NIH Public Access Author Manuscript Vaccine. Author manuscript; available in PMC 2015 January 16. NIH-PA Author Manuscript Published in final edited form as: Vaccine. 2014 January 16; 32(4): 507–513. doi:10.1016/j.vaccine.2013.11.022. Improvement of Antibody Responses by HIV Envelope DNA and Protein Co-Immunization Franco Pissani1,2,3,#,¶, Delphine C. Malherbe3,#, Jason T. Schuman4, Harlan Robins5, Byung S. Park3,6, Shelly J. Krebs2,3,¶, Susan W. Barnett7, and Nancy L. Haigwood1,2,3,* 1Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland OR 97217 2The Vaccine and Gene Therapy Institute, Beaverton OR 97006 3Oregon 4GE National Primate Research Center, Beaverton OR 97006 Healthcare, Life Sciences, Piscataway NJ 08854 NIH-PA Author Manuscript 5Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle WA 98109 6Department of Public Health and Preventive Medicine, Oregon Health & Science University, Portland OR 97239 7Novartis Institutes for Biomedical Research, Cambridge, MA 02139 Abstract Background—Developing HIV Envelope (Env) vaccine components that elicit durable and protective antibody responses is an urgent priority, given the results from the RV144 trial. Optimization of both the immunogens and vaccination strategies will be needed to generate potent, durable antibodies. Due to the diversity of HIV, an effective Env-based vaccine will most likely require an extensive coverage of antigenic variants. A vaccine co-delivering Env immunogens as DNA and protein components could provide such coverage. Here, we examine a DNA and protein co-immunization strategy by characterizing the antibody responses and evaluating the relative contribution of each vaccine component. NIH-PA Author Manuscript Method—We co-immunized rabbits with representative subtype A or B HIV gp160 plasmid DNA plus Env gp140 trimeric glycoprotein and compared the responses to those obtained with either glycoprotein alone or glycoprotein in combination with empty vector. Results—DNA and glycoprotein co-immunization was superior to immunization with glycoprotein alone by enhancing antibody kinetics, magnitude, avidity, and neutralizing potency. Importantly, the empty DNA vector did not contribute to these responses. Humoral responses elicited by mismatched DNA and protein components were comparable or higher than the responses produced by the matched vaccines. © 2013 Elsevier Ltd. All rights reserved. * Corresponding author: Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, OR 97006, Phone: (503) 690-5500, Fax: (503) 690-5569, haigwoon@ohsu.edu. #These authors contributed equally to this work. ¶Current address: U.S. Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Pissani et al. Page 2 NIH-PA Author Manuscript Conclusion—Our data show that co-delivering DNA and protein can augment antibodies to Env. The rate and magnitude of immune responses suggest that this approach has the potential to streamline vaccine regimens by inducing higher antibody responses using fewer vaccinations, an advantage for a successful HIV vaccine design. Keywords HIV; Envelope-based vaccine; DNA+protein co-immunization; neutralizing antibodies INTRODUCTION NIH-PA Author Manuscript The recent report of partial efficacy in the phase III RV144 trial underscores the challenge of designing HIV vaccines that can protect from infection. Effective vaccines may require complex regimens to elicit adaptive responses to multiple antigens. In RV144, prime-boost immunizations with recombinant ALVAC and gp120 proteins, including co-administration of these components for the last two immunizations, resulted in reduction of viral acquisition that was associated with antibodies directed to the HIV Envelope protein (Env) [1, 2]. Neutralizing antibodies (NAbs) can block SIV or SHIV infection in macaques [3–6] and appear to contribute to the control of post-infection viremia in HIV infected humans [7]. The strength of interactions occurring between polyclonal antibodies and antigen, termed antibody avidity, has recently emerged as a central feature of antibody-based vaccines [8, 9]. In addition, nonhuman primate (NHP) SIV challenge models have provided additional evidence that T cell-based vaccines can offer substantial viral control [10] but cannot prevent infection, in contrast to vaccines that include Env components [11, 12]. NIH-PA Author Manuscript