Balancing Trained Immunity with Persistent Immune Activation and the Risk of Simian Immunodeficiency Virus Infection in Infant Macaques Vaccinated with Attenuated Mycobacterium tuberculosis or Mycobacterium bovis BCG Vaccine

ABSTRACT Our goal is to develop a pediatric combination vaccine to protect the vulnerable infant population against human immunodeficiency virus type 1 (HIV-1) and tuberculosis (TB) infections. The vaccine consists of an auxotroph Mycobacterium tuberculosis strain that coexpresses HIV antigens. Utilizing an infant rhesus macaque model, we have previously shown that this attenuated M. tuberculosis (AMtb)-simian immunodeficiency virus (SIV) vaccine is immunogenic, and although the vaccine did not prevent oral SIV infection, a subset of vaccinated animals was able to partially control virus replication. However, unexpectedly, vaccinated infants required fewer SIV exposures to become infected compared to naive controls. Considering that the current TB vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), can induce potent innate immune responses and confer pathogen-unspecific trained immunity, we hypothesized that an imbalance between enhanced myeloid cell function and immune activation might have influenced the outcome of oral SIV challenge in AMtb-SIV-vaccinated infants. To address this question, we used archived samples from unchallenged animals from our previous AMtb-SIV vaccine studies and vaccinated additional infant macaques with BCG or AMtb only. Our results show that vaccinated infants, regardless of vaccine strain or regimen, had enhanced myeloid cell responses. However, CD4+ T cells were concurrently activated, and the persistence of these activated target cells in oral and/or gastrointestinal tissues may have facilitated oral SIV infection. Immune activation was more pronounced in BCG-vaccinated infant macaques than in AMtb-vaccinated infant macaques, indicating a role for vaccine attenuation. These findings underline the importance of understanding the interplay of vaccine-induced immunity and immune activation and its effect on HIV acquisition risk and outcome in infants.

KEYWORDS pediatric HIV/SIV, vaccine, immune activation, myeloid cells H uman immunodeficiency virus (HIV) and tuberculosis (TB) infections remain highly prevalent in many parts of the world and often show substantial geographical overlap. Infants represent one of the populations most vulnerable to both diseases. An HIV vaccine is not yet available, and although a vaccine to prevent TB infection, the live attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, is routinely administered to Ͼ80% of neonates globally and is effective at preventing the most severe complications of TB, BCG efficacy wanes with age (1)(2)(3). In HIV-infected infants and infants at risk for HIV, the BCG vaccine is contraindicated due to the potential of inducing disseminated BCG disease (4). Thus, both a new TB vaccine and a pediatric HIV vaccine are urgently needed to reduce infant mortality and morbidity in areas of high HIV and TB prevalence. Toward this goal, we aimed to develop a pediatric combination HIV-TB vaccine that could protect infants against HIV and TB infections. Our vaccine was intentionally designed to retain the immunogenicity of BCG but was more attenuated to exhibit an improved safety profile, while expressing HIV antigens to induce both HIV-specific and TB-specific immune responses. Further, we opted to utilize an attenuated human-adapted Mycobacterium tuberculosis for improved immune memory, rather than further edit the M. bovis backbone of BCG.
Our pediatric TB-HIV combination vaccine is based on an auxotroph mutant of Mycobacterium tuberculosis that has deletions in the panCD, leuCD, and secA2 loci that render this attenuated M. tuberculosis (AMtb) strain mc 2 6208 replication incompetent (5)(6)(7). In fact, because these bacteria are unable to synthesize essential nutrients (panthothenate and leucine), replication in unsupplemented conditions, such as a mammalian host, can occur only temporarily until the cell reserves are depleted. In contrast, BCG does not harbor auxotroph mutations and can replicate freely until thwarted by the host immune system. Using experimental simian immunodeficiency virus (SIV) infection of infant rhesus macaques, an animal model of pediatric HIV infection, we have previously demonstrated that this AMtb vaccine, in contrast to BCG, does not cause mycobacterial dissemination or TB tissue pathology (8). Further modification of the mc 2 6208 vaccine strain to coexpress SIVgag (mc 2 6435) or SIVenv (mc 2 6439) rendered this vaccine capable of inducing both TB-and SIV-specific T cell and antibody responses in infant macaques (9,10). Although these vaccine-induced SIV-specific immune responses were not sufficient to prevent oral SIV acquisition, a subset of vaccinated infant macaques with higher-avidity plasma SIV Env-specific IgG antibodies and SIV Env-specific mucosal IgA antibodies showed better control of SIV replication upon infection (9). At the same time, unexpectedly, the group of vaccinated infant macaques had a twofold-higher SIV infection risk per exposure compared to unvaccinated infants (9). Despite not being statistically significant, the difference in the per exposure risk of SIV infection was reminiscent of increased CD4 ϩ T cell activation, and thereby a potentially increased risk of HIV infection, seen in South African infants after BCG vaccination (11,12).
In human neonates, a single immunization with BCG at birth has been shown to be highly immunogenic despite the infant's reduced ability to mount potent T helper 1 (Th1) responses. Further, the introduction of the BCG vaccine has been associated with a significant drop in overall childhood mortality. In addition to protection against TB, inherent adjuvant properties of BCG can improve immunological responses to other neonatal vaccines (13,14). Only recently have we begun to understand the mechanism(s) by which BCG can exert these beneficial effects. Studies in human adults have shown that BCG vaccination induces epigenetic modifications in monocytes that can persist for several months, resulting in improved functional capacity and higher responses to both M. tuberculosis and unrelated antigens (15). This phenomenon, which has been characterized as an aspect of "trained immunity," was not restricted to monocytes and macrophages that are the target cells for mycobacteria but was also extended to T cells (16). Therefore, it has been proposed that novel TB vaccine candidates should aim to preserve these unique features of BCG. In fact, among the new TB vaccine candidates under development, several are based on additional attenuations of M. bovis within the existing BCG vaccine or on attenuated M. tuberculosis strains similar to the auxotroph AMtb vaccines used here (17)(18)(19)(20)(21). Recombinant BCG strains expressing HIV antigens are also in development as potential HIV vaccines and have been tested in mice (22)(23)(24) and in adult (25)(26)(27)(28)(29) and neonatal (30) macaques. Considering the high risk of HIV and TB infection in infants, especially in sub-Saharan Africa, we deemed it important to understand how novel TB vaccines or combination HIV-TB vaccines might impact HIV acquisition.
In a first attempt to better define the beneficial effects of mycobacterial vaccines and to weigh them against potentially negative outcomes on infant HIV infection risk, we report here studies conducted on archived tissue samples from animals immunized with the AMtb-SIV prime/modified vaccinia Ankara (MVA)-SIV boost regimen that were not challenged with SIV (10) or samples from animals included in our recent efficacy studies that were collected prior to or at the time of the first SIV challenge (9). To exclude any effects of the vaccine booster component or the SIV inserts on oral SIV acquisition, we expanded upon those historical studies by vaccinating infant macaques with AMtb vaccines only (AMtb-SIV prime plus a homologous boost) or animals primed only with AMtb-SIV, the AMtb strain without SIV cassettes, or the human infant regimen of BCG. Specifically, the current study examined the potential of our AMtb vaccine to increase functional responses of infant monocytes/macrophages and whether enhanced myeloid cell function was associated with immune activation that could potentially predispose infants to SIV infection.

