Immune perturbation following SHIV infection is greater in newborn macaques than in infants

Transmission of HIV-1 to newborns and infants remains high, with 130,000 new infections in 2022 in resource poor settings. Half of HIV-infected newborns, if untreated, progress to disease and death within 2 years. While immunologic immaturity likely promotes pathogenesis and poor viral control, little is known about immune damage in newborns and infants. Here we examined pathologic, virologic, and immunologic outcomes in rhesus macaques exposed to pathogenic SHIV at 1-2 weeks, defined as newborns, or at 4 months of age, considered infants. Kinetics of plasma viremia and lymph node seeding DNA were indistinguishable in newborns and infants, but levels of viral DNA in gut and lymphoid tissues 6-10 weeks post-infection were significantly higher in newborns versus either infant or adult macques. Two of six newborns with the highest viral seeding required euthanasia at 25 days. We observed age-dependent alterations in leukocyte subsets and gene expression. Compared with infants, newborns had stronger skewing of monocytes and CD8+ T cells toward differentiated subsets and little evidence of type I interferon responses by transcriptomic analyses. Thus, SHIV infection reveals distinct immunological alterations in newborn and infant macaques. These studies lay the groundwork for understanding how immune maturation affects pathogenesis in pediatric HIV-1 infection. Brief Summary


INTRODUCTION
Vertical HIV acquisition remains prevalent in resource-limited settings, with an estimated 130,000 infants infected in 2022, due to limitations in access to antiretroviral therapy (ART) during pregnancy 1 .Transmission occurs predominantly during labor and birth, and also during gestation or breastfeeding 2 .In newborns, untreated HIV infection is characterized by high viral loads, poor control of post-acute viremia, and rapid disease progression 3 .Without treatment, over 30% of these infants die in their first year of life, and over half die by age 2 4 .
Primate lentiviral infections in humans and nonhuman primates (NHPs) show an association with a "vicious cycle" of chronic immune activation, impaired immunological function, and viral persistence in tissues 5 .Early in infection, damage to the intestinal barrier results in microbial translocation into the blood 6 .Uptake of microbial products by myeloid antigenpresenting cells stimulates pro-inflammatory cytokine secretion, leading to systemic inflammation and immune activation 7 .In adults with HIV, activation and proliferation of CD8+ T cells occurs in a largely antigen-independent manner, and is driven by pro-inflammatory cytokines, especially IL-15 8 .Studies of untreated and ART-treated HIV infection in children and adults have identified inversion of the ratio of CD4+ to CD8+ T cells as a marker of immune activation, immune senescence, and poor prognosis [9][10][11] .Finally, alterations to the monocyte compartment during infection are associated with disease progression both in humans and in primate models [12][13][14] .Monocytes exist on a spectrum of differentiation from classical (CD14+CD16-) through intermediate (CD14+CD16+) to nonclassical (CD14-CD16+) subsets 15,16 .Increased frequencies of intermediate monocytes correlate with systemic immune activation and negatively correlate with CD4+ T cell counts during HIV infection 12 .
Microarray and deep sequencing studies of gene expression have complemented these findings, identifying distinct transcriptomic signatures associated with different HIV infection outcomes.Untreated infection is characterized by an innate antiviral response mediated by type I interferon (IFN) signaling and upregulation of IFN-stimulated genes (ISGs).Even during acute HIV infection, transcriptional differences may predict the viral set point 17 .While rapid disease progression is marked by the upregulation of certain microRNAs 18 , elite controllers and longterm non-progressors have attenuated type I IFN responses during the chronic stage, among other alterations 19,20 .Similarly, NHP studies of acute infection with simian immunodeficiency virus (SIV) and simian-human immunodeficiency viruses (SHIV) have revealed inflammasome activation, type I IFN, and IFN-g responses in tissues within days of mucosal exposure 21,22 .
Pathogenic SIV infection drives sustained type I IFN responses during chronic infection 23 , mirroring findings in human HIV-1 controllers.
Neonatal immunity is distinct from that of adults in ways that are presumed to hinder newborns' ability to control viral infection.Newborns have greater Treg-mediated suppression and Th2 bias, deficiency in Th1 responses, underdeveloped humoral immunity, and impairments in T cell priming and other innate effector functions 24,25 .CD8+ T cell responses are a major mechanism of primate lentiviral control in both human and NHP studies 26 .Weak CD8+ T cell responses are observed in HIV-infected children under 3 years of age, especially those with depleted or phenotypically altered CD4+ T cells in conjunction with high viral loads 27 .
Tregs, which are abundant in newborns, are conjectured to play multifactorial and opposing roles in HIV-1 infection by suppressing either virus-specific CD8+ T cell responses, deleterious immune hyperactivation, or both 28 .Although the relationship between immune activation and pathogenesis in infants is not well characterized, CD8+ T cell activation in infants at 1-2 months of age was predictive of disease progression in one study 29 .Newborn and infant rhesus macaques also have greater counts and frequencies of CD4+ T cells 30 , notably activated CD4+ CCR5+ memory T cells that are prime targets for infection with SIV 31,32 and may be a key target cell population during vertical HIV acquisition 33 .Among infants who acquired HIV vertically, age at the time of infection is correlated with survival rate, with mortality highest among those infected before 4 weeks of age and decreasing gradually over the first year of life 4,34,35 .This decrease in mortality may be attributable at least in part to immune maturation resulting in increased antiviral defense.
In adult macaques exposed to the pathogenic Tier 2 viral swarm SHIVSF162P3, peak viremia decreases and varies between individuals 36,37 , in some cases to undetectable levels 38 .
Adult macaques infected with this SHIV using a high dose mucosal (intrarectal) challenge, postacute plasma viremia averaged 10 6 copies/ml for the first nine weeks of infection and 3.2 log10 DNA copies per microgram in PBMC and lymphoid tissues 39 .In contrast, infant macaques infected with SHIVSF162P3 at week 4 (28 days) recapitulate the high viral loads (post-acute, persistent viremia averaging 10 7 copies/ml) and experience rapid disease progression seen in vertical HIV infection; seeding in PBMC and lymphoid tissues averages 4 log10 copies per microgram [40][41][42][43] .Many NHP studies intended to model peripartum HIV infection have assigned macaques that are several weeks [40][41][42][43] to several months old 44 .In healthy rhesus macaques, immune cell populations and cytokine responses differ from adult values and evolve with age during the first weeks and months of life 45 , underscoring the importance of understanding age in interpreting infant macaque studies of pediatric immunity and immunopathology.
Here, we asked whether SHIV infection dynamics, immune responses, and outcomes in infant macaques are influenced by age at the time of exposure.We compared disease progression and virologic outcomes, adaptive immune responses, frequencies and phenotypes of key leukocyte subsets, and transcriptome profiles during pathogenic SHIV infection in Newborn (1-2 weeks old) and Infant (15-16 weeks old) rhesus macaques that were lacking the two major MHC-I alleles for post-acute viral control 46,47 .Viral DNA copies in tissues were compared with an outgroup of adult rhesus macaques mucosally-infected with the same virus for the same length of time 39 .These longitudinal data highlight age-dependent quantitative and qualitative differences both in viral seeding and in the developing infant macaque immune system, providing a baseline for comparison to model the effect of novel HIV therapies on viral pathogenesis in the pediatric setting.