The vast variability and plasticity of Env are major obstacles to HIV vaccine design, and vaccines designed to elicit NAbs have resulted in antibodies with relatively narrow breadth and potency [13–18]. Prime-boost immunizations can increase the conformation dependence of antibodies [17] with the caveat of prolonged immunization schedules. These results emphasize the need for vaccines that rapidly elicit potent Env-specific antibodies that provide better coverage of antigenic variants. There is mounting evidence that indicates combining Env DNA and protein vaccine components may address this need. Indeed, we recently demonstrated that co-immunization with HIV-1 envelope DNA and trimeric protein accelerates the NAb response [19] and elicits T cell responses [20]. These findings have been extended by other groups who have found similar results of increased humoral responses in mice and macaques [12] as well as increased NAb breadth [21], but the contribution of each component has not been addressed yet. Here, in order to further characterize the env encoded-DNA plus gp140 protein co-immunization strategy, we used model Env immunogens from two different clades and parsed the contribution of the individual DNA and protein components by co-immunizing rabbits with either matched or mismatched subtype A and B immunogens. Our findings demonstrate that regardless of whether the immunogens were matched or mismatched, co-immunizations with DNA and protein enhanced the overall antibody response compared to immunizations with protein alone or empty vector plus protein. Importantly, our results further suggest that combining Envs derived from different sources may, in some cases, enhance antibody binding, avidity, and neutralization potency. MATERIALS AND METHODS Animals Female New Zealand White rabbits (Western Oregon Rabbit Company) were housed at ONPRC; procedures were approved by the OHSU IACUC. Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 3 HIV-1 Env Immunogens and Rabbit Immunizations NIH-PA Author Manuscript Codon-optimized SF162 (subtype B) and motif-optimized [22]Q461e2TAIV (subtype A) gp160 DNA were cloned into pEMC*, and precipitated onto gold bullets to immunize rabbits intradermally by Gene Gun (Bio-Rad) [19, 23]. Recombinant trimeric gp140 proteins (50 μg; fully characterized in [13, 24]) mixed with an equal volume of polyethylenimine adjuvant (PEI, branched; Sigma-Aldrich), were concurrently delivered intramuscularly. Blood was collected every two weeks and sera were heat-inactivated. Antibody Assays Longitudinal binding antibody responses to SF162 and Q461e2TAIV trimeric gp140 were measured by kinetic ELISA [19] with chimpanzee IgG as standard. The avidity index to both antigens was determined as described [8] by endpoint ELISA with minor modifications. Avidity of sera was determined by calculating the midpoint antibody titer after treatment with 8M Urea compared to PBS for each antigen. Surface Plasmon Resonance Assays NIH-PA Author Manuscript Antibody concentrations were determined on a Biacore T200 (GE Healthcare) at 25°C. SF162 and Q461e2TAIV trimers were immobilized at 20μg/mL in 10mM acetate buffer (pH=5.0) to flow cells 2 and 3 on a CM5 chip by amine coupling (8,860RU for SF162and 10,930RU for Q461e2TAIV). 50μg/mL Protein A (Pierce) in 10mM acetate buffer (pH=4.5) was immobilized on flow cell 4 (2,330RU). The reference flow cell was activated and blocked with ethanolamine. Samples were diluted into HBS-EP+ buffer with 0.2mg/mL BSA. An antibody standard containing polyclonal antibodies to both Q461e2TAIV and SF162 was generated by determining the concentration of a high titer sample (injected at 5 and 100μL/min for 36s) using calibration-free concentration analysis (CFCA). The data were fit using 8.526 E11 m2/s as a translational diffusion coefficient for IgGs at 25°C. Experiments were performed at dilutions 1:100 and 1:1600 to determine Env-specific and total antibody concentrations respectively. This standardized sample was then used to create a calibration curve to determine the concentration for all other samples, which were tested at dilutions 1:100 and 1:400. Samples were injected for 3min at 10μL/min. Binding responses (from a report point 10s after the end of injection) were fit to a calibration curve using the T200 evaluation software to determine