RESULTS
Increased functional responses in myeloid cell population of AMtb-vaccinated infant macaques. In human adults, the persistence of heightened monocyte function has been described as a hallmark of BCG-induced trained immunity (15,31,32). To determine whether our AMtb vaccine mc 2 6435 (expressing SIVgag) could also enhance monocyte responses and whether it could do so in the pediatric setting, we utilized archived peripheral blood mononuclear cell (PBMC) and tissue samples from SIVuninfected infant macaques that were vaccinated with strain mc 2 6435 at birth, boosted with MVA-SIV at 3 and 6 weeks of age, and monitored for 16 to 18 weeks to determine vaccine-induced SIV-and TB-specific immune responses in blood and tissues (10). As mycobacteria infect not only monocytes/macrophages but also dendritic cells (DC), we tested both monocytes/macrophages and myeloid DC (mDC) for their ability to produce cytokines in response to Toll-like receptor (TLR) ligation. Although mycobacteria are not known to signal through Toll-like receptor 7 (TLR7) or TLR8, we chose the TLR7/8 agonist R848 because monocytes/macrophages and myeloid dendritic cells from infants show reduced function compared to those from adults, and R848 has been demonstrated to potently activate infant cell populations (33,34). At 16 to 18 weeks after vaccination, in vitro R848 stimulation of PBMC resulted in higher frequencies of interleukin 12 (IL-12)-producing monocytes/macrophages (Fig. 1A) and mDC (Fig. 1B) in vaccinated animals than in naive control animals, but these differences were not statistically significant. Peripheral blood monocytes/macrophages of AMtb-vaccinated infant macaques did, however, express increased levels of the activation markers CCR5 (chemokine [C-C motif] receptor 5) and CD69 compared to naive controls (Fig. 1C). In contrast to PBMC, tissue analyses revealed clear differences in the functional capacity of myeloid cell populations from AMtb-vaccinated and unvaccinated infant macaques. It is particularly noteworthy that greater frequencies of IL-12-or tumor necrosis factor alpha (TNF-␣)-producing monocytes/macrophages (Fig. 1D) and mDC (Fig. 1E) were observed in the tonsils and submandibular lymph nodes (LN) of AMtb-vaccinated animals compared to naive controls. These results indicated that a single oral administration of an AMtb vaccine during the first week of life could enhance the function of infant monocytes/macrophages and mDC and that these responses persisted for at least 16 to 18 weeks in blood and tissues of infant macaques. As oral and intestinal tissues are presumed sites of entry and early SIV replication after oral exposure and our goal is to develop a pediatric HIV-TB combination vaccine, we deemed it important to test whether the AMtb vaccination had also resulted in CD4 ϩ T cell activation.
Prolonged CD4 ؉ T cell activation after AMtb vaccination of infant macaques. Indeed, 16 to 18 weeks after vaccination with AMtb, the frequencies of CD4 ϩ CCR5 ϩ T cells were significantly higher in PBMC, retropharyngeal LN, and colons of AMtbvaccinated animals than in naive controls (Fig. 2). In addition, frequencies of Ki-67 ϩ , PD-1 ϩ , and CD69 ϩ CD4 ϩ T cells were generally higher in retropharyngeal LN and colon cell suspensions of cells from AMtb-vaccinated infants than in those from naive controls Frequencies of IL-12-or TNF-␣-producing monocytes/macrophages and mDC, respectively, in PBMC from naive controls and AMtb-vaccinated infant macaques at 16 to 18 weeks after vaccination. (C) Frequencies of CCR5 ϩ or CD69 ϩ peripheral blood monocytes/macrophages from the same animals. (D and E) Frequencies of TNF-␣-or IL-12-producing monocytes/macrophages (D) and mDC (E) from the axillary (Ax.), mesenteric (Mes.), and submandibular (Subm.) lymph nodes (LN) and tonsils of vaccinated infant animals after in vitro stimulation with R848. Each symbol represents the value for an individual animal. The short horizontal bar in all graphs represents the median value for the group. Statistically significant differences between the groups are listed as P values and were determined by exact Mann-Whitney test. (Fig. 2). Although only PBMC, retropharyngeal LN, and colons were available for this retroactive analysis, these data further confirmed that the AMtb-induced immune activation persisted for several weeks to months after vaccination and provided evidence that SIV target cell frequencies at presumed sites of viral entry and/or early replication were concurrently increased. These findings of persistent AMtb vaccineinduced immune activation in myeloid cell and CD4 ϩ T cell populations in blood and tissues reiterated the important question of whether vaccine-induced immune activation could enhance the risk for HIV/SIV infection, as suggested by the results of the STEP HIV vaccine trial (35)(36)(37).
Study design to test the association between AMtb or BCG vaccines and oral SIV challenge outcome. We recently reported that an AMtb-SIV prime/MVA-SIV boost regimen did not protect infant macaques against oral SIV infection; furthermore, vaccinated animals appeared to require fewer oral SIV exposures to become infected compared to naive controls (9). The current study was designed to include additional control groups to more conclusively determine whether mycobacterial vaccines would predispose infant macaques to SIV infection. Vaccine-induced adaptive immune responses were previously reported (9, 10) and were not assessed in these cohorts. Here, we tested whether mycobacterial vaccines could induce innate immune responses and immune activation. As outlined in Fig. 3, control animals consisted of nine infant macaques that received placebo (phosphate-buffered saline [PBS]) immunization (group C2) and also included the six historical control animals (group C1) from our previous study (9). Similar to the vaccinees in the former study (group D), infant macaques in group E were immunized at birth with mc 2 6435 and mc 2 6439, but instead of receiving heterologous MVA-SIV boosts, the animals received the same mc 2 6435 and mc 2 6439 vaccines (homologous boost) at week 3 by the intradermal (i.d.) route, the current route of BCG vaccination. To exclude other confounding factors, such as SIV antigen expression by AMtb vaccines, group F infant macaques were vaccinated with a single dose of mc 2 6208, the parental vaccine strain of mc 2 6435/mc 2 6439 that does not contain SIV expression cassettes. A group of single-dose BCG-vaccinated infants was also included (group G) as a control for vaccine-induced enhanced myeloid cell function and to determine whether any of the effects described here for experimental AMtb vaccines may have translational potential for novel TB vaccine in human infants.
Weekly repeated low-dose oral SIV mac251 challenges were started at week 9 (Fig. 3). On the basis of our observations of persistent immune activation in blood and tissues at 16 to 18 weeks after AMtb vaccination of SIV-unexposed infant macaques, we asked whether we could detect activated myeloid cell populations or increased numbers of CCR5-positive CD4 ϩ T cells (preferred targets for SIV infection) at the time of challenge (week 9).
Increased function of peripheral blood monocytes/macrophages and mDC of infant macaques vaccinated with AMtb or BCG vaccine at the start of oral SIV challenges. To address this question, we stimulated PBMC that were collected at the time of the start of repeated low-dose oral SIV exposures (week 9) with R848. To increase our sample size, we included a group of infant macaques that received a single immunization with mc 2 6435 at birth (group H [ Fig. 3]); this group was not included in the SIV challenge analysis, because these infants were challenged with a high dose of SIV mac251 . Indeed, the frequencies of IL-12-producing monocytes were significantly higher in PBMC from AMtb-or BCG-vaccinated infant macaques than from naive control animals, whereas TNF-␣ responses were increased only in AMtb-vaccinated infants (Fig.  4A). Infant macaques vaccinated with AMtb or BCG also had significantly higher frequencies of IL-12-or TNF-␣-producing mDC than naive controls (Fig. 4A).
