Abstract Mpox poses a heightened risk of severe disease and mortality among individuals with HIV, yet the molecular mechanisms and immunopathology underlying multi-organ damage caused by the mpox virus (MPXV), particularly in the context of HIV co-infection, remain poorly understood. Here, we observe increased MPXV replication, more extensive skin lesions, and impaired humoral and cellular immune responses in SIV-MPXV co-infected rhesus macaques compared to those infected with MPXV alone. Multi-organ proteomic and phosphoproteomic analyses reveals upregulation of proteins involved in immune and inflammatory pathways in skin lesions and across multiple organs, especially in immune-related tissues. Abnormal activation of DNA replication and cell cycle signaling pathways, which may contribute to enhanced viral replication, is evident in both MPXV and SIV-MPXV co-infected groups. CDK4/6 may present a potential therapeutic target to suppress MPXV replication. These comprehensive proteomic datasets offer valuable insights into the pathogenesis of MPXV in the context of SIV co-infection and support ongoing efforts to mitigate the impact of mpox. Subject terms: Viral infection, Proteome informatics, Pox virus, HIV infections __________________________________________________________________ HIV infection is associated with increased risk of more severe Mpox, yet the underlying mechanisms aren’t well understood. Here, the authors reveal increased MPXV replication and immune dysfunction in SIV-MPXV co-infected rhesus macaques and, using proteomics and phosphoproteomics from multiple organs, identify involved pathways. Introduction Mpox (formerly known as monkeypox), previously considered endemic to several Western and Central African countries^[56]1, has been declared a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) for the second time^[57]2. The ongoing outbreak also prompted the Africa Centers for Disease Control and Prevention (Africa CDC) to declare its first-ever public-health emergency on 13^th August^[58]3. Before this, WHO had declared mpox a PHEIC by on July 23, 2022^[59]4. Mpox has sparked a global public health crisis, with over 117,600 cases and over 200 deaths reported^[60]5. In the Democratic Republic of the Congo (DRC) alone, approximately 18,000 suspected cases, many involving children^[61]6, and at least 600 deaths potentially attributable to the disease in 2024^[62]7,[63]8. Only one week in September 2024, more than 100 mpox-related deaths were recorded across African countries^[64]9. Efforts to contain the virus have been complicated by the emergence of viral variants^[65]10,[66]11, the lack of effective and widely accessible vaccines^[67]7,[68]12, and dashed hope of tecovirimat against a spreading type of mpox virus (MPXV)^[69]13, increasing the challenge of curbing further transmission both in Africa and globally. The co-infection of human immunodeficiency virus (HIV) and MPXV significantly increases the complexity of the disease and the burden on public health. During both the 2022 mpox outbreak and its recent resurgence, high transmission rates were recorded among sex workers and men who have sex with men (MSM)^[70]14,[71]15. These populations also suffer a higher risk of other sexually transmitted diseases, particularly HIV infection. Their high rates of immunosuppression make them more vulnerable to MPXV infection and associated mortality^[72]16–[73]18. In DRC, HIV prevalence is estimated at 7.5% among sex workers and 7.1% among MSM^[74]19. During the 2022 mpox outbreak, approximately 40% of MPXV cases in the MSM population were co-infected with HIV^[75]15. Similarly, an investigation in Shenzhen, China, reported about 56.5% (52/92) of enrolled mpox cases were also diagnosed with HIV^[76]11. The larger skin lesions, more prolonged illness, and higher rates of genital ulcers and bacterial superinfection were observed in the populations infected with HIV-1 and MPXV when compared to HIV-negative cases^[77]1. Notably, 15% mortality in patients with HIV-related disease characterized by CD4^+ T cell counts below 200 cells per mm³ were uncovered in a large case series^[78]17. Moreover, the safety and tolerability of drugs and vaccines targeting mpox in immunocompromised populations have also raised concerns among the public. Given the epidemiological overlap between mpox and HIV infection, the possible serious consequences of co-infection, and the limitations of medication and vaccines for HIV-infected individuals, it is both meaningful and necessary to explore the differences in symptoms, underlying mechanisms, and treatment targets between co-infected individuals and those with mpox infection alone. Omics analysis has recently become a crucial tool in studying pathogens, providing essential information for viral prevention and drug development, as demonstrated with ZIKV and SARS-CoV-2. The integrated proteomics approach, including proteome and phosphoproteome, used in research associated with ZIKV identified the proteins, the host factors involving the cellular responses to viral infection and phosphorylation sites that are specifically up- or down-regulated after ZIKV infection^[79]20. In the global responses to the COVID-19 pandemic, multi-omics (interactome, proteome, transcriptome, and bibliome) data and subsequent integrated analysis were used to identify biomarkers for understanding the pathogenesis of severe COVID-19, providing the hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies^[80]21–[81]24. Such information forms a central pillar of global pandemic preparedness programs^[82]25. However, there remains a significant gap in omics research on MPXV. Few studies exist, including limited transcriptomic analyses^[83]26–[84]29, a plasma proteomic study of MPXV-infected patients^[85]30, a pulmonary fluid proteomic study of MPXV-infected macaques^[86]31, and a multi-omics study of MPXV infection in primary human cells in vitro^[87]32. The plasma proteomic study revealed correlations between plasma proteins and disease severity, identifying strong responses among nutritional and acute-phase proteins^[88]30. Increased expression of inflammatory proteins was observed in response to MPXV in the pulmonary fluid proteomic study of MPXV-infected macaques^[89]31. Meanwhile, multi-omics studies uncovered regulation of the HIPPO and TGF-β pathways, dynamic phosphorylation of H5 affecting its binding to dsDNA, and the role of MAPKs as key regulators of differential phosphorylation in MPXV-infected cells^[90]32. Despite these findings, there is still a lack of proteomic data directly examining MPXV-induced tissue damage in vivo. Furthermore, no comprehensive studies have yet compared the multi-organ molecular and pathological differences between individuals infected with MPXV alone and those co-infected with both HIV and MPXV. Using proteomics and phosphoproteomics analysis to predict kinase targets can provide effective candidate drugs for screening antiviral agents against the monkeypox virus. To understand how HIV-MPXV co-infection leads to more severe clinical outcomes than MPXV infection alone, we inoculated rhesus macaques with simian immunodeficiency virus (SIV) to model HIV infection in humans. Furthermore, we developed rhesus macaque models for both SIV-MPXV co-infection and MPXV infection alone. Macaques co-infected with SIV-MPXV exhibited more extensive skin lesions than those with MPXV infection alone. In-depth proteomic and phosphoproteomic analyses revealed distinct protein and phosphosite profiles in the skin lesions of co-infected versus singly infected macaques. Further proteomic and phosphoproteomic analyses across eight other organs (lymph node, spleen, cerebral cortex, lung, heart, liver, kidney, and rectum) showed that SIV-induced systematic dysfunction in MPXV-infected macaques. These findings enhanced our understanding of MPXV biology and provide valuable datasets for further MPXV study. Results Chronic SIV-infected rhesus macaques develop increased lesions following MPXV infection To mimic mpox cases with and without HIV co-infection and compare their pathogenicity, we enrolled six rhesus macaques with chronic simian immunodeficiency virus (SIVmac239) infection into the SIV-MPXV (MS) group and six naïve macaques into the MPXV-alone (MP) group (Fig.[91]1a and Supplementary Data [92]S1). Prior to the MPXV challenge, the six macaques in the MS group were intravenously inoculated with 100 TCID[50]of SIVmac239 and monitored for plasma viral loads over 115 days. During this period, all six macaques exhibited persistent SIV replication in peripheral blood (Supplementary Fig. [93]S1a and Fig.