antigen-specific and total IgG concentrations. Neutralization assay Serum samples were tested for neutralizing activity in a TZM-bl assay [25] with a pre-bleed pool as negative control. Data are reported as ID50, 50% inhibitory dilution values. NIH-PA Author Manuscript Statistical Analyses Repeated Measures ANOVA followed by false discovery rate adjustment was used for longitudinal assays. Area under the curve (AUC) was calculated following the trapezoid rule after baseline subtraction. The Kruskal-Wallis test was used for comparison among multiple groups followed by Bonferroni adjustment. For SPR, a Linear Mixed Model, Repeated Measures ANOVA was followed by Tukey-Kramer adjustment. First order autoregressive covariance structure was used to account for within subject correlation. Different comparison adjustment methods and stringent or flexible adjustments were used depending on the number of comparisons. Analyses were performed with SAS V9.3 (SAS Inc). Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 4 RESULTS Co-immunization strategy of rabbits with gp160 DNA and gp140 protein NIH-PA Author Manuscript Five groups of rabbits (n=4 per group) were immunized four times on weeks 0, 4, 12, and 20 with Env (trimeric gp140) protein either alone or in combination with gp160 env DNA (Table 1). Of the five, three groups were co-immunized with plasmid DNA encoding gp160 and gp140 Env protein: (i) subtype B DNA plus subtype B protein (Matched B; SF162 [26]); (ii) subtype A DNA plus subtype A protein (Matched A; QA461e2TAIV [27]); (iii) subtype B DNA plus subtype A protein (Mismatched). As controls, two groups were immunized with subtype B protein: (iv) empty vector DNA plus subtype B protein (Empty Vector); and (v) subtype B protein alone (Protein B). At each immunization, rabbits received 50μg of gp140 in PEI adjuvant and 36 μg of DNA delivered by Gene Gun. Binding antibody responses are similar in Matched and Mismatched vaccine groups NIH-PA Author Manuscript We evaluated Env-specific binding antibody responses longitudinally by ELISA against trimeric subtype A and B antigens. Strong responses were detected in all groups after two immunizations that were maintained or boosted by subsequent immunizations (Figure 1A). We observed no difference in responses between the Empty Vector and Protein B groups (P>0.38), thus showing no adjuvant effect from the vector alone. A similar absence of adjuvant effect by the vector alone was reported previously in a DNA prime-protein boost study [28]. Overall binding potency was determined by calculating the Area Under the Curve (AUC) (Figure 1B). The Matched A and Mismatched groups developed the strongest response against the subtype A antigen compared to controls (P=0.015 and P=0.05, respectively). As expected, the Matched A group had higher subtype A binding antibodies than the Protein B group (P=0.05). Similarly, the Matched B group developed the most potent subtype Bspecific binding antibody response, significantly stronger than the Matched A group (P=0.004). Subtype A binding responses were indistinguishable between Matched A and Mismatched groups, both of which received subtype A protein. DNA+protein co-immunizations enhance avidity NIH-PA Author Manuscript We measured antibody avidity to autologous antigens two weeks after immunizations by comparing the binding titers after treatment with 8M urea or PBS (Figure 2). The Mismatched and Matched A groups developed the strongest avidity against the autologous subtype A antigen compared to the Empty Vector group (P=0.0260 and P=0.0569, respectively) and the Protein B group (P=0.0160 and P=0.0248, respectively). The Matched B group had a higher avidity towards the autologous B envelope than the Matched A group (P=0.01). Not surprisingly, these data also show that the co-immunization vaccine strategies resulted in stronger avidity for their respective cognate subtypes. Both the Empty Vector and the Protein B groups had a significantly higher avidity to the subtype B antigen than the Matched A group (P=0.0329 for both). Furthermore, the Matched B group also had a significantly higher avidity to the subtype B antigen than the Mismatched group that was immunized with subtype B DNA and subtype A protein (P=0.03). These data suggest that the protein component is the dominant partner for increasing avidity with