We next tested whether these enhanced functional responses of monocytes/macrophages and mDC were associated with systemic immune activation. Week 9 plasma samples were analyzed for a panel of cytokines and chemokines that included several proinflammatory markers. However, none of the cytokines typically associated with inflammation and immune activation, e.g., TNF-␣, IL-6, IL-1, and IL-8, were elevated in vaccinated infant macaques. In fact, out of 37 cytokines, chemokines, and growth factors tested, only macrophage chemoattractant 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1␤) were significantly increased in the plasma of vaccinated FIG 3 SIV challenge study outline. The figure shows the times of immunization (indicated by black arrows), the time of oral SIV exposures (red arrows), and lists the vaccine regimens for each of the groups. The number of animals per group is indicated, and each group is assigned a colored symbol. The route of vaccine administration is given in parentheses as follows: PO, per os; IM, intramuscular; ID, intradermal. *, animal groups have been described in reference 10; **, animal groups have been described in reference 9. animals ( Fig. 4B). Both of these cytokines represent markers of monocyte activation. Therefore, we performed further testing for soluble CD14 (sCD14) and sCD163 that are produced by activated monocytes. To account for the large variation in absolute levels between animals and to focus our analysis on vaccination-related changes, we used the ratio of plasma sCD14 and sCD163 levels at week 9 normalized to those at week 0 for Frequencies of IL-12-and TNF-␣-producing monocytes/macrophages and mDC in peripheral blood from naive controls, AMtb-vaccinated, and BCG-vaccinated infant macaques at week 9, the time when oral SIV challenges were initiated. (B) Plasma levels of MCP-1 and MIP-1␤ were measured when the infants were 9 weeks old. The plasma levels of sCD14 and sCD163 are reported as a ratio of the level at week 9 (start of SIV challenge) divided by the level at week 0 (time of immunization). Note that due to the small blood sample volume available from infants, plasma samples were not available for all infants at all time points. Each symbol represents the value for an individual animal with different vaccine groups being identified by their respective colored symbol (Ctrls, controls). Statistically significant differences between the groups are listed as P values and were determined by exact Mann-Whitney test.
Trained Immunity and Immune Activation of TB Vaccines Clinical and Vaccine Immunology each animal. Animals vaccinated with AMtb or BCG had a significant increase in sCD14 plasma levels, with the increase in sCD14 being significantly higher in BCG-vaccinated compared to AMtb-vaccinated infant animals (Fig. 4B). Vaccination was not associated with increased sCD163 plasma levels ( Fig. 4B). Consistent with our conclusion that the mycobacterial vaccine component(s), and not MVA boosting or SIV antigen expression, was driving myeloid cell activation, we observed increased functional responses and elevated plasma cytokine levels when we compared each of the AMtb vaccine groups separately to the naive control animals at week 9 (see Fig. S1 in the supplemental material).
To determine whether the elevated plasma cytokine levels were at least partially due to myeloid cell activation, we tested whether changes in plasma cytokine levels were correlated with functional measurements of myeloid cells at week 9. Indeed, the frequencies of IL-12-producing peripheral blood monocytes/macrophages and mDC were positively correlated with plasma MCP-1 levels (Fig. 5A), but not MIP-1␤ level (data not shown). This correlation was maintained when we analyzed the relationship between IL-12-positive (IL-12 ϩ ) monocytes/macrophages of BCG-vaccinated animals and naive control animals to plasma MCP-1 (Fig. 5B), but it was not statistically significant when we tested naive controls and AMtb-vaccinated infants (P ϭ 0.0940) (data not shown). We also observed positive correlations between plasma sCD14 and sCD163 week 9/week 0 ratios and week 9 IL-12 ϩ monocyte/macrophage frequencies in BCG-vaccinated infants, but not in AMtb-vaccinated infants (Fig. 5B).
Vaccine-induced CD4 ؉ T cell activation at the start of oral SIV challenges. Analysis of longitudinal blood samples showed that all AMtb-and BCG-vaccinated infant animals experienced an increase in CD4 ϩ CCR5 ϩ T cells after vaccination. However, consistent with the changing ratio of naive to antigen-experienced CD4 ϩ T cells as part of normal immune development, the frequencies of CCR5 ϩ CD4 ϩ T cells increased in all groups between week 0 and week 9 (Fig. 6A). Thus, it was important to distinguish between changes in peripheral blood immune cell populations that reflect normal  Clinical and Vaccine Immunology postnatal immune development (38) and vaccine-induced immune activation. Therefore, we report all changes in activated CD4 ϩ T cell frequencies as ratios of week 9 values compared to week 0 values for each animal. This analysis revealed that BCGvaccinated infants had significantly higher frequencies of CD4 ϩ CCR5 ϩ T cells than naive controls at 9 weeks of age ( Fig. 6A). Although CD4 ϩ CCR5 ϩ T cells were not increased in AMtb-vaccinated infants, vaccine-induced CD4 ϩ T cell activation was suggested by significantly higher frequencies of CD69 ϩ and PD-1 ϩ CD4 ϩ T cells compared to the naive controls (Fig. 6B). BCG-vaccinated infants showed a significant rise in Ki67 ϩ , CD69 ϩ , and in PD-1 ϩ CD4 ϩ T cells (Fig. 6B). Immune activation of CD4 ϩ T cells was further substantiated by higher levels of endogenous cytokines in ex vivo unstimulated CD4 ϩ T cells from vaccinated animals compared to unvaccinated infant macaques (Fig. 6C). Compared to naive controls, CD4 ϩ T cells from both AMtb-or BCG-vaccinated infants had significantly increased levels of IL-2, gamma interferon (IFN-␥), and TNF-␣ at week 9, while IL-17A levels were increased only in the BCG-vaccinated infants (Fig. 6C). It should be pointed out that this analysis was performed on ex vivo isolated cells without any stimulation and that the measured frequencies of cytokine-producing CD4 ϩ T cells exceeded those of antigenspecific (both SIV and TB) CD4 ϩ T cells (9,10), and thus are consistent with immune activation as opposed to memory responses. Furthermore, an analogous analysis of PBMC samples collected from infant macaques vaccinated with a DNA-SIV prime/MVA-SIV boost regimen did not reveal similar CD4 ϩ T cell activation (Fig. 6D). These results were confirmed by the statistical significance of separate comparisons of individual AMtb vaccine groups to naive control animals (Fig. S2), indicative of mycobacterial vaccine-driven CD4 ϩ T cell activation.
These combined data show that early vaccination with highly replication-attenuated auxotroph M. tuberculosis vaccine strains or with attenuated, but replication-competent BCG, induced activation in myeloid cells and in CD4 ϩ T cells that was clearly evident at 9 weeks after vaccination, the time of the first oral SIV challenge. While we focused our discussion of mycobacterial-vaccine-induced immune activation on myeloid cells and CD4 ϩ T cells as the cell types most relevant to SIV acquisition, vaccine-induced immune activation was also observed in CD8 ϩ T cells and in B cells (Fig. S3), underlining the global nature of systemic immune perturbation, even months after mycobacterial vaccination. Functional differences of CD8 ϩ T cells and B cells between naive controls and AMtb-or BCG-vaccinated infants could not be further explored within the scope of the current study.
