TLRs expressed on antigen-presenting cells play a central role in controlling innate and adaptive immune responses after exposure to infectious pathogens. Hence, TLRs represent potent targets for vaccine development. Here, we used a variety of TLR agonists in the context of a prime-boost vaccine regimen with HIV Gag as a model antigen and found that these immunizations can generate a dramatically different magnitude, quality, and CD4/CD8 balance. The ability to more definitely assess immune responses with multiparameter flow cytometry and to understand how vaccines or adjuvants activate innate immunity will be important for vaccine development and will provide the tools necessary for generating the particular type of T cell response best suited to protect against a given pathogen.
As described previously, heterologous prime-boost immunization with plasmid DNA followed by rAD generates HIV Gag-specific cellular immune responses in NHP (6
). Although DNA vaccines are promising, further improvements in immunogenicity are desirable. In the present study, a single immunization of HIV Gag/protein with a TLR7/8 agonist generated T cell IFN-γ responses comparable to those induced after three immunizations of plasmid DNA encoding HIV Gag protein (6
Importantly, the TLR agonist used in the primary immunization had a strong influence on both the magnitude and quality of the response after the rAD boost. To optimize vaccine delivery, we used Montanide ISA51 as it is easily combined with proteins and TLR adjuvants and has an established safety profile in humans (9
). Notably, 12 wk after a single immunization with Montanide/Gag and either the TLR7/8 agonist or CpG ODN, the magnitude of the Th1 responses was similar to responses in NHP 2 wk after four immunizations given without Montanide (8
). Mechanisms that could account for the increased efficiency and immunogenicity by the Montanide/TLR agonist include increased duration of antigen/adjuvant at the site of immunization or more synchronous delivery to the APC (8
). Thus, this type of vaccine formulation allows for more potent and durable cellular immune responses from a single immunization, making it a potentially cost effective and practical modality.
A potential caveat to this approach is that animals immunized with Montanide/Gag and the TLR7/8 agonist had local reactivity at the site of injection as early as 2 wk after immunization that resolved after 12 wk. This reactivity is not a general feature associated with the TLR7/8 agonist because repeated administration of protein and TLR7/8 agonist without Montanide elicited no local reactivity; the reactivity might be ameliorated by lowering the agonist or protein dose, or by intramuscular administration. Notably, CpG ODN enhanced T cell responses to protein and Montanide with little local reactivity. Indeed, such an approach has been used in humans to enhance CD8+
T cell responses to melanoma peptides (15
The use of TLR agonists selective for activating plasmacytoid DCs (pDCs), conventional DCs (cDCs), or both in vitro provides insight into how such subsets may be influencing T cell immunity in vivo. The findings that CpG ODN elicited potent CD4+
T cell responses suggests that activating pDCs may be sufficient for generating Th1 responses, possibly through induction of IFN-α. Whether IFN-α produced by pDCs would also affect antigen presentation through maturation of other APCs (i.e., cDCs), or whether pDCs could directly present antigen to CD4+
T cells remains an open question. With regard to the role of cDCs, the TLR8 agonist was a poor adjuvant for generating Th1 responses in vivo despite its potency for eliciting IL-12p70 in vitro (8
). It should be noted, however, that NHP immunized with Montanide/Gag and the TLR8 agonist had increased IL-2 responses after the primary immunization and higher CD8+
T cell responses after the rAD-Gag boost than animals immunized with Montanide/Gag alone. Thus, the TLR8 agonist did have functional effects on the T cell response in vivo. Although it remains possible that TLR agonists that activate cDCs only would not be sufficient for eliciting strong Th1 responses in primates in vivo, it is more likely that differences in the stability or optimal dose of the TLR8 agonist when compared with the TLR7/8 agonist could explain its limited adjuvant effect in vivo. Further studies with improved methods to activate and specifically target cDCs will clarify their role for eliciting Th1 responses in vivo. However, as the highest memory T cell responses were detected in NHP immunized with the TLR7/8 agonist, our data suggest that an adjuvant able to activate both pDCs and cDCs will be optimal for enhancing T cell immunity in vivo.
In addition to generating strong Th1 responses, most of the animals immunized with Gag and TLR7/8 agonist in Montanide also had detectable CD8+
T cell responses, providing evidence for cross-presentation. Similarly, animals immunized four times with HIV Gag protein conjugated to the TLR7/8 agonist without Montanide had detectable CD8+
T cell responses (8
). Together, these data suggest that induction of CD8+
T cell responses in NHP with a protein vaccine can be achieved with a TLR agonist capable of activating both pDCs and cDCs.
