We demonstrate that a DENV-1-specific CD4+ T cell response with specific kinetics was elicited in Flavivirus-naïve, healthy adults following vaccination with a low dose (10 PFU) of live DEN1Δ30 virus. The observed cytokine response was proinflammatory; IFNγ, TNFα and/or IL-2 production became evident 3 weeks following vaccination. We did not detect significant amounts of IL-10, a regulatory cytokine, at any particular time post-vaccination. We also noted no or low levels of DENV serotype T cell cross-reactivity in a small number of subjects. Lastly, we found that a majority of the cytokines were produced by multifunctional T cells (≥2 cytokines produced simultaneously). As has been observed in other infectious diseases, the presence of multifunctional T cells following DENV-1 vaccination suggests that live, attenuated DENV vaccines safely and effectively prompt immune responses associated with control of infection and protection from re-infection.
In natural primary DENV infections, the precise timing of infection cannot be determined, but is generally 3–14 days prior to disease onset, and disease symptoms last approximately 1 week 
. DENV-specific T cells have been observed 0–5 days prior to defervescence, when viremia levels were dropping or undetectable 
. In our vaccine platform, low level viremia (0.5 log10
pfu/ml serum) occurred 10–16 days after vaccination in 6 subjects, but DEN1Δ30 virus was undetectable in the other 5 subjects, though all seroconverted to DENV-1. Statistically significant CD4+
T cell responses to DEN1Δ30 vaccination were measurable 21 days following vaccination and pro-inflammatory cytokines remained elevated for at least 6 weeks, the latest time-point samples were available. In the 6 subjects with viremia, which occurred about 2 weeks post-vaccination, the T cell responses occurred later than expected and were only measurable 5–11 days after the onset of viremia. Similar kinetics of T cell responses were observed in studies in macaques: T cell responses were evident ~20 days following the peak of viremia (days 3 to ≥11) in macaques inoculated with a high dose tetravalent DENV vaccine, DENVax 
. Given these similarities, we suspect that the increased length of time required to elicit a measurable T cell response is a function of the attenuated DEN1Δ30 virus or inoculum size. The live-attenuated DEN1Δ30 vaccine candidate has decreased replication kinetics, which may slow the spread of the virus, resulting in lower antigenic loads and delayed or muted T cell responses.
The role of proinflammatory cytokines in the immunopathogenesis of DENV is a research area of intense interest. Proinflammatory cytokines such as TNFα, IFNγ, and IL-6 have been associated with severe secondary dengue disease in several studies 
. Far less is known about the role of these cytokines in primary disease, and in particular, mild or asymptomatic disease. In a study that included primary natural DENV infections, higher percentages of T cells (CD4+
) produced IFNγ and/or TNFα in DHF versus DF patients during the acute phase of disease 
. However, a recent study demonstrated that the pro-inflammatory cytokines, TNFα, IFNγ and IL-2, were produced by T cells (CD8+
) at higher levels in individuals who subsequently developed subclinical secondary DENV infections versus symptomatic infections 
. Human challenge studies conducted with under-attenuated DENV vaccines also correlated sustained levels of IFNγ produced by PBMCs with protection from re-challenge with the same viruses 
. These studies indicate that the production of proinflammatory cytokines may be part of a continuum between asymptomatic infection and pathogenesis.
Understanding the characteristics of T cells, including multifunctional T cells, involved in protection from DENV is important for assessing vaccine immunogenicity, safety, and efficacy. In other disease models, extensive work has focused on the role of multifunctional T cells, both CD4+
, and protection from disease or lessening of disease severity in a variety of infections: HIV, tuberculosis, malaria, leishmaniasis, influenza, and vaccinia 
. We observed significantly increased numbers of multifunctional CD4+
T cells by day 21 of infection, and these largely remained present through day 42. While we do not have data past 6 weeks to determine the duration of the multifunctional responses, our initial results parallel other models, and offer the first suggestion that multifunctional T cells may be associated with protective responses.