[94]1b) along with a decreased CD4^+/CD8^+ T cell ratio and CD4^+ T cell counts (Supplementary Fig. [95]S1b), confirming the successful establishment of a chronic and stable SIV infection model. Subsequently, all twelve macaques were intravenously challenged with 1 × 10^7 TCID[50] of MPXV clade IIb (MPXV-B.1-China-C-Tan-CQ01), isolated from the first imported mpox case in China in 2022. Fig. 1. Study design and comparison of skin lesions in rhesus macaques from the MPXV and SIV-MPXV groups. [96]Fig. 1 [97]Open in a new tab a Study design. Group A (MS, SIV-MPXV, n = 6) and Group B (MP, MPXV, n = 6). b SIV RNA and MPXV DNA loads in the plasma of monkeys before (n = 6) and after 10 dpi (n = 3). The shaded areas within the dashed lines indicate the standard deviation (SD). c,d Skin lesion counts (n = 6 before 10 dpi, n = 3 after 10 dpi) and duration from onset to resolution. Statistical differences between groups were analyzed using two-way ANOVA. ****P < 0.0001. The exact P values are both P < 0.0001 on 10 dpi and 14 dpi (c). The solid triangles indicate the time of skin lesion appearance and healing (d). e Images of skin lesions in monkeys from the MP and MS groups at 10 dpi. f Skin lesion counts at different regions in the MP and MS groups at 10 dpi (n = 6). Statistical differences between groups were analyzed using two-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, **P < 0.01. The exact P-values are: P = 0.0302 in the limbs and P = 0.0068 in the pygal. g MPXV DNA loads in skin lesions in the MP and MS groups (n = 3). Statistical differences were analyzed by a two-tailed unpaired t test. **P < 0.01 (P = 0.0022). h Histopathology of skin lesions in macaques after the MPXV challenge. The black arrows represent necrotic cell fragments and granulocytes, the gray arrows refer to fibrous cell necrosis, the purple arrows indicate inflammatory cell infiltration mainly composed of lymphocytes and granulocytes, the orange arrows point to an increase in the number of spinous cell layers, the blue arrows show loose arrangement of connective tissue, and the dark blue arrows show mild edema. Data are shown as the mean ± SD (b, c, f, g). The ‘n’ represents the number of independent biological replicates. Source data are provided as a Source Data file. After the MPXV challenge, three macaques in the MP and MS group were monitored for 10 days and euthanized, while another three macaques in either group were continuously monitored for 35 days to measure viral loads in plasma and tissues, binding and neutralizing antibodies, cellular immune response, and cytokine levels. MPXV DNA copies in both the MP and MS groups exhibited a peak on days 7 and 10, reaching 4.18 × 10^5 and 5.89 × 10^5 copies/mL, respectively. These levels are comparable to those reported in previous studies of MPXV infection in non-human primates^[98]33,[99]34, suggesting a typical viral replication pattern during the acute phase of the infection. Notably, despite the absence of significant differences in MPXV plasma loads between the two groups throughout the 35-day observation period (Fig.[100]1b), the chronic SIV co-infection was associated with more severe skin lesions and other pathological manifestations. This indicates that factors other than plasma viral load may contribute to the enhanced pathogenicity in the co-infected animals. Deaths were not observed in either group (Supplementary Fig. [101]S1c), and only mild weight loss was noted, with no significant differences between the two groups (Supplementary Fig. [102]S1d). The body temperature of all twelve macaques peaked significantly at four days post-MPXV infection (dpi) and then returned to levels similar to those at one dpi, with no significant differences between the MP and MS groups (Supplementary Fig. [103]S1e). These findings suggest that while the overall systemic manifestations, such as weight loss and fever, were not markedly affected by SIV co-infection, the local tissue damage, as evidenced by the skin lesions, was more pronounced. Skin lesions are the most common and obvious symptom of MPXV infection, and close contact with skin lesions mainly leads to human-to-human transmission of MPXV. We continuously monitored lesion counts at 1, 4, 7, 10, 14, 21, 28, and 35 days post-MPXV infection. In both groups, skin lesions were first observed at 7 dpi and reached a peak at 10 dpi (Fig.[104]1c). Notably, the macaques of MS group showed more skin lesion than those in the MP group on day 10 (group mean: 50.17 vs. 20.33) and 14 (group mean: 49.00 vs. 14.00) post-MPXV challenge (P < 0.0001 for all comparison). Disease duration was measured as the time from lesion onset to resolution and was charted for each individual animal (Fig. [105]1d). Lesions were distributed across all limbs, pygal region, soles, torso, and face of the macaques in both the MS and MP groups (Fig. [106]1e). Significant more lesions appeared on the limbs and pygal region of the macaques in the MS group than the MP group at 10 dpi. Although not statistically significant, other body parts also exhibited more lesions in the MS group (Fig. [107]1f). The increased number of skin lesions observed in the macaques with SIV-MPXV co-infection was similar to the higher incidence of skin lesions seen in HIV-positive patients infected with MPXV compared to those without HIV^[108]1. This may be due to the insufficient immune response in SIV-infected macaques, which fails to control MPXV replication. As a result, the virus spreads rapidly throughout the body, leading to more severe skin lesions. In addition, the higher MPXV DNA loads found in the skin lesions of the MS group than those in the MP group (P < 0.01, Fig. [109]1g) further confirm this. To assess the severity of pathological changes in the skin lesions of rhesus monkeys, skin samples from the limbs were collected, fixed, sectioned, stained with hematoxylin and eosin (H&E), and analyzed histopathologically. Compared to normal skin, skin lesions in both the MP and MS groups had extensive increases in the spinous cell layers, moderate to severe epidermal thickening, and loosening of connective tissue on the skin surface. However, skin lesions in the MS group displayed additional signs of pathological damage, including spinous cell necrosis, fibroblast necrosis in the dermis, nuclear condensation and fragmentation, and increased inflammatory cell infiltration (primarily lymphocytes and granulocytes) compared to the MP group (Fig. [110]1h). In summary, although there were no significant differences in MPXV plasma viral loads, survival, or body temperature, chronic SIV co-infection resulted in more severe skin lesions than MPXV infection alone in rhesus macaques. Protein and phosphosite profilings of skin lesions exhibit differences between SIV-MPXV co-infection and MPXV single infection rhesus macaques Disruptions in translation and proteostasis are frequently observed characteristics of pathogenic viruses. For a preliminary glance at SIV-MPXV co-infection induced molecular changes, limb skin lesions collected at 10 days post-MPXV infection from both the MP and the MS groups, and samples from eight other organs, were analyzed using liquid chromatography-mass spectrometry (LC-MC)-based proteomic and phosphoproteomic techniques. A total of 11,527 proteins (derived from 113,415 peptides) and 26,582 phosphosites (24,576 phosphopeptides) in 6,084 proteins were identified. To ensure high reliability, only proteins/sites present in > 50% of the samples in at least one group were included, resulting in 11,457 proteins and 26,086 phosphosites from 6056 proteins for further analysis. For lesions from each macaque, paired normal adjacent skin was named peripheral lesion (MP_PL or MS_PL) and used as control samples (Fig. [111]2a). In the macaque skin (lesion and PL), a total of 9554 proteins (derived from 65,589 peptides) and 18,565 phosphosites (16,725 phosphopeptides) were identified, and 8028 proteins and 13,718 sites were considered highly reliable. Principal component analysis (PCA), a basic unsupervised clustering method, was used to evaluate differences among groups. Though there was overlap among skin lesion and PL groups, MP_lesion and MS_lesion groups were mostly separated at protein expression level (Supplementary Fig. [112]S2a) and were totally separated at the phosphosite expression level (Supplementary Fig. [113]S2b), revealing skin lesions had different protein and phosphosite expression profilings. Comparing to MP_PL, lesions in the MP group had 29 upregulated and 26 downregulated proteins (Supplementary Fig. [114]S2c and Supplementary Data [115]S2). Comparing to MS_PL, lesions in the MS group had 67 upregulated and 29 downregulated proteins (Supplementary Fig. [116]S2d and Supplementary Data [117]S2). Combining protein alterations into a scatter plot, it is obvious that proteins only upregulated in the MS group (66 proteins) were much more than proteins only upregulated in the MP group (28 proteins; Fig.