Study design
Twelve animals were assigned to the study, regardless of sex, at approximately 1 week of age.All animals had maternal antibodies to rhesus CMV (RhCMV) prior to SHIV infection, but titers waned to below detection in 6 weeks, consistent with lack of infection (data not shown).
Groups of six rhesus macaques each were exposed to a single oral high dose (4.1x10 4 TCID50) of SHIVSF162P3 known to infect 100% of controls 42 during different stages of infancy (Figure 1).Animals in Group 1 ("Newborns") were exposed to SHIV at 1-2 weeks (7-14 days) of age, while those in Group 2 ("Infants") were exposed at 15-16 weeks of age (Table 1).Infants were allowed to age to 10 week post-SHIV (24 weeks of age) to allow for observation of changes in viremia.Blood and lymph node biopsies were sampled at age-or infection-matched intervals during the first six weeks of SHIV infection.Newborns assigned to age into Infants in Group 2 served as age-matched uninfected controls for Group 1, and blood was collected from Group 2 Infants at age-matched time points during the newborn period (<8 weeks old) in order to provide baseline measurements for comparison to the SHIV-infected Newborns.We categorized these samples as "Control Newborns."All animals were immunized with a hepatitis B (HepB) vaccine using a regimen modeled after the routine HepB immunization schedule for infants in the US 48 .
Lymphoid and gastrointestinal tract tissues were harvested at necropsy for analyses of cellular composition and viral reservoir quantification.

SHIV disease, infection dynamics, and reservoir seeding
Two newborns (one male, one female) experienced very rapid weight loss and diarrhea, accompanied by lymphodepletion, and were euthanized as required by the criteria specified in the animal care protocol on day 24 post SHIV exposure (Table 1).The other Newborns and 5/6 Infants survived to the experimental endpoint of 6 or 10 weeks, respectively.Clinical history was evaluated by assigning a disease score from 0-2 to indicate disease severity.In both the Newborn and Infant groups, 5/6 animals developed some degree of renal glomerular change or rash (disease score 1), with 2/6 Newborns and 4/6 Infants exhibiting lesions or opportunistic infections directly attributable to SHIV infection (disease score 2) (Table 1).Although there was no significant difference in disease score between groups (Mann Whitney U test, two-tailed P = 0.53, n = 6 animals per group), rapid progression prior to 6 weeks was only observed in the Newborn group (P = 0.0307, Mantel-Cox test; P = 0.0247, Gehan, Breslow, Wilcoxon test, n = 6 per group).
We measured viral RNA copies in plasma over time (Figure 2).All animals in both age groups had high peak viremia and maintained high post-acute viral loads through week 6 postinfection (p.i.), with only a transient decrease in viremia from the peak level on day 10 p.i.
(Figure 2A).Animal 38242 in the Infant group had reduced PVL at 8-10 weeks p.i., but during the first 6 weeks, there was no significant difference in cumulative plasma viral loads between Newborns and Infants (Figure 2B-C).
To compare seeding rates early in infection, we quantified viral DNA in inguinal lymph nodes at day 7 and at week 6 p.i. and found that both Newborns (that survived to week 6) and Infants had significant increases (Figure 3A-B).To determine whether levels differed between these age groups, we compared these same data (viral DNA levels in inguinal lymph nodes) on day 7 or at week 6 (Figure 3C-D), and differences were not significant at either timepoint.The Newborns that progressed quickly to disease had the highest DNA copy numbers in lymphoid tissues on day 7 (Figure 3C).Comparison of viral DNA in 15 lymphoid and gut tissues at necropsy revealed a small but significant 0.36-log10 increase in viral seeding in Newborns compared to Infants (Figure 3E).This difference remains significant (P=0.0026)if the two rapidly progressing Newborns necropsied early (black symbols) are removed from the analysis.
Pairwise comparisons of the 15 individual tissues showed that two gut (cecum, rectum) and four lymphoid samples (iliosacral, inguinal, and mixed mesenteric lymph nodes and tonsil) were significantly different between newborns and infants (Figure 3F).Necropsy tissue samples were collected at week 9 or 10 for Infants and at week 3 or 6 for Newborns (see Table 1), which are not precisely matched time points.We previously showed that, In this infant model, viral DNA levels in PBMC and lymphoid tissues peak at 6 weeks and are stable to at least 16 weeks 42 .
We compared DNA copies/million cells for 11 tissues (five lymphoid and six gut) from this study with those from six adult macaques infected with SHIV-SF162P3 and necropsied at 8 weeks post infection 39 (Supplemental Table 1).Mean Newborn DNA levels were significantly higher than Adult levels comparing all 11 tissues (P=0.0134) and a subset of five lymphoid tissues only (P=0.0029), based on Bonferroni adjusted P-values (Supplemental Table 2).Other comparisons were not significant.

