As YF immunization increases worldwide and the demand for vaccine becomes higher, an alternative to maintain ongoing vaccine production is to use the new seed lot that can provide vaccines for immunization programs. Hence, new seed lots must be prepared and tested according to the Good Clinical Practice guidelines. These guidelines include a comparative analysis of immunogenicity and reactogenicity with the already licensed vaccine.
Aiming to further contribute to the validation of the YF-17D-213/77 and the YF-17DD substrains for use in immunization programs, in the present investigation we assessed the cellular and humoral immune response triggered by these vaccine substrains. This research was conducted as a complementary parameter under Good Clinical Practice guidelines, and it could aid national immunization programs in Brazil and other parts of Latin America and Africa with respect to clinical validation studies of a new seed lot. Moreover, analysis of these YF vaccine substrains with different passage histories should reveal the extent of immunological alterations associated with the already reported genetic variability between the YF-17D-213/77 seed lot and the YF-17DD vaccine.
First, we characterized the overall cytokine-mediated immune response to YF vaccination using a recently proposed approach for identifying the frequency of high cytokine producers amongst the YF-17D-213/77 and the YF-17DD seroconverters primary vaccinees. Our findings demonstrated that the YF-17D-213/77 and the YF-17DD substrains trigger a balanced overall inflammatory and regulatory cytokine-mediated immune response among the seroconverter primary vaccinated children. These results agree with previous reports that the induction of complex immune response, including activation and modulation events as well as a mixed and balanced pro-inflammatory and regulatory cytokine profile, is relevant to the development of a nondeleterious immune response in YF-17DD primary vaccinees 
. This would be relevant in terms of guaranteeing the establishment of protective events in an eventual contact with the wild YF virus. Insights from the past decade arising from advances in our understanding of innate and adaptive immunity following YF vaccination have provided a clearer idea that an extensive inflammatory response is deleterious to establishing immunity. Hence, it is not surprising that mechanisms exist to shut off inflammation and regulate the overall immune response following vaccination and induction of an inflammatory response 
In this study, we observed that several cytokine sources are elicited by YF vaccination in the innate and adaptive arms of the immune response. Using flow cytometry, we demonstrated that the YF-17D-213/77 and the YF-17DD seroconverter primary vaccinees differ with respect to the “cytokine signatures” of innate and adaptive immunity. In fact, while the YF-17DD vaccine induces an important inflammatory cytokine signature in terms of innate immunity, particularly that mediated by TNF-α+ neutrophils and neutrophil- and monocyte-derived IL-12, which ensures the inflammatory profile in YF-17D PRNT+ primary vaccinees. Meanwhile, the YF-17D-213/77 experimental arm had a prominent inflammatory signature mainly represented by the outstanding adaptive immunity mediated by IL-12+ CD8+ T cells. However, it is important to mention that both substrains are able to trigger counter-balancing regulatory immune events of innate and adaptive immune response, as illustrated by the IL-10 production by CD4+ T cells in YF-17DD PV-PRNT+ subjects and the prominent frequency of IL-10+ neutrophils in YF-17D-213/77 PRNT+ primary vaccinees.
It is well recognized that the YF vaccines in current clinical use display an outstanding capacity for prompting antibody responses, particularly those that provide protection by neutralizing the YF virus 
. The equivalent immunogenicity of YF vaccines from the 17D-213/77 and 17DD substrains has already been demonstrated by placebo-controlled double blind randomized trial 
. Our investigation of whether the neutralizing antibody titer predicts specific cytokine-mediated immune response has shown that the serum levels of anti-YF-neutralizing antibodies are associated with distinct cytokine profiles in the YF-17DD and YF-17D-213/77 arms. However, regardless of the anti-YF PRNT antibody levels, both vaccines are able to elicit particular but relevant inflammatory events required for protection against natural infection.