this combination regimen. Env-specific antibodies are enriched by DNA+protein co-immunizations To further evaluate the relative contribution of each vaccine component on antibody production, we used surface plasmon resonance (SPR) to measure the total amount of subtype A- or B-trimeric gp140-specific antibody responses. Since the binding antibody Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 5 NIH-PA Author Manuscript titers and avidity were indistinguishable between the Empty Vector and the Protein B control groups, we used the Protein B group as control for the SPR analysis. Overall, we found that the antigen-specific responses were nearly identical and significantly higher in the Mismatched and the Matched B groups compared to the protein only group (P=0.0035 and P=0.003, respectively, Figure 3A). Consistent with the binding and avidity results, the vaccines with matched subtype components elicited higher antigen-specific responses by SPR against their cognate antigens (Figure 3B), and the Mismatched strategy resulted in comparable levels of antigen-specific responses against both subtype A and B antigens (P=0.6167). For example, the Matched A group had significantly higher subtype A antigen-specific responses than the Protein B and the Matched B groups (P=0.0035 and P=0.0421, respectively), and the Matched B group elicited significantly higher subtype B antigen-specific responses than the Matched A group (P<0.0001). Interestingly, the Mismatched vaccine elicited significantly stronger subtype A antigen-specific responses than the Matched B group (P=0.0063) and stronger subtype B antigen-specific responses than the Matched A group (unadjusted P=0.0392). Finally, we saw no difference in the responses elicited by the Mismatched vaccine and the Matched A vaccine against the subtype A antigen (P=0.9981). Taken together, our SPR results show that protein components drive strong cognate antigen-specific responses and mismatching could potentially provide an advantage in cross reactivity. NIH-PA Author Manuscript Co-immunizations increase the rate of NAb development and their potency NIH-PA Author Manuscript We measured neutralization activity against the subtype A and B viruses that were the source of immunogens in this study. Rabbits co-immunized with Mismatched DNA+Protein vaccines developed low subtype A NAbs after two immunizations (Figure 4A), and the Mismatched vaccine regimen resulted in higher subtype A NAbs than the Protein B and the Empty Vector strategies (P=0.0375 and P=0.0067, respectively). In contrast, rabbits in all groups developed NAbs against the subtype B virus after two immunizations, and subsequent co-immunizations greatly potentiated subtype B NAbs in the Matched B and Mismatched groups. The greater dynamic range observed here with clade B SF162 may be due to its high sensitivity to neutralization. The Matched B and Mismatched groups had significantly higher subtype B NAbs than the Matched A group (P=0.0007 for both), therefore showing that DNA+Protein vaccines elicited higher NAbs against their cognate antigens. The Matched B and Mismatched groups had significantly higher subtype B NAbs than the Empty Vector group (P=0.0083 and P=0.0405, respectively) and the Matched B group also had stronger subtype B NAbs than the Protein B group (P=0.0295) thereby illustrating the influence of the env DNA component. The Empty Vector and the Protein B regimens resulted in higher subtype B NAbs than the Matched A group (P=0.0295 and P=0.0083 respectively), thus showing that the autologous NAb response is mainly driven by the protein component. We performed AUC analyses to measure the overall potency of NAbs (Figure 4B). Coimmunization vaccine strategies resulted in significantly greater potency of autologous NAbs. The Mismatched group developed the strongest NAbs against the subtype A virus (P=0.034 vs Empty Vector), whereas the Matched B group developed the most potent NAbs against the subtype B virus (P=0.010 vs Matched A). No differences in subtype A or B NAbs were detected between the Mismatched and either of the Matched groups. Effect of DNA+protein co-immunization on neutralization breadth The model immunogens used in this study have not elicited heterologous NAbs with previous vaccine regimens [14, 29, 30]. Considering the improvements in avidity and neutralization