Adaptive immune responses
HepB vaccination served as a measure of adaptive responsiveness (Figure 4A).All 6   Infants made antibodies to surface antigen HBsAg after their first vaccine dose, with 5/6 (83%) responding by week 3, while 4/6 Newborns did not respond (Figure 4B).We measured the effect of SHIV on pre-existing HepB immunity by comparing the SHIV-infected Infants with an aviremic control group (Figure 4A, C) that were matched to the Infants by age and HepB vaccination history 42 .HepB antibody titers in these two groups were similar, suggesting that persistent SHIV viremia did not diminish preexisting antibody immunity to HepB in infants (Figure 4C).
Seroconversion to the infecting SHIV was observed in 2/6 (33%) Newborns and 3/6 (50%) Infants (Figure 4D).Among the Newborns, 37619 seroconverted by week 3 but did not survive beyond day 25; all other animals remained negative except for 38276, which had titers just above the limit of detection at week 6 (Figure 4D, top).Seroconverting Infants had stronger responses, with 38242 reaching titers of 10 4 by week 6 and developing moderate neutralizing activity by week 10 (Figure 4D, bottom).Time to seroconversion was similar among Newborns and Infants (Figure 4E).T cell responses were measured in PBMC, mesenteric LN, and spleen, using pools of SIVmac239 Gag and HIV-1 Clade B consensus Env peptides by IFN-g ELISPOT, and responses were weak or absent in Newborns (Figure 5A, B) and Infants (Figure 5C, C).
Newborn 37619 and Infant 38242 had weak and narrowly focused responses on just one or two peptide pools.A weak positive signal was also observed for Infant 38362 in the spleen, though not in LN and PBMC, against one Env peptide pool (Figure 5D).However, the possibility of T cells recognizing other SHIV epitopes or producing other cytokines cannot be ruled out; indeed, HIV-specific CD8+ T cells have been found to produce low IFN-g levels in adult women with acute infection 49 , suggestiong that IFN-g secretion may not be a definitive indicator for HIVspecific CD8+ T cell responses.

Leukocyte dynamics in peripheral blood
To probe the effects of SHIV infection on major immune cell populations, we performed longitudinal flow cytometric analyses of peripheral blood leukocytes.One staining panel was designed to enumerate B cells, natural killer (NK) cells, natural killer T (NKT) cells, and monocyte subsets (Supplemental Figure 1).A second panel tracked naïve and memory T cell subsets and their activation marker phenotypes (Supplemental Figure 2).A complete blood count (CBC) for each sample facilitated the quantitation of absolute cell counts from flow percentages.To analyze differences due to SHIV infection status in the newborn period, data for Newborns and Infants were aligned according to time after the start of the study, such that animals were matched in age but only the Newborns had been inoculated with SHIV.
Conversely, to determine the impact of age at SHIV exposure on immune subsets, the two groups were instead aligned according to time after SHIV exposure.For each immune parameter, the groups were compared at each timepoint using Sidak's multiple comparisons test to examine differences due to age and/or SHIV.There were no significant differences in absolute counts of white blood cells, neutrophils, lymphocytes, B cells, T cells, or monocytes between groups during either the first 6 weeks of the study or the first 6 weeks of SHIV infection (Supplemental Figure 3A-C, E-F, H).Of these groups our observations included (1) platelet and NKT cell counts in Newborns were marginally higher than in Infants on day 4 after SHIV exposure (Supplemental Figure 3D, G). (2) B cell numbers gradually increased in both groups during the first 6 weeks (Supplemental Figure 3E, top), consistent with previous findings in both human and rhesus macaque newborns 30,50 .(3) Although B cell phenotypes were not examined in detail, total B cell counts dropped during acute infection in Infants but not Newborns (Supplemental Figure 3E, bottom) and may reflect the preferential loss of memory B cells 51,52 , which are more abundant in human infants than in newborns 50 .
NK cell counts among newborns were highly variable, obscuring differences between infected and uninfected newborns (Supplemental Figure 3I, top), but at day 10 p.i. NK cell counts dipped transiently to < 200 NK cells/µl blood in both Infants and Newborns (Supplemental Figure 3I, bottom).Here, we provisionally refer to CD3 -CD8 + CD20 dim cells as CD20 dim NK cells and noted lower levels of these cells in Newborns than Infants just before SHIV infection and during acute infection and were similar in SHIV-infected and Control Newborns (Supplemental Figure 3J).In general, fewer CD20 dim NK cells expressed CD16 + than CD20 -NK cells, but CD16 expression was similar among all animals (Supplemental Consistent with a previous study 13 , absolute monocyte counts were largely stable regardless of SHIV infection or age (Supplemental Figure 3H).However, the relative frequencies of classical, intermediate, and nonclassical monocytes were altered in both Newborns and Infants during acute SHIV infection (Figure 6A).The proportion of classical monocytes was significantly reduced on day 10 of SHIV infection in Newborns, yet was unchanged in Control Newborns (Figure 6B).The magnitude of this reduction was greater in Newborns than in Infants at the same timepoint after SHIV exposure (Figure 6B We found no alterations in absolute CD4+ and CD8+ T cell counts in either Newborn or Infant macaques (Supplemental Figure 4A-B) and with no CD4/CD8 ratio inversion at 2-3 weeks as reported in adult rhesus macaque after vaginal infection with SHIVSF162P3 38 , (Supplemental Figure 4C).However, the CD4/CD8 ratio decreased in Newborns at weeks 4 and 6 after SHIV exposure compared with Control Newborns, consistent with a study showing that CD4/CD8 ratios in human infants vertically infected with HIV-1 gradually decline from normal levels of >2, but remain >1 during the first year of life 53 .
To track CD4+ and CD8+ T cell subset dynamics, we defined naïve (TN), central memory (TCM), and effector memory (TEM) T cells based on differential surface expression of CD28 and CD95 54 .We found that the CD4+ compartment was predominantly naïve in all animals regardless of infection, although Infants had somewhat lower CD4+ TN frequencies than Newborns 6 weeks p.i. (Supplemental Figure 4D).TCM comprised ~10-20% of CD4+ T cells in Control Newborns, and their frequency decreased gradually during SHIV infection in both Newborns and Infants (Supplemental Figure 4E).In contrast, CD4+ TEM cells-the smallest subset in Control Newborns-increased significantly during SHIV infection in Infants, but not Newborns, with significant differences between groups evident as early as day 4 p.i.

(Supplemental Figure 4F).
Unlike the relatively modest changes in the CD4+ compartment, CD8+ T cell naïve and memory subsets were strongly altered during SHIV infection (Figure 6E).As previously reported 54 , the majority of CD8+ T cells were naïve in the absence of infection (Figure 6F, top).
However, in Newborns but not Infants, SHIV infection heavily skewed the CD8+ T cell compartment away from naïve and towards effector memory, inverting the TN/TEM ratio by day 10 p.i. (Figure 6F and H).The magnitude of these rapid changes was maintained in most Newborns through week 4 and were still altered at week 6 p.i.No changes in the frequencies of CD8+ TCM were noted (Figure 6G).

The striking observation that acute SHIV infection causes profound expansion of CD8+
TEM in Newborns, but not Infants, led us to ask whether immune activation phenotypes differ between age groups.We monitored expression of the activation markers CD69, CD25, and HLA-DR on the surface of naïve, central memory, and effector memory CD4+ and CD8+ T cells.Surprisingly, SHIV infection did not result in overall increased expression of any of these activation markers on most T cell subsets (Supplemental Figure 4G-X).

