Abstract
Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24gag region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24gag DNA immunogens that express 7 highly Conserved Elements (CE) of 12–24 amino acids in length and differ by only 1 amino acid in each CE (‘toggle site’), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55gag DNA induced poor, CD4+ mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4+ and CD8+ T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55gag, which recognize the virus encoded p24gag protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4+ and CD8+ T cells to additional regions of Gag compared to vaccination with p55gag DNA, achieving maximal cross-clade reactive cellular and humoral responses.
Highlights
The extensive variability of HIV is a major stumbling block in vaccine design, since successful vaccines must protect against widely diverse virus strains
A refined list of 7 Conserved Elements (CE) was selected based on several criteria (Figure 1A, see Materials and Methods): a minimum length of 8 amino acid (AA); inclusion of specific epitopes that have been correlated with viral control in vivo; and exclusion of epitopes associated with high viral loads
We found that the breadth, magnitude and avidity of these cellular responses to some CE were significantly higher among patients able to control HIV-1 infection, which suggests that responses against these conserved regions are clinically relevant [37]
Summary
The extensive variability of HIV is a major stumbling block in vaccine design, since successful vaccines must protect against widely diverse virus strains. Our approach to the development of protective immunogens [35,36,37] derives from a conceptual coalescence of recent findings: Viral proteins recover ancestral amino acid (AA) states when transmitted to a new host [38], essentially recovering a more fit state in the absence of the specific immune responses found in the previous host [39,40,41]; changes in conserved AA of viral proteins can debilitate or destroy virus viability [42,43,44,45]; high avidity CTLs recognizing some conserved viral epitopes are present in controllers and long-term non-progressors (LTNP) [37,46]; CTL responses against specific viral proteins (e.g., Gag) are associated with control of viremia [32,47,48,49,50]; immunodominance obscures or prevents reactivity against other potentially more protective epitopes [51]; and some AA segments in HIV proteins are conserved throughout a given HIV-1 subtype, the entire M group of HIV-1, and even in some instances in HIV-2 and SIV [35,52]. In the present proof-of-concept study, we used DNA vectors expressing two variants of the highly conserved p24CE immunogens to characterize cellular and humoral responses in comparison to full-length p55gag immunogens in vaccinated mice
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