potency mediated by the DNA+protein co-immunizations, we tested sera after Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 6 NIH-PA Author Manuscript the final immunization for neutralization of heterologous viruses (Table 2). Tier 1B, subtype B viruses BaL.26 and SS1196.1 were modestly neutralized by sera from all rabbits in Matched B and Mismatched groups. In addition, 75% of rabbits in the Matched B group neutralized the subtype C virus ZM109F.PB4 at low titers. Matched A Rabbit #1 serum had low level neutralization of all viruses tested, but the Protein B and Matched A groups had two non-responders. DISCUSSION NIH-PA Author Manuscript There has been progress in developing HIV and SIV vaccines that can elicit strong T cell responses [10], but the components and delivery systems to invoke strong B cell responses are not fully developed [31]. It is therefore critically important to develop immunization strategies that accelerate the humoral response and enhance avidity. Earlier animal studies have shown that avidity was inversely correlated with peak post-challenge viremia [9]. Previously, we reported that co-immunizations using gp160-DNA and a recombinant HIVEnv scaffold protein induced NAbs in rabbits and Env-specific CTL in mice. We further showed that boosting in the setting of DNA priming with DNA+gp140 accelerated NAb responses in rabbits [19, 20]. Additionally, it was recently shown that DNA+protein immunization of NHPs conferred neutralization breadth and some protection from SIV challenge [12, 21]. Comparing the antibody response elicited by co-immunizations with DNA expressing model gp160 antigens plus trimeric gp140 protein, DNA vector plus protein or protein alone to determine the relative contribution of each vaccine component is a novel aspect of the current study. Moreover, we used for the first time a novel calibrationfree concentration analysis (CFCA) method to assess antigen-specific binding antibody responses in unpurified serum samples. Binding and avidity antibody data showed that the protein component strongly influences the antibody specificity, and the DNA component exerts influence in generating autologous NAbs. Mismatching the DNA and protein components resulted in comparable or higher humoral responses than Matched vaccines. NIH-PA Author Manuscript Numerous immunization studies have used Envelope immunogens to elicit NAbs in various animal models, and, these Envs have induced fairly weak NAbs developing only after multiple immunizations [8, 13, 17, 18, 29, 30, 32–34]. However, DNA vaccines are distinct from conventional vaccines because they stimulate both humoral and cellular responses against antigenic determinants expressed in vivo similar to natural exposure to the pathogen; despite their low immunogenicity, they act as intrinsic adjuvants [35]. Thus, use of DNA plasmids in prime-boost regimens is an attractive approach to increase immunogenicity, although this prolongs immunization schedules. In contrast, our DNA+protein coimmunization strategy accelerated the development of binding and neutralizing antibodies compared to vaccination with protein only. Similar results were obtained with DNA+protein co-immunizations in dengue virus and Japanese Encephalitis Virus (JEV) murine vaccine studies [36, 37]. DNA+protein co-immunizations were also successful at eliciting higher binding antibody and T cell responses against hepatitis C [38]. In addition, our results reveal that co-immunization also accelerated the development of HIV Env-specific antibody avidity, thus showing the advantage of using this approach. The protein component was the driving factor for elicitation of JE-specific NAbs when administered as a vaccine mixture with DNA [39] and as a DNA prime–protein boost vaccine [36]. Our findings also show that the protein component of the vaccine has a stronger influence on antibody specificity with higher binding and neutralizing antibody responses against the envelope cognate to the protein component. However, previous studies also showed that DNA priming improves the magnitude and quality of antibody against primary HIV-1 isolates as well as the immunogenicity of the specific Env, which is not accomplished with protein alone [40]. The ability of the DNA component to focus NAbs on Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 7 NIH-PA