Cumulative differences in blood leukocyte phenotypes
To further analuze immunological differences between Newborns and Infants, we calculated the mean area under the curve (AUC) for each parameter measured longitudinally by flow cytometry during the first 6 weeks of the study as well as the first 6 weeks of SHIV infection.For each parameter, AUCs in Newborns and Infants were directly compared using t tests, with separate analyses done to test for differences during the first 6 weeks of the study (SHIV-infected Newborns vs.Control Newborns) and the first 6 weeks of infection (Newborns vs. Infants), and significance was determined using a false discovery rate (FDR) approach.If AUCs for a parameter differed significantly between SHIV Newborns and Control Newborns during the first 6 weeks of the study, the difference was attributed to SHIV infection status because the groups were matched in age.Conversely, if AUCs differed significantly between Newborns and Infants during the first 6 weeks of SHIV infection, the difference was attributed to age at the time of SHIV exposure.We identified 9 parameters for which AUCs differed significantly between groups as a function of SHIV infection status during the newborn period (Figure 7A).In line with our findings that SHIV infection skewed CD8+ T cell and monocyte subset frequencies, especially around day 10 p.i. (Figure 6), the cumulative percentages of classical and intermediate monocytes were 25% lower and 70% higher respectively in SHIV-infected Newborns compared with Control Newborns.CD8+ T cells were even more strongly skewed, with >2-fold lower cumulative frequencies of CD8+ TN and >3-fold higher frequencies of CD8+ TEM over the first 6 weeks of SHIV infection.Interestingly, despite the paucity of significant intergroup differences in activation marker expression at any particular time point (Supplemental Figure 4G-X), AUC analysis revealed that SHIV-infected Newborns had significantly greater cumulative frequencies of CD69+ cells within several T cell subsets than their uninfected counterparts.In line with timepoint-specific comparisons, CD25+ CD4+ TN cell frequencies were significantly decreased during SHIV infection.Because this cell subset has been reported to express high levels of lymphoid tissue homing markers CD62L and CCR7 55 , their disappearance from peripheral blood may have been due to migration into lymphoid tissues during SHIV infection.Finally, SHIV infection was associated with a significant enrichment of CD16+ CD20dim NK cells; the biological importance of this observation is unknown.
In a parallel FDR analysis of intergroup differences during the 6 weeks after SHIV infection, we identified 4 parameters for which AUCs differed significantly between groups as a function of age at the time of SHIV exposure (Figure 7B).Importantly, two of these parameters-the frequencies of CD8+ TN and CD8+ TEM-were found to differ as a function of both SHIV infection status and age at SHIV exposure, indicating an age-dependent difference in the effect of SHIV.In addition, cumulative frequencies of CD25+ CD4+ TCM and TEM were 2-and 3-fold greater in Newborns than in Infants, possibly reflecting the relatively greater abundance of Tregs in the neonatal immune system.At necropsy, we analyzed the same surface markers in spleens and mesenteric LN to compare differences by age at time of SHIV exposure in the Newborns and Infants.The %CD8+ TEM were significantly greater in the spleens of Newborns (Figure 7C), and %CD8+ TN were greater in the mesenteric LN of Infants than in Newborns (Figure 7D), consistent with the ratios seen in the blood (Figure 7B).In the spleen, the % NKT cells were significantly lower in Newborns than in Infants (Figure 7E).Other comparisons were not significant.

Transcriptome analysis
To compare innate responses to SHIV infection by age, we performed transcriptome analysis in the peripheral blood using bulk RNA sequencing (RNA-seq).For Newborns and Infants, we analyzed samples on day 0 just prior to SHIV exposure, as well as day 4 and (when available) day 42 of SHIV infection (0, 4, and 42 DPI [days post-infection]).For a SHIVunexposed age-matched baseline for comparison with the Newborns, we also included Control Newborn samples on days 0, 4, and 42 from the beginning of the study (0, 4, and 42 DPB [days post-beginning]) (Figure 8A).The design of our study allowed differential expression (DE)   analyses to be performed across multiple contrasts, designed to measure different facets of infection (Figure 8A).Contrasts at time points before SHIV exposure measure age-dependent differences or cohort/batch effects.Within-group contrasts after SHIV exposure measure the impact of SHIV infection in Newborns and in Infants.Across-group contrasts at matched time points after SHIV exposure evaluate age-dependent responses to SHIV infection.
We began our analysis by examining DE genes in contrasts prior to SHIV exposure to measure baseline/background differences in gene expression.Only three genes were significantly DE (adjusted p < 0.1) between Newborns on 0 DPI and Control Newborns on 0 DPB (Supplemental Figure 5a).Therefore, these groups were combined into a single "0 DPBpooled" group for subsequent contrasts.No genes were significantly DE between 0 DPBpooled and Control Newborns 4 DPB.However, a signature of 187 DE genes was noted for Control Newborns at 42 DPB vs. 0 DPBpooled (Supplemental Figure 5e).An even stronger signature with 477 DE genes was found for Infants at 0 DPI contrasted with 0 DPBpooled (Supplemental Figure 5f).Given that 'Control Newborns' and 'Infants' are the same animals sampled at different ages, this result is consistent with age-dependent transcriptomic changes as these animals grew older in the absence of SHIV infection.In both contrasts, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed differential expression of genes associated with various processes related to immune system development and signaling (Supplemental Figure 6a-b), likely reflecting normal immunological maturation during early life.
When we evaluated within-group differences in gene expression for Newborns and Infants during SHIV infection, only five genes were found significantly DE between 0 DPI and 4 DPI in Newborns, suggesting that gene expression in peripheral blood is not yet substantially altered at this early time point in acute infection (Supplemental Figure 5b).However, by 42 DPI, we detected 129 genes DE, of which 57 (44%) were upregulated and 72 (56%) were downregulated relative to 0 DPBpooled (Supplemental Figure 5c).Similarly, for intra-group contrasts among Infants, we compared gene signatures in Infants 0 DPI with those on 4 DPI and 42 DPI.No significant genes were identified as DE in 0 DPI vs. 4 DPI.However, on 42 DPI of SHIV infection in Infants, 205 genes were DE, of which 158 (77%) were upregulated and 47 (23%) were downregulated relative to 0 DPI (Supplemental Figure 5d).
GO and KEGG analysis showed that the DE signature at day 42 DPI in Newborns consisted largely of genes involved in cell division, cell cycle regulation, and metabolism (Figure 8B).Genes in the most significantly enriched term "cell division" were AURKA, ECT2, KIF23, KIF11, BUB1B, CCNB2, BIRC5, PRKCE, CDC20, CDCA2, PTTG1, NEK2, CEP55, ZWINT, and TOP2A; most were significantly downregulated on 42 DPI compared with 0 DPBpooled.We observed upregulation of several genes, including TLR7, ISG20, CXCL10, and LIF, which were part of the significantly enriched term "immune response" (adjusted p value = 0.048), although they did not seem to cluster with any specific pathway.CXCL10 in particular is a biomarker for high viremia and rapid HIV-1 disease progression 20,56,57 , as well as immune dysfunction in infectious diseases more broadly 58 .In Infants, GO and KEGG analyses comparing day 0 vs day 42 of infection revealed differential regulation of gene networks involved in antiviral immune responses mediated by type I IFN signaling (Figure 8C).Genes contributing to enriched terms containing the phrase "type I interferon" included ADAR, BST2, IFI6, IFI27, IFIT1, IFIT3, ISG15, ISG20, MX1, MX2, OAS1, OAS2, OAS3, RSAD2, SAMHD1, SP100, STAT2, USP18, and XAF1.Notably, many of these are lentiviral restriction factors, and most were significantly upregulated on 42 DPI.The finding that CXCL10 was significantly DE in Newborns but not in Infants suggests a possible role for CXCL10 in age-dependent mechanisms of SHIV pathogenesis.Newborn and Infant transcriptional signatures were very distinct, with 97/129 (75%) of genes DE in Newborns and 175/205 (85%) of genes DE in Infants specific to each group's SHIV response on 42 DPI (Figure 8D).Of the 291 total genes DE in either group specifically on day 42 of SHIV infection relative to that group's 0 DPI baseline, only 19 genes (6.5%) were common to both groups (Figure 8D).
Finally, we examined between-group differences in gene expression at matched time points during SHIV infection.As previously noted, 477 genes were DE on 0 DPI in Infants vs. 0 DPBpooled in Newborns, indicating a substantial age-dependent difference in transcriptomic profiles at baseline (Supplemental Figure 5f).By 4 DPI, only 237 genes were found to be DE in Newborns vs. Infants, a 2-fold decrease (Supplemental Figure 5g).On 42 DPI, only 38 genes were DE, a 12.5-fold decrease from day 0 (Supplemental Figure 5h).GO and KEGG analysis of DE at 42 DPI revealed that there was no clear enrichment for particular biological processes or signaling pathways, aside from a single significant tierm, "metabolic pathways, found in KEGG" (Supplemental Figure 6d).GO and KEGG analyses showed different gene expression hierarchies between 0 DPI (Supplemental Figure 6b) and those at 4 DPI (Supplemental Figure 6c), which showed DE of genes involved in metabolism, porphyrin synthesis, and erythrocyte development.Taken together, and in light of the results from the within-group comparisons (Figure 8), these data paradoxically suggest that, although SHIV responses differ by age, SHIV infection ultimately reduces age-dependent differences in gene expression.This phenomenon is detectable as early as 4 DPI, a time point when SHIV infection has little effect on gene expression in Newborns (Supplemental Figure 5b) and no significant effect in Infants.