[118]2b and Supplementary Data [119]S2). Using KEGG enrichment analysis, we identified that in the MS group, proteins associated with the Rap1 signaling pathway were especially upregulated. The severe immunosuppression and chronic immune activation induced by SIV infection^[120]35,[121]36 may promote enhanced recruitment of inflammatory cells (e.g., neutrophils, monocytes/macrophages) to the skin lesions of the MS group. Activation of the Rap1 signaling pathway facilitates adhesion and migration of these cells^[122]37, potentially exacerbating local inflammatory infiltration and tissue destruction. Proteins only upregulated in the MP group were functionally enriched in the IL-17 signaling pathway, arachidonic acid metabolism, PPAR signaling pathway, et al. (Fig. [123]2c and Supplementary Data [124]S2). Proteins only downregulated in the MP group were functionally enriched in carbohydrate digestion and absorption, adipocytokine signaling pathway, thyroid hormone signaling pathway, and apoptosis (Fig. [125]2c and Supplementary Data [126]S2). These features in the skin lesions of the MP group reflected a more classic viral skin inflammatory response: robust IL-17-mediated neutrophil recruitment and barrier defense^[127]38, coupled with active arachidonic acid metabolism generating pro-inflammatory lipid mediators^[128]39. Concurrently, PPAR pathway activation serves to initiate anti-inflammatory responses and tissue repair^[129]40. Downregulation of metabolic pathways indicates energy redistribution toward glycolytic energy production and inflammatory/antiviral responses in infected and immune cells^[130]41. Based on the protein-protein interaction (PPI) annotations, PTGS2 (regulating arachidonic acid metabolism) showed regulatory potential in mpox infection because of its interaction with multiple proteins (Fig. [131]2d). Fig. 2. Proteomic and phosphoproteomic differences in skin lesions of rhesus macaques between the MP and MS groups. [132]Fig. 2 [133]Open in a new tab a Schematic diagram of skin lesion sampling. ‘MP’ represents the MPXV-alone group, ‘MS’ refers to the SIV-MPXV group, and ‘PL’, peripheral lesion, indicates the adjacent normal skin tissues. The lesions used for omics analysis were collected from the limbs of the monkeys. Created in BioRender. Yang, C. (2025) [134]https://BioRender.com/v95xk7r. b Scatter plots showing differentially expressed skin lesion proteins in the MP/control (Ctr) and MS/Ctr comparison groups. According to fold changes and Student’s t test P-adjust values (two-sided) in the MP/Ctr and MS/Ctr comparison groups, nine protein expression modes were identified. See also Supplementary Data [135]S2. c KEGG enrichment results (Fisher’s exact test P < 0.05, two-sided) for proteins in each expression mode in (b). See also Supplementary Data [136]S2. d PPI networks of proteins in the significantly enriched pathways in (c). e,f Kinase prediction results comparing MP to Ctr (e) or MS to Ctr (f) skin lesions. Kinases were predicted using GPS software (setting organism at Macaca mulatta and threshold at medium). Kinase activities were predicted using GSEA. Red indicates (GSEA P < 0.05 and NES > 1) activated kinases, while blue indicates inhibited ones (GSEA P < 0.05 and NES < − 1). See also Supplementary Data [137]S4. g Enriched motifs among kinase substrate phosphosites. Reliable phosphosites were submitted to GPS software to identify kinase-substrate relationships. Peptide sequences containing all kinase substrate phosphosites were uploaded to MoMo (Motif-x algorithm) to identify motifs. Phosphorylation is one of the most pivotal biological mechanisms for regulating cellular processes. Given that nearly all cell signaling pathways rely on phosphotransfer reactions and that dysregulated kinase activity is implicated in numerous diseases, kinases represent an appealing target for therapeutic intervention^[138]42,[139]43. Therefore, we also conducted the phosphorylation analysis on multi-organ samples of rhesus macaques with SIV-MPXV co-infection and MPXV infection alone. Comparing the MP_lesion to the MP_PL samples, 137 upregulated phosphosites located in proteins regulating phagosome, protein export, neutrophil extracellular trap formation, platelet activation, C-type lectin receptor signaling pathway, IL-17 signaling pathway, and tight junction (Supplementary Fig. [140]S2e and Supplementary Data [141]S3). The detected protein phosphorylation suggests significant inflammatory responses and the reprogrammed host’s baseline gene expression pattern, which may enhance the efficiency of viral genome transcription and translation, thus promoting virus propagation within infected cells^[142]44,[143]45. The proteins of MP_lesion containing 75 downregulated phosphosites were associated nucleocytoplasmic transport and thermogenesis (Supplementary Fig. [144]S2e and Supplementary Data [145]S3). Compared to the MS_PL group, the MS_lesion group had 204 upregulated and 81 downregulated phosphosites. In the MS_lesion groups, proteins containing upregulated phosphosites were functionally enriched in chemokine signaling pathway, Fc gamma receptor-mediated phagocytosis, B cell receptor signaling, endocytosis, neutrophil extracellular trap formation, platelet activation, natural killer cell- mediated cytotoxity, NF-kappa B signaling pathway, and C-type lectin receptor signaling pathway. Proteins containing downregulated phosphosites were functionally associated with toll and immune deficiency signaling pathway (Supplementary Fig. [146]S2f and Supplementary Data [147]S3). Although the pronounced upregulation of phosphosites within these signaling pathways may significantly bolster the host’s antiviral immune responses, the overall effectiveness of these mechanisms might be attenuated due to the profound immune dysregulation caused by HIV infection, especially the marked impairment of CD4^+ T-cell functionality. Referring to reported kinase-phosphosite relationships, kinase predication was performed based on phosphosite expression intensity. CDK1, FYN, TTK, LYN, SRC, YES1, CSK, GRB2, and ZAP70 were predicted to be activated, while CAMK2D, ILK, GSK3B, CAMK2D, CAMK2G, PRKG1, PRKACA, and PRKACG were inhibited in the MP skin lesions (Fig. [148]2e and Supplementary Data [149]S4). CDK1, CAMK2A, AURKB, ILK, AURKA, PLK1, TTK, and PLK4 were predicated to be activated while PIK3CD, SGK1, SGK3, RIMS1, and PLK3 were inhibited in the MS skin lesions (Fig.[150]2f and Supplementary Data [151]S4). CDK1 and TTK were predicted to be activated in both the MP_lesion and MS_lesion groups. However, CAMK2A and ILK were predicted to have opposite activity in the MP_lesion and MS_lesion groups. Eight motifs were summarized among reliable phosphosites, showing proline, arginine, lysine, and glutamate residues most commonly appeared near phosphosites (Fig. [152]2g). Referring to drug-protein relationships recorded in DrugBank, several Food and Drug Administration (FDA)-approved drugs targeting identified kinases in MPXV-induced skin lesions were listed (Supplementary Fig. [153]S2g and Supplementary Data [154]S4). These aforementioned therapeutic agents hold potential to provide novel avenues for the treatment of mpox. In summary, MPXV induced distinct protein expression profiles in skin lesions depending on the presence or absence of SIV co-infection. Chronic SIV infection enhances MPXV replication and caused systematic dysfunction in MPXV-infected macaques To understand whether SIV only exacerbates local skin lesions or widely induces systemic dysfunction in MPXV-infected macaques, samples from lymph nodes, immune tissues, the gastrointestinal tract, and other organs were collected for viral load measurements. Compared to the MP group, the MPXV DNA loads in various organs were significantly higher in the MS group, which was different from the phenomena that both the MP and MS groups had similar levels of viremia (Fig. [155]3a). For further molecular investigation, proteomic and phosphoproteomic data of eight organs, including inguinal lymph node (ILN), spleen, cerebral cortex (CC), heart, lung, liver, kidney, and rectum were processed for analyses (Fig. [156]3b). To establish baseline data for the study, we selected another three healthy rhesus monkeys (group Control) in this section of the experiment and conducted proteomic and phosphogenomic analysis on the multiple organs. Compared to MPXV-infected alone, MPXV loads in SIV-MPXV co-infection macaques were significantly increased in the heart, lung, kidney, and rectum and had upward trends in the brain and liver (Supplementary Fig. [157]S3a). Notably, MPXV burden in the inguinal lymph node and spleen of the MS group macaques were both 1.2 times those in the MP group macaques (Supplementary Fig. [158]S3b). For other organs, compared to macaques infected with MPXV alone, macaques co-infected with SIV-MPXV appear to have a higher burden of MPXV (Supplementary Fig. [159]S3c). In the eight organs for proteomic analysis, 11,481 proteins (112,559 peptides) and 26,326 phosphosites (24,352 phosphopeptides) were identified, and 11,357 proteins and 25,688 sites were considered highly reliable. MPXV single infection or SIV-MPXV co-infection had no significant effect on the count of identified proteins and phosphosites (Fig.