Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus

We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.     

Alphavirus replicon particle vaccines developed for use in humans induce high levels of antibodies to influenza virus hemagglutinin in swine: Proof of concept

A propagation-defective, single-cycle, alphavirus replicon particle (RP) system was used to produce two vaccines against human influenza virus A/Wyoming/03/2003 (H3N2). One vaccine was prepared from Venezeulan equine encephalitis virus (VEEV) strain 3014 and the other from VEEV strain TC-83. Both vaccines induced high antibody titers to the influenza hemagglutinin (HA) protein and illustrated the potential of using alphavirus RP influenza vaccines in swine.     

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1)

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4⁺ T cells and the IFN-γ expression of CD8⁺ T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-γ expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.     

The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines

The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the antiviral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN alpha/beta receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that (1) at least in regard to IFN signaling, the mechanisms which regulate alphavirus-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and (2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors.     

Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice

The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.     

Poly(γ-glutamic acid) nano-particles combined with mucosal influenza virus hemagglutinin vaccine protects against influenza virus infection in mice

Adding poly(γ-glutamic acid) nano-particles (γ-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with γ-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of γ-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and γ-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that γ-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.     

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.     

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 10⁶ pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.     

Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza

The development of safe and effective vaccines for avian influenza viruses is a priority for pandemic preparedness. Adjuvants improve the efficacy of vaccines and may allow antigen sparing during a pandemic. We have previously shown that influenza virus-like particles (VLPs) comprised of HA, NA, and M1 proteins represent a candidate vaccine for avian influenza H9N2 virus [Pushko P, Tumpey TM, Fang Bu, Knell J, Robinson R, Smith G. Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 2005;23(50):5751-9]. In this study, an H9N2 VLP vaccine and recombinant HA (rH9) vaccine were evaluated in three animal models. The H9N2 VLP vaccine protected mice and ferrets from challenge with A/Hong Kong/1073/99 (H9N2) virus. Novasome adjuvant improved immunogenicity and protection. Positive effect of the adjuvant was also detected using the rH9 vaccine. The results have implications for the development of safe and effective vaccines for avian influenza viruses with pandemic potential.     

Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine

Influenza A virus hemagglutinin (HA) and neuraminidase (NA) from A/Nanchang/933/95 were expressed by recombinant baculovirus-infected insect cell lines. HA and NA were chromatographically purified then combined in a single vaccine preparation. Immunization of mice with this preparation resulted in high titers of antibodies to both HA and NA equivalent for each antigen to titers in animals immunized with either antigen alone. Anti-NA antibody titers, measured by either enzyme linked immunoabsorbant assay or neuraminidase inhibition test were higher in the combined recombinant vaccine than in conventional monovalent inactivated vaccine. There was no difference in the anti-HA antibody titers between these two vaccine preparations. Homotypic and closely related heterotypic infections were suppressed and greater reduction in viral replication was observed following a distantly related heterotypic infectious challenge than was observed with conventional inactivated vaccine. The combined HA and NA vaccine takes advantage of the equivalent immunogenicity of dissociated HA and NA, to produce a broader and more balanced immune response to both antigens, without the HA-dominant antigenic competition that occurs with natural infection or immunization with conventional vaccine. Additionally, the recombinant baculovirus expression system offers a reliable rapid production system without the use of massive numbers of embryonated chicken eggs. These studies in a mouse model system suggest that production of a combined HA and NA vaccine from recombinant baculovirus offers an improved alternative to conventional inactivated influenza vaccine.     

Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone ® in rhesus macaques

Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone^® alone or Fluzone^® mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone^® alone, Fluzone^® + null VRP immunized animals had stronger influenza-specific CD4⁺ T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone^® + null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p < 0.0001, 1.3 log) and IgA (p < 0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone^® + null VRP immunization was 1.2 log greater (p < 0.04) than after two immunizations with Fluzone^® alone. After virus challenge, only Fluzone^® + null VRP immunized monkeys had a significantly lower level of viral replication (p < 0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone^® + null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine.     

Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine

A single-cycle, propagation-defective replicon particle (RP) vaccine expressing a swine influenza virus hemagglutinin (HA) gene was constructed and evaluated in several different animal studies. Studies done in both the intended host (pigs) and non-host (mice) species demonstrated that the RP vaccine is not shed or spread by vaccinated animals to comingled cohorts, nor does it revert to virulence following vaccination. In addition, vaccinated pigs develop both specific humoral and IFN-γ immune responses, and young pigs are protected against homologous influenza virus challenge.     

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.     

Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.     

Protection against influenza virus infection in BALB/c mice immunized with a single dose of neuraminidase-expressing DNAs by electroporation

The ability of a single dose of plasmid DNA encoding neuraminidase (NA) or hemagglutinin (HA) from influenza virus A/PR/8/34 (PR8) (H1N1) to protect against homologous virus infection was examined in BALB/c mice. In the present study, mice were immunized once with 30 microg of NA or HA DNA by electroporation. Four weeks or 28 weeks after immunization, mice were challenged with a lethal dose of homologous virus and the ability of NA or HA DNA to protect the mice from influenza was evaluated. We found that a single inoculation of NA DNA could provide protection against influenza virus challenge as well as long-term protection against viral infection. Whereas, the mice immunized with a single dose of HA DNA could not be protected. In addition, neonatal mice immunized with a single dose of 30 microg of NA DNA could be provided with significant protection against viral infection.     

Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant-combined A/Beijing/262/95 (H1N1) virus hemagglutinin or neuraminidase

Antibody (Ab) responses and protection against influenza virus infection in mice immunized intranasally with hemagglutinin (HA) or neuraminidase (NA) purified from the A/Beijing/262/95 (A/Beijing) (H1N1) virus were compared among B10 congenic mouse strains. Mice were immunized intranasally with 0.1, 0.3 or 1microg of HA or NA together with the cholera toxin adjuvant, and then boosted intranasally with 0.3 microg of the adjuvant-combined HA or NA 4 weeks later. Two weeks after the second immunization, the mice were challenged by an infection of the upper respiratory tract with the homologous virus. After 3 days, nasal wash and serum specimens were collected for virus and Ab titration. The HA immunization induced HA-specific IgG Ab responses against A/Beijing HA, which depended on the H-2 haplotype of the strain: The B10.A (H-2(a)), B10.D2 (H-2(d)), B10.BR (H-2(k)) and B10 (H-2(b)) strains were the highest, high, intermediate and low responders, respectively. The nasal IgA responses were induced in the B10.A, B10.D2 and B10.BR strains, but not in the B10 strain. In parallel with Ab responses, the B10.A, B10.BR and B10.D2 strains were conferred significant protection at any dose of primary immunization, but the B10 strain was provided protection only at 1microg of HA. On the other hand, the NA immunization induced NA-specific Ab responses, which depended on the the H-2 haplotype of the strain: the B10.A, B10.D2, B10 and B10.BR strains were the highest, high, intermediate and low responders, respectively. In parallel with Ab responses, all the strains were conferred significant protection at any dose of primary immunization. These results indicate that the MHC-restricted responsiveness of mice to HA is different from that to NA, suggesting that the use of high-HA dose or NA as a component of the nasal influenza A (H1N1 subtype) virus vaccine improves the protective efficacy against influenza among low responder populations.     

Live attenuated influenza vaccine (LAIV) impacts innate and adaptive immune responses

Influenza A infection induces a massive inflammatory response in the lungs that leads to significant illness and increases the susceptibility to secondary bacterial pneumonia. The most efficient way to prevent influenza infection is through vaccination. While inactivated vaccines induce protective levels of serum antibodies to influenza hemaglutinin (HA) and neuraminidase (NA) surface proteins, these are strain specific and offer little protection against heterosubtypic influenza viruses. In contrast, live attenuated influenza vaccines (LAIVs) induce a T cell response in addition to antibody responses against HA and NA surface proteins. Importantly, LAIV vaccination induces a response in a mouse model that protects against illness due to heterosubtypic influenza strains. While it is not completely clear what is the mechanism of action of LAIV heterosubtypic protection in humans, it has been shown that LAIV induces heterosubtypic protection in mice that is dependent upon a Type 1 immune response and requires CD8 T cells. In this study, we show that LAIV-induced immunity leads to significantly reduced viral titers and inflammatory responses in the lungs of mice following heterosubtypic infection. Not only are viral titers reduced in LAIV vaccinated mice, the amounts of inflammatory cytokines and chemokines in lung tissue are significantly lower. Additionally, we show that LAIV vaccination of healthy adults also induces a robust Type 1 memory response including the production of chemokines and cytokines involved in T cell activation and recruitment. Thus, our results indicate that LAIV vaccination functions by inducing immune memory which can act to modulate the immune response to subsequent heterosubtypic challenge by influencing both innate and adaptive responses.     

Influenza neuraminidase antibodies provide partial protection for chickens against high pathogenic avian influenza infection

Protection of chickens against avian influenza (AI) is mostly attributed to production of antibodies against the viral glycoprotein hemagglutinin, whereas less is known about the protective role of antibodies to the other surface glycoprotein neuraminidase (NA). Therefore, vaccines encoding NA antigen (e.g., DNA and alphavirus-based virus like replicon particles (VRP)) or baculovirus-expressed recombinant NA (rN2) were tested for their ability to protect against highly pathogenic AI (HPAI) in chickens. Vaccination with A/Pheasant/Maryland/4457/93 (Ph/MD) rN2 protein produced significantly higher levels of NA-inhibition (NI) activity and 88% protection from HPAI H5N2 challenge than vaccination with Ph/MD N2 DNA (25% protection). Vaccination with Ph/MD N2 VRP a minimum of two times also produced high levels of NI activity and protection against HPAI challenge (63% protection). Vaccination with VRP encoding an N2 gene that was genetically distant from the challenge virus N2 failed to protect chickens. Vaccines producing higher levels of NI activity conferred partial protection, but failed to affect viral shedding. Consideration of the homology between vaccine and challenge virus isolate NA genes may provide improved immunity if high levels of NI activity are obtained.     

Intramuscular immunization with a vesicular stomatitis virus recombinant expressing the influenza hemagglutinin provides post-exposure protection against lethal influenza challenge

Vaccines currently licensed for the prevention of seasonal influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least 2 weeks after immunization for the development of protective immunity. These vaccines do not induce protective responses quickly enough to blunt the effects of infection when administered after exposure. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly at least 24 h after delivery of the influenza challenge virus. To our knowledge this is the first vaccine that effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The induction of HA-specific immune responses by the vaccine is necessary for full protection from challenge, because animals immunized with an empty rVSV vector were not protected equally. Our results are consistent with a model in which vaccination induces an immediate antiviral cytokine response, followed by development of humoral and cellular immune responses which act to reduce pulmonary viral loads and accelerate recovery. Consistent with this model, mice vaccinated with the specific vaccine rVSV HA had high levels of IFN-α in the serum by 24 h after challenge/vaccination, developed serum neutralizing Ab to influenza 2 days prior to control animals, and had detectable anti-HA CD8 T cells present in the peripheral blood 3 days prior to control mice.     

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.