Discussion
Without treatment, vertical transmission of HIV is likely to cause severe disease in infants with high viremia, but which determinants contribute to higher viremia are not known 3 .
Here, we show in a macaque oral infection model that despite similar kinetics of viral seeding in blood and lymphoid tissues at day 7, two newborns with the highest plasma viremia succumbed to very early disease prior to the time of planned euthanasia.Although significantly higher levels of viral DNA were recorded in 15 tissues of newborns compared with infants at necropsy, seeding was variable, with significant differences in two gut and four lymphoid tissues .Viral DNA in all 11 tissues and in a subset of five lymphoid tissues was also significantly greater in newborns than in adults 39 , supporting the hypothesis that a younger age at the time of infection leads to increases in the size of the reservoir in certain tissues.
All newborn macaques in this study exhibited features consistent with increased immune damage and defective antiviral innate immunity during acute infection compared with infants infected at 4 months of age.In agreement with previous studies showing impaired vaccine responses in HIV-infected children, weaker HepB antibody responses to HepB were seen in newborns infected with SHIV compared with age-matched uninfected newborns.It is probable that SHIV weakens humoral responses to other vaccine antigens, given that reductions in vaccine-induced antibody titers after six childhood vaccines have been reported in HIV-infected infants 59 .Our data suggest that this SHIV model recapitulates this important aspect of HIV-1 pathogenesis in the pediatric setting.In contrast, we found that adaptive responses to SHIV arose with similar kinetics in Infants and Newborns, although the study timeline was too short to complete the normal course of HepB vaccination, and it was difficult to compare the potency of antibody responses, which typically take months to reach a plateau.Monocyte and T cell compartments were markedly altered in the blood during SHIV infection, especially in Newborns.First, SHIV infection skewed monocytes toward intermediate and nonclassical phenotypes, and this skewing was more pronounced in Newborns than in Infants.We also detected profound alterations in T cell subsets, with an inverted ratio of naïve to effector memory CD8+ T cells in both age groups.Analyses of leukocyte phenotype frequencies in splenic and mesenteric LN tissues at the time of necropsy revealed similar findings for CD8+ TEM CD8+ TN in Newborns and Infants.Though we did not determine what proportion of these CD8+ TEM cells were specific to SHIV epitopes, T cell responses to Gag and Env in most animals in both groups were essentially negative, suggesting that this increase reflected antigen-independent activation and expansion of CD8+ T cells as seen in adults with HIV 8 .The age-dependent increases in differentiated monocytes and CD8+ T cells in Newborns may be indicative of greater levels of systemic immune activation.Unexpectedly, we did not detect clear age-or infection-dependent differences in activation marker expression on T cell subsets, in contrast with studies documenting HLA-DR upregulation on CD8+ T cells during acute HIV-1 infection in adults and infants 29,49 .It is possible that other measures of systemic immune activation would have been informative; for instance, soluble CD14 is secreted by monocytes and macrophages stimulated by LPS, and is a biomarker of microbial translocation resulting from gut epithelial barrier damage 7 .