[160]3c,[161]d). However, SIV-MPXV organs commonly had more downregulated proteins and phosphosites than MPXV-alone ones (Fig.[162]3c and Supplementary Data [163]S5). Unsupervised clustering revealed that the tested eight organs were clearly separated based on LC-MS-identified proteins (Supplementary Fig. [164]S3d) and phosphosites (Supplementary Fig. [165]S3e), revealing that each organ had its unique characteristics. Compared to the control macaques, each organ had its unique upregulated and downregulated proteins in the MP (Supplementary Fig. [166]S4a and Supplementary Data [167]S6) and MS (Supplementary Fig. [168]S4b and Supplementary Data [169]S6) groups. To focus on the effect of chronic SIV infection on MPXV-infected macaques, proteins of each organ in the MS group were directly compared to the MP group, and proteins that were unique upregulated or downregulated in each organ were extracted for KEGG enrichment analyses (Fig. [170]3e). Compared to the MP group, we identified that the cerebral cortex had upregulated neurotrophin signaling pathway, apoptosis, and neutrophil extracellular trap formation, the heart had downregulated oxytocin signaling pathway, the kidney had downregulated ECM-receptor interaction, the liver had upregulated protein digestion and absorption and downregulated RNA polymerase, the lung had downregulated ErbB signaling pathway, base excision repair, longevity regulating pathway, Th1 and Th2 cell differentiation, and notch signaling pathway, and the rectum had upregulated N-glycan biosynthesis in the MS group (Fig. [171]3e and Supplementary Data [172]S6). These disturbances indicated that chronic SIV infection exacerbates the burden on multiple tissues of rhesus monkeys infected with MPXV. Fig. 3. Multi-organ proteomic and phosphoproteomic analysis. [173]Fig. 3 [174]Open in a new tab a MPXV DNA loads in multiple organs sampled from rhesus macaques necropsied at 10 days post-MPXV infection (dpi). The intensity of blue in the heatmap indicates the level of MPXV DNA load in each organ. The organs were categorized into four classes: lymph nodes, immune tissues, gut, and other tissues. b Study design for the proteomic and phosphoproteomic analysis using eight organs in the MP and MS groups. Created in BioRender. Yang, C. (2025) [175]https://BioRender.com/3zt44aw. c Reliable proteins in eight organs of MP and MS groups. Differentially expressed proteins were calculated using Student’s t test (two-sided, thresholds set at P-adjust < 0.05 and FC > 1.5 or FC > (1/1.5)) in each organ comparing MP to Ctr or MS to Ctr. d Reliable phosphosites in eight organs of the MP and MS groups. Differentially expressed phosphosites were calculated using Student’s t test (two-sided, thresholds set at P-adjust < 0.05 and FC > 1.5 or FC > (1/1.5)) in each organ comparing MP to Ctr or MS to Ctr. e KEGG enrichment results (Fisher’s exact test P < 0.05, two-sided) for unique upregulated or downregulated proteins in each organ comparing the MS to MP group. The red and blue arrows at the bottom of the panel respectively indicate the upregulated and downregulated proteins in the corresponding columns. f Kinase prediction results comparing the MS to the MP group in each organ. Kinases were predicted using GPS software (setting organism at Macaca mulatta and threshold at medium). Kinase activities were predicted using GSEA. The ‘*’ symbol indicates the marked kinase had significantly changed activity (GSEA P < 0.05 and |NES | > 1). Source data are provided as a Source Data file. Using the phosphosite expression levels, kinases were predicted in each organ in the MP and MS groups (Fig. [176]3f, Supplementary Fig. S4c, d). comparing the MS to MP groups, the observed differences, particularly in cell cycle control (PLK1, PLK4, AURKA, AURKB, CDK1, BUB1, BUB1B, TTK, NEK2), immune signaling (SYK, ZAP70, LYN, FYN, SRC, YES1, GRAP2), PI3K/MTOR pathways, and cytoskeletal organization (CAMK2A, CAMK2G, ILK, SRC), strongly suggested alterations in cellular proliferation, immune responses, metabolic homeostasis, and structural integrity. In summary, although exacerbated skin lesions were nearly the only apparent physical signs, chronic SIV infection caused systemic dysfunction, promoted inflammatory responses, and affected cell mitosis and cytoskeleton regulation. Chronic SIV infection impairs the immune responses and functional heterogeneity in the spleen and lymph node In the absence of antiviral treatment (ART), HIV infection is characterized by the gradual loss of CD4^+ T cells and progressive immune deficiency that leads to opportunistic infections, and ultimately death^[177]46. Early CD8-cell depletion studies in experimentally SIV-infected rhesus macaques demonstrated that CD8^+ T cells are critical for controlling virus replication in vivo^[178]47,[179]48. Cellular immune responses against cross-reactive orthopoxviruses of rhesus monkeys were evaluated at 28 dpi to understand the impact of chronic SIV infection. The vaccinia virus (VACV), a poxvirus with 90% sequence homology to MPXV^[180]49, was used to assess specific cellular immune responses, as its induced T-cell response is largely cross-reactive with MPXV epitopes. Flow cytometry was performed to analyze VACV-specific cellular immune responses in the MP and MS groups. The results showed that stimulation of peripheral blood mononuclear cells (PBMCs) with VACV resulted in an obvious decrease in the proportion of VACV-specific CD8^+ T cells producing interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) in the MS group compared to the MP group (Fig. [181]4a and Supplementary Fig. [182]S5a). In contrast, there were no significant differences in the proportion of IFN-γ, IL-2, and TNF-α secreting CD4^+ T cells between the MP and MS groups. These findings indicate that chronic SIV infection impairs specific CD8^+ T cell responses against MPXV in rhesus monkeys, while having no significant effect on CD4^+ T cell immunity. Fig. 4. Impaired humoral and cellular immune responses to MPXV infection in chronically SIV-infected rhesus macaques. [183]Fig. 4 [184]Open in a new tab a VACV-specific CD4^+ and CD8^+ T-cell immune responses measured by TNF-α, IFN-γ, and IL-2 intracellular cytokine staining (ICS) assays at 28 days post-MPXV infection (dpi, n = 3). Statistical differences were analyzed using two-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05, ****P < 0.0001. The exact P-values of IFN-γ, IL-2, and TNF-α level differences in CD8^+ T cell immune responses between MPXV (MP) and SIV-MPXV (MS) groups are: P < 0.0001, P = 0.0107 and P < 0.0001. b Neutralizing antibody titers against MPXV of the MP and MS groups (n = 6 before 10 dpi, n = 3 after 10 dpi). Statistical differences were analyzed using two-way ANOVA and Šídák’s multiple comparisons test (*P <  0.05, P = 0.0139). c Plasma cytokine and chemokine levels of the MS and MP groups at 10 dpi, and of SIV-infected alone macaques at two months post-SIV infection with stable SIV viral load (n = 6). Statistical differences were analyzed using two-way ANOVA with Tukey’s correction for multiple comparisons. **P < 0.01, *** P < 0.001, ****P < 0.0001. The exact P-values were shown in the Supplementary Notes for the Figure legends of Fig.4c. d,e Expression trends of differentially expressed proteins (one-way ANOVA P < 0.05) among control (Ctr), MP, and MS groups in the inguinal lymph node (ILN, d) and spleen (e). f,g Kinase prediction results among Ctr, MP, and MS groups in the ILN (f) and Spleen (g). Kinases were predicted using GPS software (setting organism at Macaca mulatta and threshold at medium). Kinase activities were predicted using GSEA. The green to red color indicates kinase activity (GSEA NES values). h Sankey plots showing relationships of drugs and kinases in the ILN and spleen. Red symbols indicate activated kinases, while blue symbols indicate inhibited ones. Data are shown as the mean ± SD (a–c). The ‘n’ represents the number of independent biological replicates. Source data are provided as a Source Data file. To further assess