Challenge outcome in AMtb-or BCG-vaccinated infant macaques using a repeated low-dose oral SIV exposure model. Similar to our recent study (9), in which unvaccinated infants had an SIV infection risk per exposure of 0.24, the SIV infection risk per exposure for naive controls was 0.25 in the current study. Thus, 4 of 9 naive controls (44%) became infected after 1 SIV exposure, with 1 animal each requiring 3, 4, 5, or 6 exposures, and 1 animal failing to become persistently infected after 10 oral SIV exposures (not persistently infected [NPI]) (Fig. 7A). In contrast, AMtb-vaccinated infants in groups E and F tended to become infected after fewer SIV exposures, with 8 of 11 animals (73%) becoming infected after only one or two exposures (Fig. 7B) and 1 group F animal requiring four exposures. As one infant each in groups E and F remained uninfected after 10 SIV exposures, the SIV infection risk per exposure (0.34) in infant macaques vaccinated with AMtb vaccines was lower than in our previous study using the AMtb-SIV prime/MVA-SIV boost regimen (exposure risk of 0.5) (9). Similar to AMtb, five of seven BCGvaccinated infants (71%) became infected after a single oral SIV exposure, while one animal required two exposures and one animal remained uninfected for an estimated probability of infection per oral SIV exposure of 0.35 (Fig. 7C).
Given the overall similarity in challenge outcome between the previous (9) and current studies, we combined the animals of both studies to assess the risk of SIV infection. These results translated into a per exposure risk of SIV infection of 0.25 for naive controls (groups A and C) and 0.34 for infant animals receiving any kind of AMtb vaccine (groups D, E, and F). As one unvaccinated infant (group C), two AMtbvaccinated infants (groups D to F) and one BCG-vaccinated infant (group G) had to be categorized as NPI, there was only a 1.4-fold increased risk of SIV infection per exposure in vaccinated animals compared to naive control animals. Comparing the AMtbvaccinated animals (n ϭ 19) or the BCG-vaccinated animals (n ϭ 7) to the control animals (n ϭ 15), we did not detect statistically significant differences in the number of SIV exposures until infection (P ϭ 0.6 and P ϭ 0.8, respectively; Fig. 7D). Yet, descriptively, after only two low-dose oral SIV exposures, 79% of AMtb-vaccinated and 88% of BCG-vaccinated infants were infected compared to 47% of naive controls. Furthermore, BCG-vaccinated animals (group G) exhibited significantly higher acute viremia (weeks 1 to 3 postinfection) than AMtb-vaccinated animals or naive controls (Fig. 7E), and they tended to have higher viremia during the period after the acute phase of infection (weeks 5 to 7 postinfection; Fig. 7F).
To exclude genetic factors associated with increased susceptibility or resistance to SIV infection or with disease outcome, we determined the tripartite motif-containing protein 5 (TRIM5␣) and major histocompatibility complex (MHC) class I genotypes of SIV-challenged animals. The TRIM5␣ genotypes TFP/TFP and TFP/Cyp confer resistance to SIV infection, while the genotypes Q/Q and Q/CypA are associated with increased susceptibility to infection (39,40). The MHC alleles Mamu A*01, B*08, and B*17 alleles have been associated with improved control of viral replication and better disease outcome, whereas allele B*01 is associated with higher viremia (41)(42)(43)(44)(45)(46). Genotypes could not be evaluated prior to enrollment because animals were assigned to the different groups within 3 days of birth. Animals that required more challenges or were resistant to SIV infection showed no bias toward protective TRIM5␣ alleles or MHC class I alleles, as calculated in the susceptibility score (Table 1). Conversely, animals infected after fewer SIV exposures were not more likely to express alleles associated with greater susceptibility to infection or with reduced control of viremia. Although none of the animals in groups D and E expressed the protective Mamu A*01 allele, "MHC protection" refers to the ability to control virus replication better in infected animals; there are no reports, to our knowledge, that conclusively show that the Mamu A*01 genotype protects against SIV infection. Similarly, TRIM5␣ alleles have not been associated with resistance to infection with the pathogenic strains SIV mac239 or SIV mac251 (47). Therefore, Genotypes associated with increased susceptibility or disease outcome received a score of Ϫ1. All other alleles were assigned a score of 0. The susceptibility score represents the sum of all scores for an individual animal (e.g., animal 42376 has MHC 1 ϩ 0 ϩ 1 ϩ 0 and TRIM5␣ of 0 for a score of ϩ2). e In addition, group C includes the six naive control animals (group C1) described by Jensen et al. (9). f NPI, not persistently infected.  (9). i Note that group H is not included in this table, because animals received a high-dose SIV challenge and were used for week 9 immune activation measurements, but not for SIV risk calculations. Animals in groups A and B were not exposed to SIV.
we concluded that the genotype of each animal was not a principal cause of enhanced SIV mac251 acquisition in vaccinated animals.
Collectively, although statistically not significant, the SIV challenge results suggested that infant macaques vaccinated with AMtb vaccines or BCG showed a trend toward enhanced SIV acquisition compared to vaccine-naive controls.
Persistence of vaccine-induced immune activation post-SIV challenge. Although BCG-vaccinated animals had higher viremia after infection, CD4 ϩ T cell loss did not differ significantly between these animals, naive controls, and AMtb-vaccinated infant animals (data not shown). Histological analysis also revealed no differences in the integrity of the mucosal epithelium between the naive controls and AMtb-or BCGvaccinated groups (data not shown). It is also important to note that mycobacteria could not be recovered from the lung or tracheobronchial lymph nodes of any of the animals at the time of euthanasia. Despite similar clinical outcome, differences in activation of myeloid cells and CD4 ϩ T cells between naive controls and AMtb-or BCG-vaccinated infants were still apparent post-SIV infection. BCG-vaccinated animals had higher frequencies of CCR5 ϩ monocytes/macrophages in the submandibular lymph nodes and in bronchoalveolar lavage (BAL) fluid than naive control infants did (Fig. 8A). HIV and SIV infections have been associated with increases in CD14 ϩ CD16 ϩ cells (48,49), and while the frequency of activated CD14 ϩ CD16 ϩ peripheral blood monocytes in naive controls increased from a median of 4.2% at week 9 to 24.5% after SIV infection (Fig. 8B), even higher frequencies of this cell population were observed in blood and tissues of AMtb-and BCG-vaccinated infants. At the time of euthanasia, BCG-vaccinated animals also had higher frequencies of activated CD4 ϩ T cells in lymphoid tissues than naive controls did (Fig. 9), but differences were not consistently observed in mucosal tissues. In contrast, tissue CD4 ϩ T cell activation generally did not Trained Immunity and Immune Activation of TB Vaccines Clinical and Vaccine Immunology differ significantly between AMtb-vaccinated infants and naive controls, except for higher frequencies of CCR5 ϩ and Ki-67 ϩ CD4 ϩ T cells in the ilea of AMtb-vaccinated infants (Fig. 9). Similarly, the activation of CD8 ϩ T cells and B cells was more pronounced in BCG-vaccinated animals ( Fig. S4 and S5). Differential immune activation of human myeloid cells by BCG and strain mc 2 6435 in vitro. Our data suggested that immune activation of myeloid cell populations might be more pronounced after BCG than AMtb vaccination, and thus, we wanted to confirm this effect in human myeloid cells. This direct comparison was also important because the AMtb and BCG vaccines were administered at different doses and routes in the infant macaque studies, making a quantitative comparison of immune responses between these two groups difficult. However, the main goal of our studies was to determine whether enhanced myeloid cell function was induced by both vaccine regimens, and thus likely mediated by mycobacteria. To assess for common signaling properties, we first used the HEK-Blue human TLR reporter cell assay to evaluate the TLR2, TLR4, or TLR8 signaling by M. tuberculosis-SIV vaccine strain mc 2 6435, BCG, and Mycobacterium smegmatis mc 2 155. The highest TLR2 signal was observed after stimulation with M. smegmatis, which is thought to be the least immunogenic of the mycobacteria tested (Fig. 10). However, in contrast to the other strains, the replication of M. smegmatis is not attenuated, and the higher replication rate may have resulted in stronger signaling. None of the mycobacterial strains induced signaling via TLR4 or TLR8 in a 24-h time period.