Stark differences in transcriptomic signatures were associated with SHIV infection in
Newborns and Infants, raising the possibility that antiviral immunity is impaired in Newborns due in part to underdeveloped type I IFN responses, which was intact and readily detectable in Infants infected at 15-16 weeks of age.In contrast, newborns had a transcriptional signature dominated by the downregulation of genes involved in mitotic, cell cycle, and activation processes; the near-absence of a viral infection response; and upregulation of CXCL10, a biomarker of greater disease severity.Given the central importance of type I IFN signaling in mounting an effective innate antiviral defense during acute infection 22,60 , its presence in 4month-old macaques yet profound lack in newborns may explain, in part, the age-dependent difference in rapid disease in this model as well as pathogenesis seen in children infected with HIV-1 in utero or at birth compared with those infected at older ages.The timing of the development of type I IFN-mediated immune function during infancy should be defined more precisely in future studies, given evidence that type I IFN may contribute to rapid progression in SIV-infected infants 61 and resolution of innate activation is associated with lack of pathogenesis in adapted hosts 23,62 .
We also examined the relationship between immune maturation over the course of early infancy and damage associated with pathogenic lentiviral infection.The strengths of our study lie in the comprehensive immunologic and transcriptomic profiling, as well as in the study design and protocol with attention to age-matched controls.Second, to our knowledge, this study is the first to examine and compare transcriptome profiles during lentiviral infection in newborns and infants at different ages.Despite these strengths, this study had limitations.To assure that all newborns and infants were infected with a single oral exposure to mimic peripartum exposure, the SHIV dose was possibly higher than the HIV dose a human infant might receive from exposure to maternal blood or vaginal secretions.Because the study was terminated early in infection, we cannot determine from our findings how virologic outcomes and disease severity may compare between age groups over a longer period of observation.Finally, we did not examine transcriptomes in gut and lymphoid tissues, the major sites of viral replication and host defense.Age-dependent dynamics of immunopathology at these anatomic sites should be elucidated in future studies.
Taken together, our findings suggest that rapid viral seeding, combined with defective innate defenses, slow or defective adaptive responses, and elevated immune activation in newborns contribute to age-dependent differences in pathogenesis.We conclude that SHIV, like HIV, is more acutely damaging in newborns than in infants infected at older ages, reinforcing the validity of this model for understanding mechanisms of pathogenesis in vertically-acquired HIV infection.Ultimately, therapeutic intervention as early as possible after birth is more likely to have a durable effect on disease progression by limiting damage to the immune system.

Materials and Methods
Animal model and sex as a biological variable.Twelve newborn male and female outbred rhesus macaques of Indian origin (Macaca mulatta) were obtained from the breeding colony within days of birth, underwent veterinary evaluation, were adapted to bottle feeding in the ABSL-2 infant nursery, and transferred to ABSL-2+ containment as soon as possible after 1 week of age.Sex was not considered as a biological variable.Both sexes of newborns were included in the study, but were not balanced between groups because they were assigned over a period of two birthing seasons, and sex is random.The Newborn group included 4 females and 2 males; the Infant group included 2 females and 4 males.No differences could be attributed to sex, and we believe that the results are expected to be the same.Animals were excluded from the study if they possessed Mamu-B*08 and -B*17 MHC Class I alleles 46,47 .
Animals were housed as age-matched pairs, and were monitored for clinical signs of disease by regular evaluation of body weight, peripheral lymph node size, appetite, behavior, and stool quality.Animals were euthanized under IACUC guidelines using standard methods consistent with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for Euthanasia 63 .

Hepatitis B immunization. All animals received the HepB vaccine Recombivax HB (Merck) by
intramuscular injection on the first day of the study (age 7-14 days).Infants received an additional dose at week 6.

SHIV virus exposure.
A challenge stock of SHIVSF162P3 virus was generated in activated, CD4+enriched macaque splenocytes inoculated with SHIVSF162P3 obtained from the AIDS Reagent Program (no.6526).Virus production and in vitro and in vivo quantification methods are described in detail in an earlier publication 42 .Each animal received a total of 2 ml (4.1 × 10 4 TCID50, rhesus PBMC) of undiluted just thawed cell-free SHIVSF162P3 virus by swallowing in awake animals, two 1 ml doses 10-15 min apart.Newborns were exposed to SHIV at 1-2 weeks of age (day 0 study time point.Infants were exposed at 15-16 weeks of age (week 14 study time point).
Blood and tissue harvest and processing.Peripheral blood was collected into EDTA blood tubes and centrifuged in the collection tubes at 750 x g for 30 min at 4 °C with no brakes; plasma supernatant was stored at −80 °C.The remaining blood fraction was resuspended in sterile PBS to double the original volume, and PBMC were isolated by centrifugation in SepMate tubes (StemCell Technologies) over Lymphocyte Separation Medium (Corning) and cryopreserved in LN2.At each biopsy, two adjacent lymph nodes were taken to obtain a sample for paraffin embedding and to make a cell suspension that was cryopreserved.At necropsy, blood, cerebrospinal fluid (CSF) was collected into a 2 ml vial and stored at −80 °C. 100 µg samples of solid tissues were excised and frozen at −80 °C in 2 ml tubes pre-filled with 1.4 mm zirconia beads (Spex SamplePrep) for tissue homogenization with a bead beater, nucleic acid extraction, and vDNA detection by quantitative PCR.Any remaining spleen or mesenteric lymphoid tissue was processed to make single cell suspensions and cryopreserved.
Viral nucleic acid quantitation in plasma, cells, and tissue homogenates.Nucleic acid from plasma, CSF, or lymph node biopsy cell pellets was purified using a Maxwell 16 instrument (Promega, Madison, WI) per the manufacturer's protocol, using the LEV Viral Total Nucleic Acid Kit for plasma and CSF and the LEV Whole Blood DNA Kit for lymph node cells.Complete PCR and RT-PCR protocols for viral RNA, viral DNA in cell pellets, and viral DNA in tissues were published previously 42 .
Evaluation of pathology.At necropsy, rhesus macaque tissues and body fluids were collected fresh, frozen, or fixed in 10% formalin and subsequently processed for histologic evaluation.
Necropsies and microscopic evaluation of tissues were performed by veterinary pathologists blinded to animal group assignments and viral load data.Pathogens were identified and confirmed by H&E morphology and histochemistry, IHC, and PCR.Adenovirus, cytomegalovirus, and Enterocytozoon bieneusi were visualized by IHC using the Vectastain ABC Kit, Peroxidase Standard (Vector Laboratories, Burlingame, CA, USA).Adenovirus was detected with a mouse anti-adenovirus mAb (MAB8052, 1:500 dilution, EMO Millipore, Temecula, CA, USA).Cytomegalovirus was detected using an antibody gifted by Dr. Peter A.
Barry, UC Davis School of Medicine (1:750 dilution) as previously reported 64 .E. bieneusi were visualized using a mouse anti-measles matrix protein mAb (MAB8910, 1:1000 dilution, EMO Millipore, Temecula, CA, USA).Spironucleus species (sp) were confirmed in tissue by nested PCR amplification of small subunit ribosomal DNA using methods developed by Bailey et al. 65 .
Cryptosporidium sp, SHIV giant cell disease, flagellated protozoa, Pneumocystis sp, Malassezia sp, and attaching and effacing E. coli were all diagnosed by H&E stain.Additional diagnostic methods included Gomori methenamine-silver stain for Pneumocystis sp and Periodic Schiff reaction for Malassezia sp.Intracellular argyrophilic bacteria were visualized by Warthin-Starry stain.Disease scores of 0, 1, or 2 were assigned to each animal according to the criteria outlined in Table 1.