the impact of chronic SIV infection on humoral immune responses against MPXV infection, the plasma neutralizing antibody levels against MPXV were initially measured using a 50% plaque reduction neutralization titer (PRNT[50]). Neutralizing antibodies against MPXV peaked at 28 dpi (Fig. [185]4b). The antibody titer in plasma from the MS group macaques was significantly lower than that in the MP group (1:59 vs. 1:100, P < 0.05), indicating an impaired humoral immune response against MPXV due to chronic SIV infection. By 180 dpi, the PRNT[50] neutralizing antibody titers were 1:46 and 1:22 in the plasma of the MP and MS groups, respectively, reflecting the persistence of humoral immunity after MPXV infection. In addition, total IgG antibody levels in plasma collected at 28 dpi were tested against six MPXV antigens by ELISA, including A35, B6R, H3L, M1R, A29, and E8L. The results showed no significant difference in specific binding antibodies to these six key MPXV antigens between the two groups (Supplementary Fig. [186]S5b). Plasma cytokine and chemokine levels in plasma at 10 dpi of the naïve, SIV-alone, MPXV-alone, and SIV-MPXV macaques were also detected. The MS group monkeys had significantly higher levels of B cell activating factor (BAFF), C-X-C motif chemokine 10 (CXCL10), and C-C motif chemokine ligand 5 (CCL5) compared to both the MP and SIV groups (P < 0.05 for all comparisons, Fig. [187]4c). The plasma level of suppression of tumorigenicity 2 (ST2) in the MS group was higher than that in the MP group but lower than in the SIV group, indicating that MPXV infection inhibits ST2 production (P < 0.05 for all comparisons). In addition, the growth/differentiation factor-15 (GDF-15) showed no significant difference between MPXV-infected monkeys with or without SIV infection (P > 0.05). We found high levels of IL-6 and IFN-γ in the plasma of macaques infected with SIV-MPXV, and the concentrations of C-reactive protein (CRP) and serum amyloid A1 (SAA1) in the MS group were 1.7- and 1.2-fold those in the MP group, respectively, which showing intense inflammatory responses in the co-infected macaques. The significantly low levels of cellular and humoral immunity against MPXV in the macaques highlight the substantial damage caused by SIV and MPXV to the immune system. Further combining the proteomic analyses of immune organs, differentially expressed proteins were calculated among control, MP, and MS macaques for ILN and spleen, respectively. Then, differentially expressed proteins were separated into eight expression clusters in the ILN (Fig. [188]4d) and seven clusters in the spleen (Fig. [189]4e), respectively. In the lymph nodes, control, MP, and MS macaques had gradually decreased T cell receptor signaling pathway and Th1, Th2, and Th17 cell differentiation (cluster 4). Adhesion pathways were downregulated in the MS ILN (cluster 5) but were not decreased in the MP ILN (cluster 1; Fig. [190]4d and Supplementary Data [191]S7). In the spleen, the control, MP, and MS groups had gradually higher complement and coagulation cascades, NF-kappa B signaling pathway, RIG-I-like receptor signaling, pyrimidine metabolism, and natural killer cell-mediated cytotoxicity (cluster 1) and gradually downregulated ECM-receptor interaction and focal adhesion (cluster 6). DNA replication seemed enhanced in both the MP and MS spleens (cluster 7; Fig. [192]4e and Supplementary Data [193]S7). Researchers have also confirmed the downregulation of cell-cell adhesion molecules in cells infected with MPXV, which may lead to the entry of MPXV into cells during the infection process^[194]32. Comparing the differentially expressed phosphosites between MP/control (Ctr) and MS/Ctr, both lymph node and spleen showed unique up- and down-regulated phosphosites in the MP and MS groups (Supplementary Fig. [195]S5c, [196]d and Supplementary Data [197]S8). In the lymph node, MPXV infection alone especially upregulated phosphosites associated with mTOR signaling pathway and AMPK pathway, while SIV-MPXV co-infection especially upregulated phosphosites associated with DNA replication, ribosome, and protein processing (Supplementary Fig. [198]S5e and Supplementary Data [199]S8). In the spleen, the upregulated protein in the MP group were associated with ribosome biogenesis in eukaryotes, homologous recombination, and antigen processing and presentation. While the MS group showed more upregulated proteins related to the regulation of actin cytoskeleton, proteasome, and arginine and proline metabolism (Supplementary Fig. [200]S5fand Supplementary Data [201]S8). Using kinase prediction, the kinases (PLK1, PLK2, CDK3, CDK4, CDK6, CDK16, and CDK18) associated with DNA replication were identified to be activated in lymph node and spleen in both MP and MS groups (Fig. [202]4f, [203]g and Supplementary Data [204]S9). The MAPK3 was especially activated in the spleen in the SIV-MPXV co-infection group (Fig. [205]4g and Supplementary Data [206]S9). Based on DrugBank, drugs targeting altered kinases in the MP or MS immune organs were also listed (Fig. [207]4h and Supplementary Data [208]S9). Discussion As an epidemic declared a PHEIC twice by the WHO^[209]2,[210]4, the mpox outbreak poses significant health risks, including death, in Africa^[211]9,[212]50–[213]52 and other affected regions^[214]5,[215]17. HIV infected individuals might exhibit serious infection consequences due to immunocompromised^[216]1,[217]17,[218]53–[219]56, complicating the clinical management of MPXV and raising significant challenges for treatment. Despite reports of HIV-infected individuals in varying advancements contracting MPXV from different countries, no widely accessible animal model recapitulates well the disease progression observed in patients. Herein, we established rhesus macaque models of both SIV-MPXV co-infection and MPXV infection alone to mimic human infection conditions. Chronic SIV infection made more severe symptoms in rhesus macaques infected with SIV-MPXV co-infection compared to those infected with MPXV alone, including more counts and severe lesions (Fig. [220]1c–h), higher MPXV loads in the multi organs (Figs. [221]1g, [222]3a, Supplementary Fig. [223]S3a–c), and impaired humoral and cellular immune responses against MPXV infection (Fig. [224]4a, [225]b). Our animal models simulated the immune status of people living with HIV (PLWH) who have not undergone ART. It suggests that the diagnosis and treatment of untreated or uncontrolled HIV viral load patients co-infected with MPXV need to be strengthened, as their immune suppression caused by HIV infection may result in a loss of normal immune response to MPXV invasion. Fortunately, in those PLWH with normal immune function or suppressed HIV, clinical presentations, complications and prognosis were reported to be similar^[226]57, and the levels of MPXV-specific binding and neutralizing antibodies, and MPXV-specific T cell response were comparable^[227]58,[228]59 with those without HIV infection. This suggests that in clinical practice, more finely tailored treatment strategies may be required for HIV-infected patients co-infected with the MPXV, taking into account their varying HIV virus suppression statuses. We provided the first proteomic and phosphoproteomic profiling of multi-organ in rhesus macaques of MPXV infection with and without SIV. Due to the low mortality rate of MPXV clade IIb^[229]11,[230]60, most omics research on MPXV has been limited to in vitro cell experiments and the peripheral blood of infected individuals^[231]31,[232]32. Proteomic profiling provides detailed insights into protein expression levels, post-translational modifications, and dynamic changes in host cells during viral infection, which relies on advanced proteomic analysis techniques. In this study, we observed that SIV-MPXV co-infection specifically induced the regulation of numerous key pathways involved in various aspects of the viral life cycle and host homeostasis (Figs. [233]2c, [234]3e). And each organ displays its unique protein expression landscapes. Based on cytokine/inflammatory factor profiling and multi-organ proteomic and phosphoproteomic analyses, we demonstrated that MPXV infection triggers systemic immune-inflammatory responses across multiple organs, distinct from the single-target-organ pathology. This systemic inflammatory response involves the dysregulated expression of multiple related pathway proteins across multiple organs. IL-17 pathway-associated proteins were found to be significantly upregulated in the skin lesions of the MPXV infection alone, but no abnormality was detected in the SIV-MPXV co-infection group (Fig. [235]2c, [236]d). IL-17 can stimulate epithelial cells to produce antimicrobial peptides, defensins, and