We then incubated human PBMC with strain mc 2 6435 or BCG and measured the induction of activation markers and cytokines. Heat-killed mycobacteria (heat-killed M. tuberculosis [HK-Mtb]) and R848 were included as controls; all responses were corrected for baseline values in unstimulated cultures. Peripheral blood monocytes/macrophages, but not mDC, stimulated with BCG showed higher induction of the costimulatory molecules CD40, CD80, and CD86 than mc 2 6435-stimulated cells did (Fig. 11). Consistent with stronger myeloid cell activation, frequencies of IL-12-and TNF-␣-producing monocytes/macrophages and mDC were also higher after BCG stimulation than after mc 2 6435 stimulation (Fig. 12A), and these results were confirmed by increased proin- Trained Immunity and Immune Activation of TB Vaccines Clinical and Vaccine Immunology flammatory cytokine secretion into culture supernatants, reiterating the possible impact of vaccine attenuation on cellular activation (Fig. 12B). These in vitro results with adult PBMC confirm that mycobacterial exposure also induces myeloid cell activation in human cells and recapitulates the differences in the degree of activation observed in infant macaques between BCG and our auxotroph vaccine strain mc 2 6435. Further, these experiments were normalized for bacterial dose, suggesting that the in vivo studies using different dose and route immunization regimens did not artificially bias immune activation readouts.

DISCUSSION
Vaccines to prevent HIV-1 and TB infections are especially needed for vulnerable infant populations in resource-poor countries where limited access to therapies results Trained Immunity and Immune Activation of TB Vaccines Clinical and Vaccine Immunology in high morbidity and mortality rates. Several new TB vaccine candidates are currently being pursued, many of which take advantage of the potent adjuvant activity of mycobacteria and the nonspecific activation of innate and adaptive immune cells, termed "trained immunity," which can provide bystander protection against other pathogens (32,50). These advantages of mycobacterial vaccinations are especially attractive features for pediatric vaccines that need to stimulate the immature infant immune system (51). Trained immunity was originally described in BCG-vaccinated adults and refers to the persistence of enhanced monocyte function to mycobacterial antigens and heterologous antigens for several months (15). Recently, it was demonstrated that NK cells and T cells of BCG-vaccinated adults can also mediate heterologous immunity (15,16,52). While enhanced monocyte function in BCG-vaccinated human adults was associated with NOD2-dependent epigenetic changes (15), the mechanisms mediating trained immunity in NK cells and T cells of BCG-vaccinated adults have not been defined. Multiple mechanisms contribute to heterologous immunity. In fact, the phenomenon of vaccine-induced heterologous immunity has been known for many years and is not limited to BCG, but has also been reported for measles vaccine (31,53). So far, only live attenuated vaccines have been shown to confer trained immunity (54), suggesting that persistent antigen expression might be essential for its induction. Among the current TB vaccine candidates, the MTBVAC (a mutant of M. tuberculosis) and the recombinant BCG vaccine VPM1002 represent live attenuated vaccines with the potential to confer "trained immunity," and both vaccines have already been shown to exhibit improved safety compared to BCG, while maintaining immunogenicity (55,56).
Our AMtb vaccine strain mc 2 6435 (ΔpanCD ΔleuCD ΔsecA2) is similar in concept to MTBVAC and VPM1002, but it undergoes only limited replication in the mammalian host (8,57). However, we cannot exclude the possibility that certain bacterial byproducts persisted in vivo and stimulated innate immune responses. The data of the current study clearly demonstrated that our AMtb vaccines enhanced the functional responses of monocytes/macrophages and mDC and that these heightened responses persisted for at least 16 to 18 weeks after a single AMtb immunization at birth. These results are consistent with a recent study showing that African infants in Guinea-Bissau had greater responses to various TLR agonists at 4 weeks after BCG vaccination (58). Consistent with findings in BCG-vaccinated adults (16), we also observed vaccineinduced activation of CD4 ϩ and CD8 ϩ T cells and of B cells. Our results in infant macaques, which mirrored results from the human pediatric study (58), imply that the phenomenon of mycobacterial-vaccine-induced trained immunity in adults (16) might also be applicable to infants. However, we have not specifically tested for the induction of heterologous immunity and limited cell numbers did not allow us to directly test whether our AMtb vaccines induced epigenetic modifications in myeloid cell populations similar to those observed in BCG-vaccinated adults (15). Given our narrow mechanistic understanding of trained immunity in myeloid and other cell populations, additional research is needed to enhance our understanding of the complex interactions between vaccine components, innate immune cell activation, and adaptive immune responses. Such knowledge will be essential to design more-effective and safe vaccine strategies, both for infant and adult populations.
The potent effects of BCG, and similarly AMtb, vaccines likely explain the improved immune responses to pediatric vaccines administered to previously BCG-immunized infants (13,14). Not surprisingly though, the increased functional capacity of myeloid cell populations was associated with persistent immune activation. In comparison to control animals, vaccinated infant macaques had increased levels of MCP-1 in plasma that positively correlated with in vitro TLR responses, suggesting that systemic immune activation was driven by myeloid cells, which are target cells for mycobacteria present in live attenuated TB vaccines.