Enzyme-linked immunosorbent assay (ELISA).
Antibody responses were determined by measuring binding of plasma IgG to recombinant HIV-1 SF162 gp140 trimer and described previously 42 .The gp140 trimer was produced as described 66 by transient transfection in Expi293F cells and purified over a Galanthus nivalis lectin-coupled agarose (GNA) column (Vector Laboratories, Burlingame, CA), followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare Life Sciences).Peak trimer fractions were pooled, concentrated, and frozen at −20 °C.Heat-inactivated plasma samples were assayed in duplicate for neutralization against single round of entry SHIVSF162P3 Env-pseudoviruses using TZM-bl reporter cells.

Enzyme-linked immunospot assay (ELISPOT). T cell responses in blood and tissues were
tested in an interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assay as previously described 42 .
Flow cytometry.To evaluate CD8+ T cells and monocytes in blood and tissues, 2 staining panels were developed and samples were run on a Becton-Dickinson LSR II flow cytometer.Data was analyzed in Flowjo according to the gating strategies defined in Supplemental Figures 3 and 4 for Panels 1 and 2 respectively.

RNA isolation and deep sequencing (RNA-seq).
From 500 µl peripheral whole blood collected in EDTA tubes, total cellular RNA was isolated using the QIAamp RNA Blood Mini Kit (Qiagen, catalog number 52304).RNA was eluted in 30 µl nuclease free water, split into two aliquots of 23 µl and ~5 µl, and immediately frozen at -80C.Frozen RNA aliquots were shipped on dry ice to MedGenome (Foster City, CA) for sample quality control, barcoded cDNA library construction, and bulk deep sequencing (RNA-seq) using the Illumina platform.Barcoded cDNA Encyclopedia of Genes and Genomes [KEGG]), and Reactome databases for functional annotation of significant genes, to identify pathways and cellular processes that are DE in contrasts of interest.Significantly enriched terms (adjusted p value < 0.1) that were represented by at least 3 query genes were reported in Figure 8 and Supplemental Figure 6.
Statistics.Newborn rhesus macaques were assigned randomly to the Newborn or Infant study groups as they accrued, regardless of sex.Historical studies using this animal model showed that group sizes of 6 were sufficient to provide 80% power to detect a 1-log difference in plasma

35
A The start of the study is defined as the first timepoint at which blood was collected from each animal.For Newborns, this is the day of SHIV exposure (day 0).For Infants, this is the day they would have been exposed to SHIV had they been assigned as Newborns -i.e., the day 0 sample from the age-matched uninfected control group ("Control Newborns").
B Disease score criteria: 0 -No significant lesions and clinical history OR diarrhea and typhlocolitis consistent with colony infants without SHIV. 1 -One or more non-specific findings such as rash or minimal or mild glomerular change.Findings cannot be definitively assigned to SHIV-related immune suppression.2 -One or more of the following: opportunistic infections, lymphoid depletion, glomerulonephropathy consistent with SHIV-related disease in infant macaques.
C Like the other four Newborns, animals 37619 and 37650 were orally exposed to a single high dose of SHIVSF162P3 at 1-2 weeks old.However, except for CD4, CD8, and CD20 counts, detailed longitudinal monitoring by flow cytometry was not performed for these two animals.

Figure 3K )
Figure 3K) except at week 4 where higher levels of CD16 were observed in SHIV-infected Newborns compared with Control Newborns (Supplemental Figure 3L, top).The implications of these differences are unclear and merit further study.
), suggesting an age-dependent difference in the impact of SHIV on monocyte differentiation.The decrease in classical monocytes accompanied increases in both intermediate and nonclassical monocytes (Figure 6C-D) during SHIV infection in both Newborns and Infants.Neither intermediate nor nonclassical monocytes differed significantly in frequency based on age at the time of SHIV exposure (Figure 6C-D).
viral loads.For viral quantitation in plasma and tissues, measurement of T cell responses by ELISPOT, and pathology evaluation, experimenters were blinded to the animal group assignments.No blinding was possible for longitudinal flow cytometry experiments, transcriptome analysis, and ELISA assays.Nonparametric tests were used to compare groups for viral loads in plasma and tissues, and immunological metrics as measured by flow cytometry and ELISA.The specific statistical approach used for each hypothesis test is noted in the figure legends.P values less than 0.05 were considered significant.Study approval.Macaque studies were performed at the Oregon National Primate Research Center (ONPRC) in Beaverton, OR, USA in compliance with all ethical regulations for animal testing and research.The ONPRC is accredited by AAALACi, and adheres to the Guide for the Care and Use of Laboratory Animals and the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals.The study protocol was approved by the OHSU West Campus Institutional Animal Care and Use Committee.

Figure 1 .
Figure 1.Study design.Group 1 ("Newborns," n = 6 animals) were orally exposed to a single high dose of SHIVSF162P3 at 1-2 weeks of age, while those in Group 2 ("Infants", n = 6 animals) were exposed at 15-16 weeks of age.During the first 6 weeks of the study, the Group 1 Infants served as uninfected age-matched controls for comparison with Newborns (NB), and are termed "Control Newborns."All animals were given HepB vaccine (light blue X) on the first day of the study and, for Infants, a second dose 6 weeks later.Blood was sampled at the time points indicated in order to measure antibody responses and plasma viral loads (orange circles), complete blood counts and leukocyte phenotyping by flow cytometry (red diamonds), and RNA isolation for transcriptome analysis (purple squares).Inguinal lymph node biopsies (blue triangles) were taken at 1 week and, for Infants, 6 weeks after SHIV exposure.Animals were euthanized at the time points indicated (dagger) after SHIV exposure, or sooner if clinical endpoints were met due to onset of SHIV disease, and tissues were harvested for viral load quantitation and measurement of T cell responses.Time points in the study were assigned specific labels to indicate Newborn (NB), Control Newborns (CNB), Infant (INF); time point labels indicate days post-beginning of the study (DPB) or days post-infection (DPI).

