Recent studies have suggested that a heterologous prime-boost vaccination approach in which the same antigen is delivered sequentially by different types of vaccines was more effective in eliciting higher immune responses than the homologous prime-boost using same type of vaccines [14
]. In the current study we have built upon previous work [15
] in evaluating humoral responses generated using HIV-1 gp120 antigen as a model antigen delivered by different prime-boost regimens. A more rigorous comparison of immunizations was conducted with two or five protein vaccinations, five DNA vaccinations, or a combination of DNA and protein vaccinations in a rabbit model. We demonstrated that all regimens studied were capable of eliciting an equivalent binding antibody response. However, sera generated by each of these immunization regimens proved to differ greatly in more important characteristics including specificity, functionality, and avidity. This finding will have significant impact on the future development of vaccines.
Different vaccine delivery approaches have been developed based on the available technology at any given time in history. Efficacy and safety have been the main final parameters driving the development of different vaccine delivery approaches. However, little work has been done to compare the detailed parameters among different vaccine delivery approaches. Unfortunately, immune correlates of protection are not well understood, even for licensed human vaccines. While antibodies are well recognized for playing a major role in protection for many successful vaccines, it is frequently not clear what specific mechanism contributes to such protection. This situation became even more complicated with the discovery of a newer generation of vaccination approaches, such as DNA- or viral vector-based vaccines, and the use of prime-boost strategies employing different types of vaccines; there have been very few studies examining how these newer immunization regimens affect the quality of the final antibody response.
Due to the challenge of developing an HIV vaccine, many novel approaches have been developed and tested with the goal of raising an optimal antibody response to the HIV-1 envelope glycoprotein. The current study utilized some of these novel approaches in order to conduct a detailed analysis on the quality of antibody responses elicited by different prime-boost vaccination strategies.
We hypothesized that the measurement of binding antibodies against a protein antigen by a polyclonal animal serum may not reflect the difference in detailed antibody profiles of such immune sera. It is well-known that the gp120 form of HIV-1 envelope protein-based immunizations typically leads to antibodies targeted to the immunodominant V3 loop. We used this region as the first model antigen determinant to identify a number of differences among different prime-boost immunization regimens. As expected, all immunization regimens used in our study generated antibodies to this domain, all of which were shown capable of outcompeting binding to theV3-directed mAb, 447-52D. However, it became apparent that immunization regimens that included a DNA-based immunization elicited antibodies that were more capable of outcompeting binding to 447-52D. This manifested itself at a functional level and in DNA-immunized animals as it was observed that these animals were more capable of neutralizing the V3 sensitive isolate, NL4-3. Similarly, only sera from animals that received a DNA prime demonstrated neutralizing activities against the CD4-sensitized primary isolate, JR-FL. Through the use of V3 peptide adsorptions, we further demonstrated that this neutralizing activity was mediated by antibodies recognizing the exposed V3 loop after CD4 treatment.
Every animal in our study generated neutralizing antibodies against SF162, an isolate which is extremely sensitive to neutralization by V3 antibodies. These results indicate that even for the V3 antigen, there are different fine specificities, which can mediate different degrees of neutralization breadth. Additional evidence for this has also been recently reported when different V3-specific human monoclonal antibodies with different levels of breadth on neutralizing activities were identified [30
]. Results from our study indicated that the less effective protein-based immunizations generated mainly V3-directed neutralizing antibodies only against the highly sensitive SF162 isolate. Other types of V3-directed neutralizing antibodies, such as those capable of neutralizing NL4-3 or other heterologous primary isolates, may require a DNA priming step. Therefore, the DNA priming step either provides a more relevant antigen conformation or generates antibodies with some biophysical quality that is superior than those generated through the protein alone vaccination approach.
Further investigation into epitopes outside of the V3 loop also yielded interesting results. While little to no antibodies targeted to the CD4-induced (17b-like) or glycan (2G12-like) epitopes were seen through immunization with any regimen, a significant number of antibodies capable of outcompeting the CD4 binding site monoclonal antibodies, F105 and b12, were observed. However, these CD4 binding site-directed antibodies were only seen in animals that had received some form of DNA-based immunization. Also interesting to note is data suggesting that the fine specificities of these antibodies can be shifted with different types of protein vaccine boost. Vaccination regimens that included the JR-FL monovalent gp120 formulation as either DNA alone or in a DNA prime-protein boost format, elicited detectable levels of F105 or b12 competition in almost every instance. However, when the JR-FL DNA prime was followed by a polyvalent gp120 protein boost, only a single rabbit was capable of outcompeting binding to F105 and b12. This may reveal a shift in specificity to CD4 binding sites of subtypes other than clade B, or potentially, but less likely, loss of CD4 binding site-directed antibodies altogether. The fact that antibodies to this domain are being generated at all is potentially important based upon evidence that broadly neutralizing activity in some individuals is mediated by antibody recognition of this domain [33
]. This fact alone makes the use of DNA vaccines an attractive platform for HIV vaccine development.
While using competition assays to dissect antibody specificity revealed interesting trends, the best evaluation for these comparisons is the neutralizing activity elicited by each regimen. Consistent with previous reports [15
], animals that received a heterologous DNA prime-protein boost were better capable of neutralizing relevant primary isolates. Neutralization of primary isolates was almost completely lacking in rabbits that received antigen by only a single vaccine modality. In addition to the issue of the method of immunization, and in concurrence with previous data [18
], a polyvalent formulation of envelope antigens appears to play an important role in eliciting a broader neutralizing antibody response than immunization with only a single envelope antigen. This may be the result of too much focus on a single envelope leading to a more potent, but less broadly neutralizing antibody response.
Additional data demonstrating that immunization with some form of DNA-based vaccine increases serum avidity to the vaccine antigen is also enlightening. It may be possible that the smaller amount of antigen being produced from the initial DNA immunizations results in a higher avidity antibody response. This may prove to be an important facet of a potential vaccine in light of recent data indicating that serum avidity inversely correlates with viral load after challenge [29
]. This result points to the need for future studies to understand how DNA immunization may affect the generation of B cells that produce high avidity antibodies.
In summary, results from this study have confirmed prior work [15
] showing that a heterologous DNA prime-protein boost approach elicits a higher quality antibody response as indicated by the fine specificity, the avidity, and the ability to neutralize heterologous HIV isolates. However, when the individual antibody specificities that are being elicited are examined, very little difference can be detected between animals who receive a heterologous DNA prime-protein boost and those that receive only DNA immunizations. This is in spite of significant differences in the ability to neutralize primary HIV-1 isolates. This indicates that much work remains to understanding changes in the fine specificity of antibodies being elicited between immunization regimens and in the identification of potentially new neutralizing epitopes present on the viral glycoprotein. This finding will have an important impact on the future study of antibody profiles related to the development of highly effective vaccines.