Despite the obvious benefits of improved myeloid function in the context of the maturing infant immune system, persistent immune activation can afford greater susceptibility to HIV infection. Thus, in areas of high HIV prevalence, TB vaccine-induced immune activation could potentially increase the risk of HIV infection. In fact, it has been reported that BCG vaccination in South African newborns induced an increase in CCR5 ϩ and in HLA-DR/CD38 ϩ -expressing CD4 ϩ T cells (11,12), raising the question of whether BCG-induced CD4 ϩ T cell activation might increase the risk of HIV-1 acquisition in the pediatric population. Further, immune activation in the intestine or other sites of virus entry has been associated with increased HIV or SIV acquisition in humans and in nonhuman primates (59)(60)(61)(62)(63)(64). This conclusion is supported by our findings that infant macaques vaccinated with AMtb or BCG vaccines had increased CD4 ϩ CCR5 ϩ T cells in blood and in tissues that serve as presumed entry sites for SIV after oral exposure. Indeed, as in our recent SIV challenge study (9), AMtb-or BCG-vaccinated infants in the current study on average required fewer oral SIV exposures to become systemically infected than unvaccinated animals did, and they had a 1.4-fold higher risk of SIV infection per exposure than naive controls of similar age. The differences in per challenge probability of infection did not reach statistical significance between naive controls and AMtb-or BCG-vaccinated infant macaques. To conclusively demonstrate a 1.4-fold increased risk of SIV infection would require 100 animals per group to achieve 80% power assuming a 25% per challenge probability of SIV infection in naive controls (http://www.scharp.org/tools/RLDwebCalc/RLD.php), a prohibitive study design for multiple reasons. Further, each experimental group included one animal that remained uninfected after 10 exposures, which is not unusual in studies with outbred rhesus macaques, especially protocols like the present one employing low-dose SIV challenge regimens (65,66). Thus, despite the lack of a formally demonstrated statistically significant difference in acquisition rates between vaccinees and controls, the facts that (i) both AMtb-and BCG vaccine-primed infants needed fewer SIV exposures to become infected, (ii) this challenge outcome was observed regardless of vaccine boost, administration route, or known genetic susceptibility factors for SIV, and (iii) compelling statistically significant differences in viremia and in immune activation, the trend toward enhanced SIV acquisition in vaccinated infant macaques may have high biological significance for human infants who receive the BCG vaccine at birth and are at risk for HIV acquisition from breast-feeding.
The importance of vaccine-induced immune activation in relation to HIV infection risk was brought to the forefront by the unexpected results of the STEP HIV vaccine trial (35). Since then, there have been numerous investigations of the mechanisms responsible for increasing HIV susceptibility in STEP vaccine recipients. Some studies have suggested that vaccination with the adenovirus 5 (Ad5) vector activated Ad5-specific memory T cells, increasing the number of target cells for HIV (63,(67)(68)(69). However, others have not been able to confirm these results (70,71). Two recent nonhuman primate studies also suggested that the outcome of SIV challenge is associated with immune activation at the time of challenge. One study showed that PBMC IFN-␥ enzyme-linked immunosorbent spot assay (ELISPOT) responses inversely correlated with the number of SIV exposures needed to establish infection in adult macaques (72). Another study comparing various vaccine modalities showed that regardless of the vaccine strategy, the number of activated CD4 ϩ T cells in the rectal mucosa predicted early viremia and risk for rectal SIV infection (73). Both studies established a link between immune activation at the time of challenge and SIV exposure risk, although a statistically significant difference in challenge outcome between the groups was not demonstrated (72,73). Similarly, in a recent low-dose rectal SIV infection study, animals with higher numbers of Ki67-positive CD4 ϩ T cells in the colorectal mucosa required fewer SIV exposures to become infected (74). These results are particularly reminiscent of our findings, namely, increased activated CD4 ϩ T cells at SIV entry sites and SIV infection after fewer exposures in vaccinated infants.
Recent in vitro studies provide further evidence that exposure of human CD4 ϩ T cells to M. tuberculosis complex strains including M. bovis BCG or M. tuberculosis H37Rv derivatives can enhance HIV infection. In the latter study, increased susceptibility was not associated with increased HIV coreceptor expression, but rather with a TLR2dependent mechanism (75). Although we could confirm that mc 2 6435 signals through TLR2, it was beyond the scope of our study to test whether AMtb or BCG vaccination in infant macaques altered TLR2 expression or TLR2 signaling in CD4 ϩ T cells. We are planning to explore this mechanism in future studies because TLR2-dependent mechanisms have been implicated in enhanced HIV infectivity (76,77). The latter study also showed that exposure of human CD4 ϩ T cells to nonpathogenic M. smegmatis mc 2 155 did not result in increased HIV susceptibility (75). Thus, the identification of the specific mycobacterial components that are responsible for immune activation could lead to the design of safer pediatric TB vaccines that do not contain these mycobacterial components (78). Alternatively, genetic factors known to influence TB pathogenesis, such as single nucleotide polymorphisms in CCL2 (chemokine [C-C motif] ligand 2) (79), TLR (80), or cytokine genes (81), could play a role. These are important questions, but they were beyond the scope of the current study that was focused on risk factors for oral SIV acquisition. It is noteworthy that we observed differences between AMtb and BCG vaccine-induced immune activation in the in vivo studies and could confirm these differences in myeloid cell activation in human PBMC. Mycobacterial strain-specific differences in response magnitudes suggest that the attenuation of the mycobacterial vaccine strains might influence host immune responses, HIV/SIV susceptibility, and disease outcome.
Combined, the data from human studies, adult macaque studies, and the infant macaque studies here, strongly suggest the need to conclusively determine the risk of HIV infection in human infants that are vaccinated with BCG or novel TB vaccine candidates. In areas with a high incidence of TB, even a small increase in the rate of HIV acquisition would have a major impact on pediatric HIV infections. Importantly, a recently published study reported that higher frequencies of activated CD4 ϩ T cells in BCG-vaccinated human infants from the MVA85A TB vaccine trial had an increased risk for TB disease (82). This is the first report that CD4 ϩ T cell activation is associated not only with higher HIV infection risk but also with predisposition to TB disease. Considering the high susceptibility of neonates and infants in many resource-poor countries to both HIV and TB, our data reiterate the need to include pediatric populations in clinical trials testing novel TB vaccines and to incorporate immune activation measurements into vaccine safety assessments. Several of our analyses imply that vaccineinduced immune activation was less pronounced in infants vaccinated with the auxotroph AMtb vaccine than in those vaccinated with BCG. Therefore, an important goal for future pediatric TB vaccine development should include the design of AMtb or BCG vaccine strains with reduced nonspecific immune activation to achieve a balance between potent priming of innate immune responses to induce long-lasting vaccineinduced memory T and B cell responses while avoiding persistent immune activation that could enhance susceptibility to HIV. Concern for such risk might be reduced once (i) new therapeutics, such as long-lasting broadly neutralizing antibodies, become available as treatment options for breast-feeding infants with HIV-infected mothers, (ii) more-frequent HIV testing of mother and child during the breast-feeding period becomes the standard of care, and/or (iii) better adherence of HIV-infected mothers to antiretroviral drug regimens is achieved.

MATERIALS AND METHODS
Animals. Infant rhesus macaques (Macaca mulatta) were vaginally delivered by colony dams from the SIV-negative and type D retrovirus-free colony at the California National Primate Research Center (CNPRC; Davis, CA) and reared in the nursery. Infant macaques of the same experimental group were cohoused in groups of two or three animals, depending on the group size. We strictly adhered to the Guide for Care and Use of Laboratory Animals (83) and the standards outlined by the American Association for Accreditation of Laboratory Animal Care; all animal protocols were reviewed and approved by the University of California, Davis Institutional Animal Care and Use Committee prior to study initiation. Animals were randomly assigned to groups and were between 3 and 7 days of age at the first immunization (week 0 in Fig. 3). For all vaccinations and blood draws, animals were immobilized by 10 mg of ketamine-HCl (Parke-Davis, Morris Plains, NJ) per kg of body weight, injected intramuscularly. Complete blood counts (CBC) were performed on each peripheral blood sample by the CNPRC Clinical Laboratory.