Another major emphasis of this study was to determine how different TLR agonists would alter the quality of memory T cell responses in a prime-boost regimen. 2 wk after primary immunization, animals that received Montanide/Gag with CpG ODN had a striking increase in the frequency of IFN-γ–producing cells compared with animals immunized with Montanide/Gag and the TLR7/8 agonist. However, these responses quickly diminished and, by 12 wk, were similar in both groups. The rapid decrease in the number of IFN-γ–producing cells in NHP immunized with CpG ODN may be explained by the quality of the CD4+
T cell responses 2 wk after primary immunization. These animals had a high frequency of effector memory cells producing only IFN-γ, which would likely undergo cell death (16
). Alternatively, the dramatic decrease in CD4+
/IFN-γ–producing cells in this vaccine group may be the result of redistribution of such cells to nonlymphoid organs. By 10 wk after primary immunization, the quality of the memory CD4+
T cell responses was nearly identical in animals immunized with CpG ODN or the TLR7/8 agonist. However, the total number of IL-2–producing cells was higher when the TLR7/8 agonist was administered (), and this vaccine group had the highest memory T cell responses after the rAD-Gag boost. Collectively, these data suggest that although CpG ODN elicit better short-term effector cells in the primary phase, they are less efficient for inducing “boostable” memory cells than the TLR7/8 agonist. Despite the extensive analysis used in the present study, we did not identify a phenotypic or functional marker of T cells that can predict this boosting potential. However, it is possible that the increased frequency of IL-2–producing cells accounted for the enhanced magnitude and quality of the responses seen with the TLR7/8 agonist.
It is important to note that NHP immunized without a TLR adjuvant had an IL-2–dominated primary CD4+ T cell response (with or without TNF-α). After rAD-Gag boosting, these animals did not show a sustainable increase in IFN-γ–producing cells. This suggests that the primary immunization “imprints” the CD4+ T cell responses and limits their capacity to further differentiate into CD4+/IFN-γ–producing effector cells. This has direct relevance for designing vaccines against diseases requiring both humoral and cellular responses, such as malaria or HIV. In this regard, if a primary immunization with a protein antigen were given with an oil-based or alum adjuvant without a TLR agonist, there would be efficient induction of antibody responses, but the potential of limiting the cellular response after a viral boost.
After the rAD-Gag boost, there was no increase in the frequency of the total CD4 memory responses in all groups of animals immunized with any of the TLR adjuvants. It is possible that the rAD-Gag boost induced further differentiation of the primary Th1 response, resulting in an increased frequency of IFN-γ–producing cells that readily undergo cell death (16
), thereby offsetting the expansion of existing or the generation of new antigen-specific cells. Because high doses of rAD-Gag efficiently induce IFN-γ–producing cells, it may be possible to enhance the magnitude of the total CD4 memory responses by using lower doses or by administering rAD by a different route.
In contrast with the CD4 response, the magnitude of the CD8 response dramatically increased in all NHP after the rAD-Gag boost. Primary CD8+
T cell responses were only detected from animals immunized with Montanide/Gag and the TLR7/8 agonist; in this group, the increased magnitude after the rAD-Gag boost may reflect expansion of these cells. However, it is likely that the presence of a Gag-specific CD4+
T cell response significantly enhances the CD8 generation, even in animals without detectable primary CD8+
T cells (17
). This would extend to animals immunized with Montanide/Gag and CpG ODN or the TLR8 agonists, which had dramatically higher levels of Gag-specific CD8+
T cells after rAD boosting than animals that were not primed or those that were primed with Montanide/Gag alone.
Finally, the Montanide/Gag plus TLR7/8 agonist priming and rAD-Gag boosting induced a high frequency of polyfunctional cells producing IL-2, IFN-γ, and TNF-α; the high responses were sustained over 1 yr. Hence, these data suggest that activating both pDCs and cDCs with the TLR7/8 agonist elicits polyfunctional Th1 and CD8+
T cells. Generating a high frequency of IL-2–producing Th1 and CD8+
T cells may indeed be desirable for an HIV vaccine. In this regard, individuals infected with HIV have better control of infection if their CD4+
T cells sustain their proliferative and/or IL-2–producing capacity (20
). A recent study extended this finding by demonstrating that long-term nonprogressors had a significantly biased T cell response with more “polyfunctional” CD8+
T cells simultaneously capable of multiple effector functions when compared with progressors (27
). Similarly, in the experimental mouse model of Leishmania major
infection, vaccines that induce a high frequency of CD4+
T cells simultaneously producing IL-2, TNF-α, and IFN-γ confer the best protection (unpublished data). Together, these results emphasize that multi-functional T cell responses will be beneficial for infections in which Th1 or CD8+
T cells are mediating effector functions. In the current study, we could not determine whether the immune responses generated by the TLR agonist–HIV protein vaccines would be sufficient to mediate protection because HIV Gag responses do not protect against SIV challenge. Future studies using SIV rather than HIV Gag protein together with TLR agonists are planned to assess whether this type of vaccine regimen confers protection.
Overall, these data highlight the remarkable heterogeneity of T cell responses elicited by prime-boost immunization regimens. An important question for vaccine development is whether the quality of the immune response is a better predictor of efficacy than the magnitude of the response. To this end, we show that remarkably different types of T cell responses (cytokine profile, CD4/CD8 balance, as well as magnitude) can be generated through the use of different TLR agonists. Our data provide a framework for using protein-based vaccines with TLR agonists in combination with replication-defective viral-based boosting to induce long-lived polyfunctional Th1 and CD8+ T cell responses. The ability to generate a variety of durable T cell responses will be a focus of future vaccine studies for infections such as HIV, Mycobacterium tuberculosis, Plasmodium falciparum, or L. major.