mucins, thereby enhancing the integrity of the skin barrier and resisting viral invasion^[237]61,[238]62. It is also a potent chemotactic factor and activating protein for neutrophils^[239]63,[240]64, indicating a robust immune response mounted by epithelial cells upon MPXV infection. However, the persistent chronic immune activation and inflammatory state induced by SIV infection may lead to the functional exhaustion of T helper (Th17) cells in rhesus macaques, rendering them hyporesponsive or unresponsive to stimulation by the MPXV. In this study, we also observed a significant decrease in the expression of proteins related to Th17 cell differentiation in the inguinal lymph nodes of rhesus monkeys infected with SIV (Fig. [241]4d). This lack of effective IL-17-mediated early defense might allow for less controlled viral replication. In addition, MAPK3 was predicted to be activated in the spleen of the SIV-MPXV co-infection group (Fig. [242]4g). This kinase promotes the production of inflammatory cytokines (e.g., IL-6, CXCL10), forming a positive feedback loop with pre-existing hyperinflammatory phenotype (Fig. [243]4c) that further depletes normal immune function. More proteins containing upregulated phosphosites enriched in inflammation related pathways, such as chemokine signaling pathway, Fc - γ receptor-mediated phagocytosis, and B-cell receptor signaling pathway related proteins, were more highly expressed in the skin lesions of co-infected rhesus macaques. The dysregulated expression of these proteins associated with these multiple inflammation pathways or activation of phosphate kinases may exacerbate the inflammatory levels in SIV-MPXV co-infected rhesus macaques. MPXV infection alone and SIV-MPXV co-infection trigger organ signaling reprogramming. Elevated expression of DNA replication-related pathway proteins was observed in both lymph nodes and spleens of MPXV-infected and SIV-MPXV co-infected rhesus macaques (Fig. [244]4d, cluster 2). Concomitant upregulation of CDK-associated kinases (e.g., CDK4/6/16/18) in these organs (Fig. [245]4f, [246]g) suggests viral exploitation of host cell cycles by driving cells into the DNA synthesis phase (S-phase)^[247]65,[248]66, thereby hijacking nucleotide metabolism and replication machinery to facilitate MPXV genome replication. Furthermore, PLK1—a mitotic kinase stabilizing DNA replication forks and regulating replication initiation^[249]67—showed synergistic co-activation with CDKs (Fig. [250]4f, [251]g). This pronounced CDK-PLK axis activation implies that co-infection optimizes the viral replication niche through dual regulation of cell cycle progression and replication fidelity^[252]68. In the ILN and spleen, downregulated focal-adhesion pathways (Fig. [253]4d, cluster 5; Fig. [254]4e, cluster 6) likely compromise tissue architectural stability, causing proliferation-microenvironment decoupling that exacerbates inflammatory damage. Notably, in skin lesions, the co-activation of CDKs and PLKs was exclusive to SIV-MPXV co-infection (Fig. [255]4f, [256]g), indicating a replication-favorable environment correlating with higher viral DNA copies in lesions. In addition, CDK6—a central regulator of lymphocyte activation^[257]69,[258]70—exhibited sustained activation in SIV-MPXV co-infection, potentially driving aberrant T/B-cell proliferation. However, proteomics revealed downregulated T-cell receptor signaling and Th-cell differentiation in lymph nodes (Fig. [259]4d, cluster 4), indicating that hyperproliferating immune cells fail to undergo functional effector differentiation. Targeting the CDK-PLK axis, we identified clinical agents (e.g., palbociclib^[260]71, Fig.[261]4h). Inhibiting this pathway may block viral co-option of host replication machinery and restore adhesion pathway function. This study has several limitations. First, although we conducted the study using an appropriate number of rhesus monkeys in accordance with animal ethics and statistical requirements, the multi-organ samples used for proteomic and phosphoproteomic analyses were derived from only nine rhesus macaques (three per group). While the results demonstrate good consistency, the conclusions drawn from this sample size require further validation and exploration through additional experimental data and clinical population characterization. Furthermore, future studies should validate the upregulated protein-associated IL-17 signaling pathway, DNA replication, and cell cycle identified through proteomic analysis, as well as the potential therapeutic drugs predicted from kinase prediction. In addition, further proteomic analysis in SIV-infected rhesus macaques is necessary to explore the interaction between SIV and MPXV in disrupting host protein homeostasis, building upon the findings of this study. In Fig. [262]4d and Fig. [263]4e, we presented results using ANOVA P < 0.05 rather than P-adjust < 0.05. While this approach retains more proteomic features for biological interpretation, future studies should validate key targets using adjusted p-values (e.g., FDR < 0.05) to ensure statistical robustness. Finally, a limitation of this study is the inability to present the full scope of bioinformatics results. Future MPXV-related research can supplement these findings through expanded data mining approaches. Overall, this dataset provides a resource for elucidating critical host-MPXV interactions in future studies. In summary, our study revealed the immune damage, more severe skin lesions, multi-organ inflammatory response, and organ signaling reprogramming experienced by HIV and MPXV co-infected populations, based on a non-human primate model. These findings emphasize the urgent need to strengthen interventions for HIV-infected and high-risk populations, as well as to adjust treatment strategies for patients clinically affected by co-infections. The multi-organ proteome and phosphoproteome landscape of MPXV infection, with and without HIV co-infection, provides a comprehensive understanding of the pathogenic characteristics of HIV and MPXV co-infection. This dataset offers detailed and robust support for identifying potential diagnostic and therapeutic targets. In addition, we established an effective non-human primate model of SIV-MPXV co-infection, which serves as a valuable preclinical platform for evaluating the safety and efficacy of anti-MPXV drugs and vaccines in HIV-infected individuals. Methods Virus and cells Low-passage stocks of MPXV (MPXV-B.1-China-C-Tan-CQ01) were cultured in VeroE6 cells as previously described^[264]72. The culture conditions included Dulbecco’s Modified Eagle Medium (DMEM, Gibco, C11995500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 30044333) and 1% penicillin-streptomycin (PS, Gibco, 15140122), with the culture environment maintained at 37 °C and 5% CO[2]. Simian immunodeficiency virus (SIVmac239) was a gift from Dr. Preston Marx and was expanded using rhesus macaque PBMCs (rmPBMCs). The VACV Tiantan strain was propagated and titrated in chicken embryos prior to use. The Env sequences of SIVmac239 were sequence-verified, and two challenge stocks were deposited at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. The titers of SIVmac239 and MPXV were determined in rmPBMCs and VeroE6 cells, respectively, using a standard 50% tissue culture infection dose (TCID[50]) assay. Nonhuman primates and virus challenge The macaque study was conducted in an ABSL-3 facility, adhering to protocols approved by the Institutional Animal Care and Use Committee (IACUC) under the guidelines of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Approval Number XJ23004). All animals were single-housed and cared for in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Six rhesus macaques (3 males and 3 females), aged 6–8 years, were inoculated with SIVmac239 at a dose of 100 TCID[50]via the intravenous (i.v.) route. On day 115 post-challenge, these 6 rhesus macaques, along with 6 new rhesus macaques (3 males and 3 females, aged 6–8 years), were challenged with 1 × 10^7 TCID[50] of MPXV through the i.v. route. Half of the rhesus macaques were necropsied on day 10 post-MPXV infection for tissue viral load assessment and histopathology. The animals were monitored for mortality, moribund conditions, and specific disease signs, including fever, poxvirus lesions, appetite loss, dyspnea, eating behavior, and edema. All immunologic and virologic assays were performed in a blinded manner. Manipulations involving MPXV were conducted under BSL-3 conditions. Euthanasia was conducted by bloodletting through the femoral artery under the condition of Zoletil 50 anesthesia (5 mg/kg). Quantification of viral RNA and DNA loads SIV RNA and MPXV DNA loads were both determined