Vaccine strains. The vaccine strains described in this study were derived from the human-adapted M. tuberculosis strain H37Rv as described previously (7,8,10). Briefly, the parental H37Rv strain was engineered with panCD and leuCD locus deletions to produce a highly attenuated double auxotroph strain mc 2 6206 unable to synthesize the essential nutrients pantothenate and leucine (57). The mc 2 6206 strain was further attenuated by deletion of the secA2 locus, encoding components of a nonessential secretion system important for bacterial growth, host immune response restriction, and secretion of virulence factors, resulting in strain mc 2 6208. Strain mc 2 6208 was further engineered to coexpress full-length SIVmac239 Gag (mc 2 6435) or Env (mc 2 6439) multimer cassettes that were codon optimized for expression by mycobacteria. MVA-SIV was kindly provided by B. Moss and P. Earl (NIAID, NIH, Bethesda, MD) (84)(85)(86).
Immunizations. Figure 3 schematically shows the vaccine regimens applied to historical animals (groups A, B, C1, and D) and animals of the current study. Briefly, naive controls (groups A, C1, and C2) were immunized at weeks 0, 3, and 6 with sterile phosphate-buffered saline (PBS) (9). Group B (n ϭ 8) and group D animals (n ϭ 8) were orally immunized with 10 9 CFU each of strains mc 2 6435 and mc 2 6439 at birth before receiving systemic boosts intramuscularly (i.m.) (divided over four injection sites) at weeks 3 and 6 with recombinant modified vaccinia virus Ankara expressing SIVmac239 Gag and Pol (MVA vJH4) and MVA-SIV Env (MVA-SIV; each at 10 8 infectious units) as described previously (9). The per os (p.o.) immunizations were done by slowly releasing vaccine with a needleless 1-ml syringe at different sites within the mouth, including the cheek pouches, the sublingual mucosa, and the top of the tongue. Therefore, vaccine uptake could occur locally and after swallowing. In the current study, group E animals (n ϭ 6) were primed using the same regimen but received an intradermal (i.d.) boost of a divalent cocktail of 10 7 CFU each of strains mc 2 6435 and mc 2 6439 at week 3. We chose to boost group E animals by the i.d. route because the current TB vaccine BCG is administered by this route. Group F infants (n ϭ 5) were orally immunized at birth with 10 9 CFU of mc 2 6208 without the SIV expression plasmid. Group G animals (n ϭ 7) received 10 5 CFU of BCG Danish by the i.d. route (Statens Serum Institute, Denmark, Copenhagen) as clinically advised for human newborns. Group H infants received a single p.o. immunization of mc 2 6435 at birth. In order to assess the effects of mycobacterial immunizations alone, animals in groups F, G, and H did not receive booster immunizations. SIV challenge. As described previously (9), low-dose exposures to virulent SIV mac251 (500 TCID 50 ) were administered orally under light anesthesia beginning at week 9 to naive controls (group C) and vaccinated infant macaques (3 or 6 weeks after the last immunization in group D or group E infants, respectively, and 9 weeks after vaccination of infants in groups F and G). The p.o. SIV exposures were performed analogously to the p.o. immunizations in volumes of 1 ml. Animals were challenged weekly until SIV viral RNA levels in plasma indicated persistent infection. We defined persistent infection as 3 or more consecutive weeks of plasma viremia greater than the highest limit of detection over the course of the study (Ն65 copies of SIV RNA/ml of plasma). Due to the low-dose nature of the challenge model, some animals exhibited episodes of very low transient viremia or measurable viral RNA levels at or below the limit of detection. Animals that failed to become infected after 10 low-dose SIV exposures were right censored for statistical analysis. All animals were monitored for a minimum of 10 weeks after SIV infection and were euthanized before they met clinical criteria established for retrovirus-infected animals (87).
Sample collection and processing. EDTA-anticoagulated blood samples were longitudinally collected prior to all interventions throughout the study period. Infant blood sample volumes were based on infant weight and ranged from 0.5 to 3.5 ml per bleed over the course of the study. Plasma was isolated from whole blood by centrifugation and stored in multiple small aliquots at Ϫ80°C. At euthanasia, bronchoalveolar lavage (BAL) fluid samples were collected. Recovered BAL fluid samples were centrifuged to pellet cells which were resuspended in complete RPMI 1640 medium for flow cytometric analysis, while supernatant was aliquoted and stored separately at Ϫ80°C.
At euthanasia, multiple tissues, including tonsil, lymph nodes (LN) (axillary, mesenteric, retropharyngeal, and submandibular), spleen, and intestinal tissues (colon and ileum) were also collected. Tissues were divided into multiple aliquots and (i) formalin fixed/paraffin embedded, (ii) preserved in RNAlater, or (iii) placed in complete media for fresh tissue analysis as previously described (10,65). Single-cell suspensions for cellular immunity assays were prepared using density gradient centrifugation (whole blood, spleen), gentle homogenization (lymph nodes, tonsil), or collagenase digestion and gradient centrifugation (mucosal tissues) as described previously (10,65). For the analysis of archived samples, cryopreserved peripheral blood mononuclear cell (PBMC) and tissue cell suspensions from historical animals (9,10) were quickly thawed at 37°C, immediately washed two times in 37°C serum-free AIM V medium (Thermo Fisher Scientific) supplemented only with antibiotics and allowed to rest overnight at 37°C in 5% CO 2 . The cells were washed the next day prior to staining for flow cytometric analysis.
SIV RNA analysis. Longitudinal plasma samples were used for virological analysis by reverse transcription-PCR (RT-PCR) for SIV RNA essentially as described but with manual RNA extraction for samples with limited volumes (88). Note that limits of detection depended on input plasma volume and ranged from 3 to 65 copies/ml, with a mean of 26 and median of 30. Samples showing transient low viremia followed by SIV RNA-negative time points were retested to confirm the initial PCR results. Data are reported as the number of SIV RNA copy equivalents per milliliter of plasma.
TRIM5␣ genotyping. The TRIM5␣ genotype of macaques was determined by sequence analysis of the C-terminal B30.2/SPRY domain of the TRIM5 gene as described previously (9). The genotypes of TRIM5 TFP , TRIM5 Q , and TRIM5 CypA were identified, and animals were classified as resistant, intermediate, or sensitive as described previously (39,40).
Flow cytometry. The expression of phenotypic markers, activation markers, and cytokines was evaluated using flow cytometry for T cells, B cells, and monocytes/macrophages in single-cell suspensions from blood and tissues. The cells were stained using rhesus macaque cross-reactive antibodies for phenotype ( ) markers alongside a fixable LIVE/DEAD discriminator (Invitrogen). All antibodies were obtained from BD Biosciences, except for PD-1 and IL-17 (eBioscience). The cells were fixed in 1% paraformaldehyde, transferred to PBS, and stored in the dark at 4°C until flow cytometric analysis, which was performed within 24 h. All staining panels were evaluated using fluorescence-minus-one (FMO) controls. Per each sample, 300,000 live, singlet events were acquired on an LSRII instrument. Using FlowJo software (TreeStar, Ashland, OR), cell populations from all staining panels were analyzed by first removing cell debris, multiplet events, and nonviable cells. Monocyte populations were phenotypically defined as CD3 Ϫ CD20 Ϫ CD14 ϩ HLA-DR ϩ , and myeloid dendritic cells were defined as CD3 Ϫ CD14 Ϫ CD20 Ϫ CD11c ϩ HLA-DR ϩ . Within the lymphocyte gate, T cells were defined as CD3 ϩ cells and further categorized by expression of either CD4 or CD8 markers to identify classical T cell subsets. B cells were defined within the lymphocyte gate as CD3 Ϫ CD14 Ϫ CD20 ϩ . Intracellular cytokine responses were reported based on Boolean gating strategies and after subtraction of background responses from unstimulated controls.