It is important to bear in mind that both the cellular and humoral arms of the immune system are required for effective protection against YF natural infection 
. Insights from innate immunity research have led to a better appreciation of how existing vaccines work and have also contributed to the rational development of new vaccines 
. In fact, previous studies have demonstrated that the Toll-like receptors (TLR) involved in the NK-cell activation following vaccination with the YF-17DD substrain seems to play a potential role in the development of long-lasting protective memory to the YF virus 
. Progress has been made in defining the contribution of these receptor-ligand interactions to YF-vaccine–mediated protection. Impaired cytokine production was observed when dendritic cells from TLR-deficient mice were stimulated with the 17D-204 vaccine 
Some of the studies that address the complex interactions between the virus and the immune system suggest that the ability of YF-vaccines to rapidly produce neutralizing PRNT antibodies as well as the CD8+
T cell effectors. A cardinal property of memory CD8+
T cells is their ability to undergo rapid proliferation upon reencountering the priming antigen. This is an important component of protective immunity; a higher proliferative potential implies a larger pool of secondary effectors. Akondy et al 
have define the attributes of a human CD8+
T cell response that generates high-quality immune memory by performing a comprehensive analysis of the CD8+
T cells elicited after vaccination with the efficacious yellow fever live virus vaccine. The YF-specific CD8+
T cells displayed broad specificity, high magnitude, multiple functions, robust proliferative potential, and long-term persistence, all characteristics of protective cellular immunity.
To our knowledge, there are no systematic data on the magnitude and relevance of the antigen-specific CD8+
T-cell response necessary for protection against YF infection. However, evidence has shown that both the CD8+
T-cell and the antibody responses can be critical parameters of protective immunity upon YF-17D-204 vaccination. Using the systems biology approach, recent studies have identified relevant aspects of the innate immunity that can be used to predict both neutralizing antibody and CD8+
T-cell responses to YF-17D-204 vaccination 
. In the case of the present investigation, it is noteworthy that the YF-17D-213/77 substrain, a close relative of the YF-17D-204 substrain, prompts a cytokine-mediated immune response with important contribution from CD8+
T cells. On the other hand, the YF-17DD (derived from the YF-17D substrain with independent passage in tissue culture prior to propagation in embryonated chicken eggs) presents a more robust involvement of the innate immune compartment. These particularities in the source of inflammatory cytokines of innate and adaptive immunity can represent events associated with the genetic variability incorporated in the YF-17D-213/77 seed lot and the YF-17DD vaccine during the passage history of the original YF Asibi strain.
The high immunogenicity of YF vaccines has been confirmed by studies in adults and children. The YF vaccine immunogenicity in adults can reach over 95% seroconversion among subjects that were previously seronegative and it probably persists for at least 35 years 
. However, the seroconversion rates range from 77.5% to 90% in children, with distinct seroconversion rates being observed according to the age at vaccination 
. Despite the great public health success of YF vaccination programs, the lack of seroconversion in some vaccinees is a problem, since these subjects are not protected against natural YF infection. Our present evaluation of the cytokine-mediated immune response of a group of nonseroconverters after YF-17D-213/77 or YF-17DD vaccination confirmed that, besides the lack of relevant titers of anti-YF neutralizing antibodies detected by PRNT, the PV-PRNT−
subjects from both experimental arms exhibited relevant gaps in the inflammatory cytokine response of circulating leukocytes upon antigen-specific in vitro
recall. Interestingly, the gaps could be selectively observed in the most relevant inflammatory component induced by each YF substrain; i.e., pro-inflammatory innate immunity in the YF-17DD arm and IL-12 response of CD8+
T cells in the YF-17D-213 nonseroconverters. Most importantly, our findings demonstrate that these deficiencies in the cytokine-mediated immune response can be repaired upon revaccination of the subjects that were seronegative after primary vaccination. This phenomenon has been discussed in more details elsewhere 
In conclusion, despite the particular sources of cytokine-mediated immune response in the innate and adaptive compartments, both vaccines (YF-17DD and YF-17D-213/77) trigged a balanced cytokine signatures, thereby corroborating their already reported attenuated phenotype 
. These findings suggest that both the YF-17DD reference vaccine and the YF-17D-213/77 seed lot trigger an overall balanced cytokine-mediated immune response, which supports their universal use for immunization purposes when it comes to preventing YF outbreaks worldwide. It is important to notify that these hypotheses should be taken with the appropriate prudence considering the qualitative and observational nature of the present investigation.