by real-time quantitative PCR (qPCR) carried out on an ABI 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). For SIV, viral RNA was extracted using QIAamp Viral RNA Mini Kit (QIAGEN, 52906). The primers and protocol are as described in the previous study^[265]73,[266]74. The detection limit for viral RNA in plasma was 100 copies/mL. Regarding MPXV, the total DNA in tissues and plasma was extracted using QIAamp DNA Mini Kit (QIAGEN, 51306) and QIAamp DNA Blood Kit (QIAGEN, 51106), respectively. The following primers and combination were used for detection of the gene encoding F3L: 5’- CTCATTGATTTTTCGCGGGATA -3’(forward), 5’- GACGATACTCCTCCTCGTTGGT -3’(reverse), and 5’-(FAM)- CATCAGAATCTGTAGGCCGT -(MGB) -3’(probe). The cycling protocol was as follow: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The detection limit for viral DNA in plasma was 100 copies/mL, and for viral DNA in tissues was 10 copies/μg of total DNA. Cytokine and chemokine measurements The plasma levels of BAFF, CXCL10, CCL5, GDF-15, and ST2 were measured using an automated enzyme-linked immunosorbent assay (ELISA)-based microfluidic system (ELLA; ProteinSimple) with dedicated cartridges, following the manufacturer’s instructions. The absolute concentration of each analyte was determined using a standard curve. The assay was conducted with Simple Plex Runner Software and analyzed using Simple Plex Explorer ELISA. The plasma levels of IL-6 and IFN-γ were measured using an NHP ProcartaPlex 8-Plex Plate (eBioscience, PPX-08) according to the manufacturer’s instructions. The data were collected on the Luminex FLEXMAP 3D® System (Luminex Corporation, Austin, USA) and analyzed by MILLIPLEX® Analyst 5.1 software (MerkMillipore, Boston, MA, USA). Enzyme-linked immunosorbent assay (ELISA) MPXV antigens (A35, B6R, H3L, M1R, A29, and E8L [40886-V08H, 40902-V08H, 40893-V08H1, 40904-V07H, 40891-V08E, and 41042-V08H, purchased from Sino Biological]) were diluted to 1–10 μg/mL in carbonate buffer, coated onto ELISA plates, and incubated overnight at 4 °C. The following morning, the plates were washed three times with 1 × PBS-T and then blocked with 1% BSA at 37 °C for 2 h. After blocking, the BSA was removed, and the plates were washed three times. A dilution series of plasma samples, prepared with PBS-T + 1% BSA, was then added to the plates. After 1 h of incubation at 37 °C, the plasma was removed, washed with PBS-T, and goat anti-monkey IgG H&L (Abcam, ab112767), diluted 1:10,000 in blocking buffer, was added for a 1 h incubation at 37 °C in the dark. The secondary antibody was then washed off three times, and freshly prepared TMB substrate was added to the plates. After 15–30 min of incubation at room temperature, the reaction was stopped by adding a stop solution. Absorbance was measured at 450 nm, with background subtraction at 630 nm, using the SpectraMax® iD5 Multi-Mode Microplate Reader (MOLECULAR DEVICES). Endpoint titers were reported as the highest reciprocal plasma dilution that produced an absorbance exceeding the negative control values by more than 2.1-fold. The C-reactive protein (CRP) and serum amyloid A1 (SAA1) levels were measured using the Monkey CRP SimpleStep ELISA® Kit (Abcam, ab260062) and Monkey serum amyloid A1 (SAA1) ELISA Kit (CUSABIO, CSB-EL020656RH), according the manufacturer’s manual. The concentrations of CRP and SAA1 in the samples were determined by comparing the absorbance values to the standard curve. Plaque reduction neutralization test (PRNT) VeroE6 cells were seeded in 24-well plates and incubated overnight at 37 °C with 5% CO[2]. Plasma samples were inactivated at 56 °C for 30 min, then serially diluted in DMEM containing Glutamax and 2% FBS. The 100 μL diluted plasma samples were mixed with an equal volume of MPXV solution and incubated at 37 °C for 60 min before being transferred to the VeroE6 cells. The 24-well plates were then incubated at 37 °C with 5% CO[2] for 3 days. The PRNT[50] titers were calculated using the Spearman-Karber method. Histopathological analysis Skin lesions collected from the limbs of six infected monkeys were fixed in 4% neutral-buffered formaldehyde. The fixed tissues were then embedded in paraffin, trimmed, processed, and sectioned. Sections were mounted on glass slides, stained with H&E, and examined under a microscope. All slides were evaluated by a veterinary pathologist, and the severity of the microscopic findings was ranked using a four-step grading system for comparison between groups. Flow cytometry analyses For whole blood cell counts, the following monoclonal antibodies were used to stain ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood: Mouse Anti-Human CD3 (SP34-2, BD Bioscience, 552851, 1:6), Mouse human CD4 Antibody (OKT4, Biolegend, 317408, 1:21), and Mouse Anti-Human CD8 (RPA-T8, BD Bioscience, 555367, 1:6). CD4^+ T cell counts were measured using BD Trucount tubes according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). For VACV-specific T cells response in MPXV infected monkeys, a total of 1 × 10^6 rmPBMCs were incubated with R10 medium (RPMI-1640 medium containing glutamine, adding 10% FBS and 1% penicillin-streptomycin, background control), vaccina or virus (2 × 10^6 PFU/ml), 10 ng/ml phorbol myristate acetate (PMA, positive control, Invitrogen, 00-4970-93,) at 37 °C and 5% CO[2] for 3 h, and then incubated with Brefeldin A (BFA, Solarbio, IB0270) for 15 h. Cells were washed by PBS containing 2% FBS, and fixed and incubated with Fixable Viability Dye (Invitrogen, L-23105) at room temperature in the dark for 20 min. Then washed the cells by PBS containing 2% FBS, and incubated with anti-monkey CD3 antibody (SP34-2, BD Bioscience, 563916, 1:50), CD4 antibody (OKT4, Biolegend, 317444, 1:50), CD8 antibody (SK1, BD Bioscience, 612754, 1:500) at room temperature for 30 min in the dark. Cells were then fixed and permeabilized with the Cytofix/Cytoperm kit (BD Bioscience, 554714) and incubated with fluorescently conjugated antibodies to IFN-γ (B27, BD Bioscience, 557995, 1:250), IL-2 (MQ1-17H12, BD Bioscience, 559334, 1:50) and TNF-α (MAb11, BD Bioscience, 554512, 1:250) for 30 min at room temperature in the dark. Cells were washed twice with FACS buffer (BD Bioscience, 566385) and fixed with 250 mL paraformaldehyde. Fixed cells were acquired and analyzed by a BD LSR Fortessa flow cytometer. Data was analyzed using FlowJo v10.1. Statistical analysis All statistical analyses about experimental data were performed using GraphPad Prism 10, with results expressed as mean ± standard deviation (SD). Group comparisons for experimental results were carried out using two-tailed unpaired t tests or two-way ANOVA, with significance set at P < 0.05. Protein extraction for proteomic and phosphoproteomic detection Each macaque tissue sample was added with four volumes of lysis buffer (1% SDS [Solarbio, S8010-500g], 1% protease inhibitor cocktail [Merck Millipore, 539134-10 ML], and 1% phosphatase inhibitor [Merck Millipore, 539133-1SET]) and homogenized. The tissue samples were heated at 95 °C for 30 min for virus inactivation. After sonication six minutes on ice using a high-intensity ultrasonic processor (Scientz, JY92-IIN), the remaining debris was removed by centrifugation at 12,000 × g at 4 °C for 10 min. Finally, the supernatant was collected, and the protein concentration was determined with the BCA kit (Beyotime Biotechnology, P0011) according to the manufacturer’s instructions. Trypsin digestion For each sample, a solution containing an equal amount of protein was taken and made into an equal volume using the lysis buffer. Each protein sample was combined with an equal amount of cold acetone (ThermoFisher Scientific, c204433) and vortexed. Then, four more volumes of pre-chilled acetone were added. The mixture was precipitated at − 20 °C for 2 h. Supernatant was removed by centrifugation at 4500 × g at 4 °C for 5 min. The remaining precipitate was washed 2-3 times with cold acetone, air-dried, redissolved in 200 mM TEAB (Sigma-Aldrich, T7408-500 mL), and dispersed through ultrasonication. For the initial digestion, trypsin (Promega, V5117) was added at a 1:50 mass ratio (trypsin-to-protein), and the digestion was carried out overnight. The sample underwent reduction with 5 mM dithiothreitol (Sigma-Aldrich, D9163-25G) for 60 min at 37 °C, then alkylation using 11 mM iodoacetamide (Sigma-Aldrich, V900335-5G) for 45 min at room temperature in darkness. For the second round of digestion, trypsin was added at a 1:100 mass ratio (trypsin-to-protein) for 4 h. Desalting of peptides was achieved using a Strata X SPE column (Phenomenex, 8B-S100-AAK). Phosphopeptide enrichment Peptide mixtures were first incubated with immobilized-metal affinity chromatography (IMAC) microspheres (ThermoFisher Scientific, A32992) suspension with vibration in loading buffer (50% acetonitrile/0.5% acetic acid [Sigma-Aldrich, 695092-100 ML]). To remove the non-specifically adsorbed peptides, the IMAC microspheres were washed with 50% acetonitrile/0.5% acetic acid and 30% acetonitrile/0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, 302031-100 ML), sequentially. To elute the enriched phosphopeptides, the elution buffer containing 10% NH[4]OH (Sigma-Aldrich, 338818) was added, and the enriched phosphopeptides were eluted with vibration^[267]75. The supernatant containing phosphopeptides was rapidly acidified by 10% TFA, and then subjected to peptide desalting by Pierce™ C18 ZipTips (ThermoFisher Scientific, 87784). The clean phosphopeptides were collected and dried using a SpeedVac (Eppendorf, concentrator plus) for LC-MS/MS analysis. The phosphopeptide enrichment process consulted the work of Humphrey et al.