In vitro activation of rhesus macaque blood and tissue myeloid cell populations. Mononuclear cell suspensions were cultured in complete RPMI 1640 medium in 5-ml fluorescence-activated cell sorting (FACS) tubes with R848 (1 g/ml; InvivoGen) and incubated for 5 h at 37°C/5% CO 2 . At 1 h, brefeldin A (3 g/ml) was added. Unstimulated cells were used in parallel as controls, and any background signal was subtracted from treated cell responses accordingly. Cells were analyzed for activation markers and cytokine production by flow cytometry as described above.
M. tuberculosis isolation. Multiple tissues (lung, bronchial LN, axillary LN, spleen) were snap-frozen and stored at Ϫ80°C until shipment to the USDA ARS National Animal Disease Center (Ames, IA) to recover viable mycobacteria using three different culture methods: (i) the fast indicator tube test (MGIT), (ii) Middlebrook 7H12 medium (BacTec), and (iii) solid culture media to determine CFU. Because vaccine strains are auxotrophic, pantothenate-and leucine-supplemented media were used to ensure maximal bacterial recovery. A tissue was considered positive if one of three culture methods yielded mycobacterial growth. M. tuberculosis positive-control samples were run in parallel for quality assurance, and the analysis was performed in a blind manner.
HEK TLR reporter cell assay. 293HEK-derived cell lines with an NF-B-inducible secreted embryonic alkaline phosphatase (SEAP) reporter engineered to individually express human Toll-like receptor 2 (TLR2), TLR4, or TLR8 were purchased from InvivoGen (San Diego, CA). The Null-1 parental cell line was used as a control. According to the manufacturer's instructions, the cell lines were passaged twice in growth media (Dulbecco modified Eagle medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 4.5 g/liter glucose, 2 mM L-glutamine, 50 U/ml penicillin, 50 g/ml streptomycin [all from Gibco], and 100 g/ml normocin [InvivoGen]) before the cell lines were expanded under antibiotic selection for plasmid maintenance (1ϫ HEK-Blue Selection for TLR2 and TLR4 cell lines and 30 g/ml blasticidin and 100 g/ml zeocin for the TLR8 cell line; all InvivoGen). Cell lines were used for reporter detection experiments between passages 8 and 10, at which time the cells were resuspended in HEK-Blue detection media (InvivoGen) and plated at 50,000 cells/well on 96-well plates with a flat bottom. Human embryonic kidney (HEK) TLR2, TLR4, and TLR8 cells were stimulated in triplicate with previously titrated (i) recombinant human TNF-␣ (1 g/ml), (ii) heat-killed M. tuberculosis (HK-Mtb; 100 g/ml), (iii) ultrapure lipopolysaccharide from Escherichia coli K-12 (LPS-EK) (1 g/ml), (iv) R848 (5 g/ml) (items 1 to iv all from InvivoGen), (v) AMtb-SIV vaccine strain mc 2 6435 (multiplicity of infection [MOI] of 5), (vi) M. bovis bacillus Calmette-Guérin (MOI of 5), (vii) M. smegmatis mc 2 155 (MOI of 5), or (viii) sterile molecular-grade water (Gibco). The plates were incubated at 37°C and 5% CO 2 and monitored longitudinally for 24 h. TLR-specific signaling was quantified by measuring the colorimetric change induced by HEK-Blue detection medium substrate hydrolysis by SEAP secreted into supernatant by spectrophotometry at 650 nm. The reported values are the colorimetric readings after 15 h of stimulation minus background from Null-1 control cell wells and are representative of longitudinal results through 24 h.
Human peripheral blood. The use of human peripheral donor blood for research use was approved by the University of North Carolina at Chapel Hill Institutional Review Board. Blood samples were collected from deidentified healthy adult donors following informed written consent and preserved in EDTA before use within 24 h of collection. The BCG immunization status and purified protein derivative (PPD) reactivity of the blood donors was not disclosed. Mononuclear cells were purified from peripheral blood (PBMC) using density gradient centrifugation over lymphocyte separation medium (MP Biomedical). The cells were resuspended in RPMI 1640 medium (Cellgro) supplemented with 10% heatinactivated FBS (Gibco) and L-glutamine-penicillin-streptomycin antibiotic cocktail (Sigma) and were used immediately for functional assays.
In vitro human myeloid cell activation. PBMC (2 ϫ 10 6 cells/ml) from eight healthy human adult donors were cultured in complete RPMI 1640 medium in 5-ml FACS tubes with (i) strain mc 2 6435 (MOI of 5), (ii) BCG (MOI of 5), (iii) HK-Mtb (5 g/ml; HKMT [InvivoGen]), or (iv) R848 (1 g/ml; InvivoGen) and incubated for 5 h at 37°C and 5% CO 2 , with brefeldin A being added for the last 4 h of culture. Unstimulated cells were used in parallel as controls, and any background signal was subtracted from treated cell responses accordingly. Cells were analyzed for activation markers and cytokine production by flow cytometry as described above.
In vitro cytokine production by human PBMC. Human PBMC (1 ϫ 10 6 ) from eight healthy adult donors were incubated in 1 ml of complete RPMI 1640 medium in 12-well plates with strain mc 2 6435 (MOI of 5), BCG (MOI of 5), or sterile PBS. Culture supernatants were harvested after 18 h, centrifuged, fractioned into small aliquots, and stored at Ϫ80°C until ELISA analysis. Each assay was titrated to determine optimal dilutions. Human ELISAs targeting TNF-␣ and IL-12p70 were purchased from eBioscience, and culture supernatants were assayed at a dilution of 1:10 and undiluted, respectively. Human high-sensitivity IL-6 and IFN-␥ ELISAs were purchased from R&D Systems and assayed at 1:500 and 1:1 dilutions, respectively. Culture supernatants plus eight dilution standards were assayed in triplicate wells. Reported concentrations have been adjusted first for reference wavelength values and background absorbance from cell-free media before replicates were averaged and multiplied by the relative dilution factor, when applicable.
Statistical analysis. Data were analyzed using GraphPad Prism and InStat software (version 6; GraphPad Software, Inc., La Jolla, CA) and SAS version 9.3 (Cary, NC). The probabilities of infection per SIV exposure for naive controls or vaccinated animals were estimated in each group by the number of infected animals divided by the total number of exposures until infection or censoring and were used to estimate the relative risk of infection per exposure. The risk of infection per cumulative number of exposures was estimated using the Kaplan-Meier method and compared between vaccine groups with an exact log rank test. Nonparametric, exact Mann-Whitney tests were used to compare distributions of variables not subject to censoring between two groups. We applied individual exact Mann-Whitney tests because each pairwise comparison was of interest and because the group sizes were small. The exact Wilcoxon signed-rank test was used for within group comparisons of the same variable at two time points. Correlations between two continuous variables were evaluated by Spearman rank test. All statistical tests were two-sided, and P values of Յ0.05 were considered statistically significant. Figures do not show P values that did not reach statistical significance, and no adjustment was applied for multiple testing.