We observed a trend of more multifunctional T cells in non-viremic vaccinees relative to viremic vaccinees. While these data did not reach statistical significance because of the limited number of available subjects, they suggest that multifunctional CD4+
T cells may be indicators of individuals who are better able to control DENV infection, and therefore may have less severe clinical disease. This is consistent with findings from HIV studies: individuals with lower viral loads have larger percentages of multifunctional T cells, in particular IFNγ+
(triple producers) or IFNγ+
(double producers) CD4+
T cells, while those who have higher viral loads and more severe disease progression produce greater numbers of IFNγ+ CD4+
T cells 
. However, HIV causes a chronic infection while DENV results in an acute infection. This difference might affect the nature of the T cell response. More work is clearly required to discover whether the correlation between levels of viremia for DENV and production of multifunctional T cells is a reason some individuals may develop more severe DENV disease. Of note, no significant differences in neutralizing antibody titers were observed between the viremic and nonviremic subjects or high (Subjects 2, 3, 5, 6, 8, 14) and low (Subjects 4, 7, 10, 12, 18) T cell responders; all groups generated robust antibody responses. Future work should also address whether memory T cell responses maintain specific multifunctional properties or reduce the number of cytokines produced, and whether memory T cell populations are correlated with neutralizing antibody outcome.
The role of IL-10 in DENV disease is still contradictory as it has been correlated with severe dengue disease, despite having multiple regulatory functions that aid in DENV clearance 
. In general, IL-10 is a regulatory cytokine, produced by CD4+
T cells, activated CD8+
T cells, and activated monocytes, that can reduce IFNγ-related antiviral effects 
. We detected little to no IL-10 production at each specific time-point suggesting that it is either not produced at the times sampled following DENV-1 inoculation or our flow assays did not capture the cell population producing it. The role of IL-10, if any, following natural DENV exposure or vaccination therefore requires further investigation.
Our work also contributes to the understanding of memory T cell cross-reactivity following DENV infections, which is thought to contribute to increased disease severity 
. While we observed small increases in the number of cross-reactive CD4+
T cells producing TNFα, IL-2 or IL-10, especially on day 42, the magnitude of the responses were much lower than those observed for homologously stimulated T cells (stimulated with DENV-1 antigen). Similar to the rationale outlined above for delayed T cell responses, the low dose and/or attenuation of the infecting DENV-1 vaccine strain may decrease the antigenic load affecting the overall T cell response, including cross-reactivity. Higher viral loads, as seen in natural exposure, may be required to activate lower affinity T cells such as memory T cells, which cross-react to a different DENV serotype 
. Alternatively, the difference between our data and other reports could be the result of the small subject numbers or differences in study populations. The issue of T cell cross-reactivity is important to clarify as it potentially affects dengue vaccine safety in Flavivirus-experienced populations. Future work will continue to address whether the dose of virus affects the development of cross-reactive T cell responses and whether cross-reactivity varies amongst populations.
T cells play an important role in resolving of DENV infections, CD8+
T cells are active participants in clearing DENV infections 
. We likely did not observe robust CD8+
T cell responses because the inactivated DENV antigens we used are not ideal antigens for CD8+
stimulation from frozen PBMCs which are not able to process full-length antigens efficiently 
. Low CD8+
responses have been observed using this type of antigen by other groups 
. Future work using specific DENV Class I peptides will help define the role of CD8+
in the overall T cell response to monovalent live vaccines.
Longitudinal DENV vaccine specimens provide a unique opportunity to determine T cell profiles in primary, mild DENV infections in a controlled, clinical setting. Further analysis using these and other specimens from DENV monovalent vaccine clinical trials will enable us to determine whether there are differences among serotypes, since each DENV serotype may stimulate a unique cytokine production pattern in T cells. We can also test the effect of antigenic load on the immune response using specimens from different doses of the same vaccines. These are nearly impossible goals to achieve in natural settings as the time of infection and infectious dose cannot be determined, and sometimes even the infecting serotype is difficult to determine due to cross-reactive immune responses. To best determine the role of T cell responses in protection, future work will need to compare specimens from controlled settings to field-setting specimens from individuals with known clinical outcomes. A detailed understanding of a protective versus aberrant immune response following DENV infection will greatly enhance the development and evaluation of an efficacious tetravalent DENV vaccine.