^[268]76 and Zhang et al.^[269]75 and made minor adjustments, which has been used in our previous studies^[270]77,[271]78. LC-MS/MS Analysis for the proteome Tryptic peptides were dissolved in solvent A (0.1% formic acid [Fluka, A117-50]) and directly loaded onto an Evotip (EV2011, EvoSep, Denmark) following the manufacturer’s guidelines. Peptide separation was performed on the EvoSep One liquid chromatography system (EvoSep, Denmark), utilizing the preset 60-SPD (Samples per Day) method. The EvoSep One system was equipped with ReproSil-Pur Basic C18 column (resins: 120 Å, 1.9 μm, Dr. Maisch GmbH, Ammerbuch, Germany; column: 150 μm × 15 cm). The mobile phase included consisted of solvent A and solvent B (0.1% formic acid in acetonitrile). Peptides were ionized using a capillary source and analyzed on the timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany) in data independent parallel accumulation serial fragmentation (dia-PASEF) mode. A 1.75 kV electrospray voltage was applied, and a TOF detector was used for analysis. Charge states were set as 0–5, cycle time as 1.6 s, accumulation time as 70 ms, and ramp time as 70 ms. The full MS scan range was set as 300–1500 m/z, and 20 PASEF scans per cycle and an MS/MS isolation window was set as 7 m/z within the 400–850 m/z range. LC-MS/MS Analysis for the phosphoproteome For phosphoproteome analysis, peptides were prepared similarly as the proteome by dissolving in solvent A (0.1% formic acid) and loaded onto an Evotip as the manufacturer’s instructions. The EvoSep One liquid chromatography system with the preset 30-SPD method was used for peptide separation, with the mobile phase consisted of solvent A and solvent B (0.1% formic acid in acetonitrile). The EvoSep One system was equipped with ReproSil-Pur Basic C18 column (resins: 120 Å, 1.9 μm, Dr. Maisch GmbH, Ammerbuch, Germany; column: 150 μm × 15 cm). Peptides were ionized using a capillary source and analyzed on the timsTOF Pro2 mass spectrometer in dia-PASEF mode. A 1.7 kV electrospray voltage was applied. Precursors and fragments were analyzed with a TOF detector. Charge states were set as 0–5, cycle time as 2.44 s, accumulation time as 100 ms, and ramp time as 100 ms. The full MS scan range was set as 100–1700 m/z, and 22 PASEF scans were acquired per cycle. The MS/MS isolation window was set as 20 m/z in the range of 395–1395 m/z. Proteome database search DIA data were processed using the DIA-NN search engine (v1.8), with spectra matched to the UniProt Macaca Mulatta (Rhesus macaque) FASTA file (Proteome ID: UP000006718; downloaded on 2024/06/12) and a reverse decoy database. Trypsin/P was specified as the cleavage enzyme, allowing one missed cleavage. Peptide length was set as 7–30. Cysteine carbamidomethylation was included as a fixed modification, and N-terminal methionine excision was noted. FDR was controlled to less than 1% at the peptide and protein levels. Phosphoproteome database search Phosphoproteomic data analysis followed the same procedure as the proteome using the DIA-NN search engine, again matched to the UniProt Macaca Mulatta (Rhesus macaque) FASTA file (Proteome ID: UP000006718; downloaded on 2024/06/12) and a reverse decoy database. One missed cleavage was allowed for trypsin/P. Peptide length was set as 7–30. Variable modifications included methionine oxidation and phosphorylation of serine/threonine/tyrosine residues, with fixed modifications including Cysteine carbamidomethylation and N-terminal methionine excision. FDR was controlled to less than 1% at the peptide and protein levels. KEGG Pathway enrichment analysis KEGG pathway enrichment analysis was performed on differentially expressed proteins (DEPs) to identify significant functional enrichment. KEGG annotations were obtained from [272]http://www.kegg.jp/^[273]79. The proteome enrichment background was established by mapping all reliable proteins to KEGG annotations, generating a curated set of 4999 annotated proteins (Supplementary Data [274]S2, [275]S6, [276]S7). Similarly, the phosphoproteome enrichment background was established by mapping proteins containing reliable phosphosites to KEGG annotations, generating a set of 2,600 phosphoproteins with pathway information (Supplementary Data [277]S3/[278]S8). Fisher’s exact test (fisher_exact function in scipy.stats, a Python package) was used to determine statistical significance, and pathways with P < 0.05 were considered significant. Protein-protein interaction network The STRING database (v11.5)^[279]80 was queried to identify protein-protein interactions, and high-confidence interactions (confidence score > 0.7) were visualized using Cytoscape (v3.8.2)^[280]81. Kinase prediction Kinase prediction was conducted using GPS software (version 5.0), following kinase-phosphosite interactions detailed by Song and Ye et al.^[281]82. All reliable phosphosites were submitted to the GPS software. Setting the organism at Macaca mulatta and the threshold at medium, the GPS software derived kinase-phosphosite relationships. Kinase activity was inferred using GSEA with a P-value < 0.05 and NES > 1 indicating activation, and NES < − 1 indicating inhibition^[282]83. Motif analysis Using GPS software, kinase-phosphosite relationships were identified among reliable phosphosites. Then, 21 amino acid peptide sequences around the GPS-identified substrate phosphosites were extracted, with phosphorylated residues in the middle. The peptide sequences around kinase substrate phosphosites and the UniProt Macaca Mulatta FASTA file were uploaded to MoMo (Motif-x algorithm, [283]https://meme-suite.org/meme/tools/momo) for motif analysis, setting peptide length at 21 amino acids and the other parameters as default^[284]84. Drug prediction Potential drugs targeting differentially expressed proteins, phosphoproteins, or kinases relevant to MPXV infection were predicted using the DrugBank database^[285]85. Data management for proteomics and phosphoproteomics Proteins present in ≥ 50% of detected samples in at least one group were considered reliable. Protein quantification values were normalized using each sample’s median intensity, with missing values imputed based on the lowest values in the protein matrix. Similarly, phosphosites with a localization probability of ≥ 0.75, detected in ≥ 50% of samples in at least one group, were deemed reliable. Phosphosite quantification was normalized using sample median intensity, and missing values were replaced with the lowest value in the phosphosite matrix. Statistical comparisons between two groups were conducted using Student’s ttest, while differences among three or more groups were assessed using one-way ANOVA. The P-values were corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) correction. Proteins/Phosphosites with adjusted P-values (P-adjust) < 0.05 were considered differentially expressed. Fold change (FC) > 1.5 or FC < (1/1.5) were used to define upregulated or downregulated proteins/phophosites, respectively. Reporting summary Further information on research design is available in the [286]Nature Portfolio Reporting Summary linked to this article. Supplementary information [287]Supplementary Information^ (2MB, pdf) [288]Peer Review file^ (2.4MB, pdf) [289]41467_2025_62919_MOESM3_ESM.pdf^ (138KB, pdf) Description of Additional Supplementary Files [290]Supplementary Data 1^ (10.8KB, xlsx) [291]Supplementary Data 2^ (3.1MB, xlsx) [292]Supplementary Data 3^ (5.6MB, xlsx) [293]Supplementary Data 4^ (332KB, xlsx) [294]Supplementary Data 5^ (40.3MB, xlsx) [295]Supplementary Data 6^ (232.5KB, xlsx) [296]Supplementary Data 7^ (432.4KB, xlsx) [297]Supplementary Data 8^ (55.1KB, xlsx) [298]Supplementary Data 9^ (125.1KB, xlsx) [299]Reporting Summary^ (140.8KB, pdf) Source data [300]Source Data^ (36.7KB, xlsx) Acknowledgements