Author Manuscript conserved regions [28] and enhance avidity against Env protein vaccines [41] may have mediated this effect. Similarly we demonstrate here that the DNA component also contributes to the antibody response, because co-immunizations enhance antibody binding, antibody avidity, and potency of NAbs, and accelerate the rate of NAb development. The DNA+protein combinations elicited higher antigen-specific responses towards their cognate antigens, as demonstrated by binding and neutralizing antibody data, but the Mismatched group had comparable or at least in one case better responses than the Matched groups towards their cognate antigens. Indeed, the Mismatched vaccine displayed strong binding titers against antigens of both subtypes. It also improved subtype A NAbs, as shown by the Mismatched group having the highest titers of subtype A NAbs, while maintaining strong subtype B NAbs. Because this study is one using model antigens that principally target V3 [13, 24]), we did not explore V2 responses, and we can only speculate if the results that we obtained can be generalized for transmitter/founder Envs or other primary Envs. Nonetheless, these results are corroborated by a previous DNA prime–protein boost vaccine study showing that a polyvalent heterologous protein boost elicit a broader NAb response than a homologous boost [41]. NIH-PA Author Manuscript In conclusion, our findings show that DNA+protein co-immunization accelerates and enhances binding and NAb responses and that the DNA empty vector component does not contribute. Our results also underscore the role of intrinsic Env immunogenicity in inducing NAb breadth, as despite enhancing the overall antibody response, the effect of DNA+protein co-immunizations using model antigens on NAb breadth was less impressive. Uncleaved gp140 trimers have been shown to be less stable and display aberrant conformations compared to the new cleaved BG505 SOSIP.664 gp140 trimer[42], and thus may also contribute to this effect. The current study begins to address one obstacle to eliciting potent, broad NAbs through Env immunizations by shortening the vaccine regimen. We further highlight the importance of considering intrinsic Env immunogenicity in the selection of future immunogens. This co-immunization approach has translational potential for HIV vaccine design when used with newly discovered or engineered Env immunogens. Acknowledgments We thank Leonidas Stamatatos and George Sellhorn for the gp140 trimeric proteins used in this study. We are grateful to Biwei Guo, Shilpi Pandey, Zachary Brower, and Chelsea Smith for technical assistance. We thank Ann Hessell and Julie Overbaugh for their contribution to the manuscript. We also thank William Sutton for helpful discussions. TZM-bl and 293T cell lines were obtained from the NIH AIDS Research and Reference Reagent Program. This work was supported by National Institutes of Health grants P01 AI087064 (H.R. and N.L.H.), P51 OD011092 (N.L.H. and B.P.), and NIH 5 T32 AI7472-17 (F.P.). NIH-PA Author Manuscript References 1. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, et al. Immunecorrelates analysis of an HIV-1 vaccine efficacy trial. The New England journal of medicine. 2012 Apr 5; 366(14):1275–86. [PubMed: 22475592] 2. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. The New England journal of medicine. 2009 Dec 3; 361(23):2209–20. [PubMed: 19843557] 3. Baba TW, Liska V, Hofmann-Lehmann R, Vlasak J, Xu W, Ayehunie S, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. 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Page 11 Highlights for Review NIH-PA Author Manuscript We co-immunized rabbits with clade A or B HIV gp160 plasmid DNA plus Env gp140 trimer DNA+Protein co-immunization was superior to immunization with protein alone Co-immunization enhanced antibody kinetics, magnitude, avidity, neutralizing potency The protein component drove the antibody avidity and neutralizing responses Mismatched vaccines elicited comparable/better humoral responses than matched ones NIH-PA Author Manuscript NIH-PA Author Manuscript Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 12 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1. Autologous Envelope-binding antibody response NIH-PA Author Manuscript (A) Longitudinal analysis of binding antibody titers measured by kinetic ELISA against autologous (vaccine) subtype A (Q461e2TAIV, left) and B (SF162, right) trimeric gp140. Arrows indicate co-immunization timepoints. (B) Area Under the Curve analysis of longitudinal binding curves, expressed as relative units. Each symbol represents an individual rabbit. P values are indicated (Kruskal-Wallis test followed by Bonferroni adjustment). Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 13 NIH-PA Author Manuscript Figure 2. Potency of antibody avidity to autologous Envs NIH-PA Author Manuscript Avidity indices were determined by 8M urea displacement ELISA two weeks after immunization against subtype A (Q461e2TAIV, left) and B (SF162, right) vaccine gp140 Envs. P values were determined by Repeated Measures ANOVA followed by false discovery rate adjustment. For autologous subtype A avidity indices: Mismatched vs Empty Vector, P=0.0260; Matched A vs Empty Vector, P=0.0569; Matched A vs Protein B, P=0.0248 and Mismatched vs Protein B, P=0.0160. For autologous subtype B avidity indices: Matched B vs Matched A, P=0.01; Matched B vs Mismatched, P=0.03; Empty Vector vs Matched A, P=0.0329 and Protein B vs Matched A, P=0.0329. NIH-PA Author Manuscript Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 14 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. Subtype A and B autologous Envelope-specific antibodies Subtype A (Q461e2TAIV) and B (SF162) Envelope-specific antibodies present in rabbit antisera two weeks after immunization were assessed by surface plasmon resonance and reported as percent of total IgG. (A) Total Subtype A and B Envelope-specific IgG responses in each vaccine group. (B) Subtype-specific Envelope IgG response (Subtype A Q461e2TAIV, closed bars; Subtype B SF162, open bars) within each vaccine group. P values are indicated (Linear Mixed Model Repeated Measures ANOVA with Tukey-Kramer adjustment). Vaccine. Author manuscript; available in PMC 2015 January 16. Pissani et al. Page 15 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 4. Neutralization potency against vaccine antigens Rabbit antisera were tested for neutralization of autologous subtype A (Q461e2TAIV, left panels) and B (SF162, right panels) viruses by TZM-bl neutralization assay. (A) 50% neutralization (ID50) of rabbit immune sera displayed longitudinally. Arrows indicate coimmunization timepoints. P values were determined by Repeated Measures ANOVA followed by false discovery rate adjustment. For autologous subtype A NAbs: Mismatched vs Protein B, P=0.0375; Mismatched vs Empty Vector, P=0.0067. For autologous subtype B NAbs: Matched B vs Matched A, P=0.0007; Mismatched vs Matched A, P=0.0007; Matched B vs Empty Vector, P=0.0083; Mismatched vs Empty Vector, P=0.0405; Matched B vs Protein B, P=0.0295; Empty Vector vs Matched A, P=0.0295 and Protein B vs Matched A, P=0.0083. (B) Area Under the Curve analysis of longitudinal neutralization data, expressed as relative units. Each symbol represents an individual rabbit. P values are indicated (Kruskal-Wallis test followed by Bonferroni adjustment). NIH-PA Author Manuscript Vaccine. Author manuscript; available in PMC 2015 January 16. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Table 1 Vaccine. Author manuscript; available in PMC 2015 January 16. Vaccine DNA component Protein component Protein B None Subtype B (SF162) Empty Vector/Protein B pEMC* Subtype B (SF162) Matched B Subtype B (SF162) Subtype B (SF162) Mismatched Subtype B (SF162) Subtype A (Q461e2TAIV) Matched A Subtype A (Q461e2TAIV) Subtype A (Q461e2TAIV) Pissani et al. Co-immunization strategies Five groups of rabbits (n=4) were co-immunized with different combinations of gp160 envelope DNA (36 μg via Gene gun, intradermal) and gp140 trimeric protein (50 μg, intramuscular) in presence of PEI adjuvant. Rabbits were vaccinated at weeks 0, 4, 12 and 20. Page 16 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Table 2 Viral isolates BaL.26 (B) SS1196.1 (B) JRCSE (B) YU-2 (B) QH0692 (B) TRO.11 (B) ZM109F.PB4 (C) Pissani et al. Heterologous neutralization activity of rabbit immune sera 10 Protein B Vaccine. Author manuscript; available in PMC 2015 January 16. 27 15 23 14 27 15 71 57 31 30 30 17 34 37 21 49 73 20 25 21 11 24 41 58 78 19 22 13 Matched B Vaccine groups 18 Mismatched Matched A 62 49 59 42 141 19 Serum ID50 <10 10–20 21–50 51–100 >100 Page 17