Figure 2 .
Figure 2. Viral loads during the acute phase following high dose challenge with SHIV do not vary by age at time of exposure in infant macaques.(A) Top, plasma viral load (PVL) data for Newborns (exposed to SHIVSF162P3 at 1-2 weeks of age).Bottom, PVL data for Infants (exposed to SHIVSF162P3 at 15-16 weeks of age).Individual animals are represented by unique symbols.Symbols with darker colors indicate viral load in cerebrospinal fluid (CSF) at necropsy.(B) PVL between Newborns and Infants, with data censored at 6 weeks, and time points were excluded from analysis if PVL was measured for only one group.Data represent mean +/-SEM.(C) Area under the curve was computed by taking the mean PVL measurement at each timepoint during the first 6 weeks after SHIV exposure, resulting in no significant difference between the groups (unpaired t test with Welch's correction, two-tailed P > 0.05, n = 6 animals per group).

Figure 3 .
Figure 3. Viral DNA in tissues of Newborns and Infants.(A-D) Viral DNA copies in inguinal lymph nodes from Newborns and Infants were compared at day 7 and week 6 using one-way AVOVA with Tukey's multiple comparsons.Two Newborns euthanized before week 6 were excluded from panels A and D because there was no 6 week sample for comparison.(A-B) Newborns and Infants both had significantly more viral DNA at week 6 compared with day 7 after SHIV exposure.Newborns: P = 0.007.Infants: P = 0.0005.(C-D) Viral DNA copies in inguinal LN were not significantly different between age groups at day 7 or week 6 after SHIV exposure.Day 7: P = 0.756.Week 6: P = 0.998.E) Viral DNA quantitation in lymphoid and gut tissues at time of death including all animals in the study; black symbols indicate animals euthanized at week 3 (Newborns) and week 9 (Infants).Newborns had a median of 0.36 logs more viral DNA copies per 10 6 cells in each tissue than Infants (Wilcoxon matched-pairs signed rank test on log10-transformed data, two-tailed P = 0.0006).F) Means +/-SEM of individual tissues for Newborns and Infants (Unpaired t test, Benjamini, Krieger, and Yekutielli two stage

Figure 4 .
Figure 4. Acute SHIV infection impairs humoral responses to Hepatitis B vaccine in Newborns.(A) Plasma IgG binding titers to hepatitis B (HepB) surface antigen recombinant protein (HBsAg) in Newborns (top), Infants (middle), and a comparison group of approximately age-matched infants without detectable SHIV viremia (bottom).These animals, termed "Aviremic Infants", were exposed to SHIVSF162P3 on the same day as their first HepB vaccine dose, followed 30 hours later by a neutralizing antibody treatment that halted infection, resulting in undetectable viremia.Blue dotted lines at x = 0 and x = 6 denote the first and second HepB vaccine doses; Newborns only received the first dose.Yellow shaded areas indicate time periods after SHIV exposure.(B) Kaplan-Meier analysis of the percentage of animals in each group with HBsAg antibodies.Log-rank test, P = 0.0115, n = 6 animals per group.(C) Comparison of HBsAg binding titers in Group 2 Infants and Aviremic Infants at either 14 weeks (top) or 24 weeks (bottom) after the first dose of HepB vaccine (unpaired t tests, two-tailed P = 0.8225 and P = 0.8473 for weeks 14 and 24 respectively, n = 6 animals per group).Animal symbols are consistent throughout the manuscript.Data represent mean +/-SEM.(D) Plasma IgG binding titers to HIV-SF162 Env gp140 glycoprotein in Newborns (top) and Infants (bottom).Black arrow indicates the only sample with neutralizing activity against SHIVSF162P3 (ID50 = 1200).(E) Kaplan-Meier analysis of the percentage of animals that developed binding antibodies to SF162 gp140 by the indicated time points.Log-rank test, P = 0.8354, n = 6 animals per group.

Figure 5 .
Figure 5. T cell responses to Gag and Env peptide pools.T cell responses to SIVmac239 Gag and Clade B consensus Env 15-mer peptide pools were measured in PBMCs by IFNγ ELISPOT necropsy in Newborns (A, B) and in Infants (C,D).Symbol shapes in the Key correspond to individual animals and colors indicate tissue type.Concanavalin A (ConA) and/or Staphylococcal Enterotoxin B (SEB) were used as positive stimulation controls.Background signal was determined using no-antigen controls.MesLN, mixed mesenteric lymph nodes.SFC, spot-forming centers.

Figure 7 .
Figure 7. Between-group differences in leukocyte counts and phenotypes during SHIV infection in early life.(A) For each of 42 parameters measured by flow cytometry, area under the curve (AUC) during the first 6 weeks of the study was computed for each group.Groups were compared by one t test per AUC comparison (total t tests: 42) and p values were adjusted for multiple comparisons by the False Discovery Rate (FDR) approach.(B) AUC during the first 6 weeks of SHIV infection was computed for each group, using one t test per AUC comparison (total t tests: 42); P values were adjusted for multiple comparisons by the FDR approach.Discoveries were determined using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, Q = 0.05.Each parameter was analyzed individually, without assuming a consistent SD.Discoveries are denoted by colored symbols and corresponding labels.In each plot, the horizontal line indicates the -log10 transformed threshold of significance at the Q = 0.05 level.Statistically significantly different cell types in spleen and mesenteric lymph nodes collected at necropsy include: (C) percent CD8 effector memory in the spleen, (D) percent CD8 naïve in the mesenteric lymph nodes, and (E) percent NKT cells in spleen.P values are shown on each graph.Symbols identify animals as defined in Figure 2.

Figure 8 .
Figure 8. RNA-seq reveals distinct transcriptomic signatures in Newborns and Infants on day 42 of SHIV infection.(A) Contrasts of interest are shown with arrows.DPB, days postbeginning of study.DPI, days post-SHIV infection.0 DPBpooled is the combined sample group of all uninfected newborns at the beginning of the study (N = 12 animals).Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome analyses of DE genes in Newborns (B) and Infants (C) on 42 DPI vs. 0 DPI (adjusted p < 0.1).Where many terms were significant (adjusted p < 0.1), top significant terms are shown.No pathways were significantly enriched in the Reactome database.(D) Venn diagram of DE genes in Newborns 42 DPI vs. 0 DPBpooled (purple), Infants 42 DPI vs. Infants 0 DPI (pink), and background transcriptional signatures in Control Newborns (green) and Infants (gray) at different timepoints in the absence of SHIV infection.