose of the H5 HA protein. However, cats given the commercial H5N1 vaccine required 2 doses before increased H5 HA‐specific antibody titers were observed. Despite boosting, cats receiving the commercial H5N1 vaccine had HI titers lower than those given one injection of recombinant protein. Cats vaccinated with plasmid DNA encoding HA failed to develop HA‐specific antibody responses. Finally, sera from cats that received the protein vaccine neutralized wild type H5N1 influenza virus (VN/120304). The results of this study indicate that recombinant H5 HA protein‐based vaccines may be more effective that either inactivated influenza virus or DNA vaccines in cats.

Immunogenicity of Avian H5N1 Influenza Virus Recombinant Vaccines in Cats

Confirmed reports of large domesticated cats becoming infected with highly pathogenic avian influenza (HPAI) H5N1 virus have raised questions about both the risk of infection for these animals, and their potential as vector or reservoir hosts in an influenza pandemic. With this in mind, we examined the immunogenicity of the hemagglutinin (HA) of H5N1 strain A/Vietnam/1203/04 using several different vaccination strategies. Data from ELISA assays showed that vaccination with a single dose of recombinant H5 HA protein induces a robust antibody response against both whole inactivated virus and recombinant HA antigen. Moreover, a single dose of the recombinant H5 HA protein induced hemagglutination inhibition titers >or=40, which is indicative of protective immunization. Cats receiving the IND H5N1 vaccine required two doses before similar H5 HA-specific antibody titers were observed, and despite boosting, these animals had HIA titers that were lower than or equivalent to those in the group receiving one injection of recombinant protein. In contrast, cats vaccinated with plasmid DNA encoding HA failed to develop HA-specific antibody responses above those seen in cohorts receiving an unrelated control plasmid. The results of this study indicate that recombinant H5 HA protein-based vaccines can rapidly induce high serum antibody titers, and may be more effective than either inactivated influenza virus or DNA vaccines in cats.