Durable but impaired humoral and cellular immune responses against monkeypox virus in people with HIV over 18 months

Abstract

Background

Monkeypox virus (MPXV), a zoonotic orthopoxvirus (OPXV), re-emerged as a global public health concern following the 2022 multi-country outbreak that disproportionately affected people with human immunodeficiency virus (HIV). In July 2023, the U.S. National Institutes of Health classified MPXV as an AIDS-defining opportunistic infection, underscoring the urgency of understanding immune features in immunocompromised populations. Existing studies provide limited data on the durability and coordination of MPXV-specific humoral and cellular immunity in people with HIV (PWH). Moreover, whether concomitant MPXV infection modulates HIV-specific immunity in PWH remains unclear. We aimed to evaluate the durability of immune responses against MPXV, compare immune profiles between PWH and people without HIV (PWoH), and assess the impact of MPXV coinfection on HIV-specific immunity.

Methods

We conducted a prospective immunological analysis involving 28 PWH and 13 PWoH with confirmed MPXV infection. Peripheral blood samples were collected at 1–2 weeks, 1–6 months, and 12–18 months post-infection. Plasma IgG levels against MPXV antigens were measured using ELISA. Poxvirus- and HIV-specific memory T (T_m) cell responses were assessed using activation-induced marker and intracellular cytokine staining assays via flow cytometry.

Results

MPXV infection elicited durable humoral and cellular immune responses for up to 18 months, regardless of HIV status. However, PWH demonstrated attenuated MPXV-specific antibody responses, lower frequencies of poxvirus-specific T_m cells, and reduced polyfunctionality compared to PWoH. Notably, immune coordination in PWH was impaired, as evidenced by weakened concordance between humoral and cellular responses, disrupted interactions between CD4⁺ and CD8⁺ T_m cell subsets, and diminished consistency among antibody levels. Additionally, HIV-specific T_m cell responses remained stable in antiretroviral-treated PWH, irrespective of prior MPXV infection.

Conclusions

MPXV infection induced long-lasting cellular and humoral immunity in both PWH and PWoH. However, HIV-mediated immune dysregulation compromised the strength and coordination of these responses.

Supplementary information

The online version contains supplementary material available at 10.1186/s12967-026-08341-5.

Introduction

Monkeypox virus (MPXV), a member of the orthopoxvirus (OPXV) genus, shares strong antigenic similarity with variola and vaccinia viruses [1, 2]. Once a rare zoonosis endemic to parts of Africa, mpox (monkeypox) emerged in non-endemic regions in 2022 and rapidly spread worldwide, marking the largest orthopoxvirus outbreak in humans since smallpox eradication [3]. The World Health Organization (WHO) declared mpox a Public Health Emergency of International Concern (PHEIC) in July 2022 and reaffirmed it in August 2024 [4, 5]. By May 31, 2025, 146,051 confirmed cases and 359 deaths had been reported globally [6]. The outbreak predominantly affected men who have sex with men (MSM), among whom an estimated 38–50% were people with human immunodeficiency virus (HIV) [7]. This group (PWH) with uncontrolled viremia or severe CD4⁺ T cell depletion faces increased risks of severe disease and death, which highlights the critical role of host immunity in shaping MPXV outcomes [7, 8].

Although smallpox vaccination provides partial cross-protection against MPXV, its global cessation after smallpox eradication in 1980 has left most individuals without poxvirus-specific immune memory [9, 10]. Consequently, antiviral immunity depends on responses generated through natural infection. MPXV infection can induce both humoral and cellular immune responses, which form the basis of protective antiviral immunity [11]. However, chronic immune activation and T/B cell dysfunction in PWH may impair the generation and maintenance of MPXV-specific immune responses [12–14]. Previous studies have demonstrated that PWH and people without HIV (PWoH) differ in their immune responses against MPXV infection, especially the timing and durability of antibody production [15, 16]. However, data comparing immune responses between PWH and PWoH beyond the acute phase are limited, with only two studies extending assessments up to 6 and 12 months [15, 16]. The long-term persistence of these differences remains uncertain. Additionally, as viral coinfections can alter existing immune responses through mechanisms such as antigenic competition and bystander activation, it is unclear whether acute MPXV infection perturbs pre-existing HIV-specific immunity.

In this study, we conducted a comprehensive assessment of antibody responses to MPXV antigens and poxvirus-specific memory T (T_m) cells in both PWH and PWoH at multiple time points after MPXV infection. Among PWH, participants were stratified by CD4 count to investigate the impact of immune reconstitution on immune responses against MPXV. Additionally, we analyzed immune coordination by examining the interactions between humoral and cellular responses, as well as the correlations among T cell subsets and antibody levels. Furthermore, we evaluated HIV-specific T_m cell responses to determine whether MPXV coinfection modulates pre-existing HIV-specific responses, thereby informing personalized immune surveillance and reinfection risk assessment.

Methods

Study design, participants, and sample collection

This prospective cross-sectional study was conducted at Beijing Ditan Hospital from September 2023 to October 2024, enrolling 41 laboratory-confirmed MPXV-infected individuals, including 28 PWH and 13 PWoH. Blood samples were collected at 1–2 weeks, 1–6 months, and 12–18 months after symptom onset. Immune responses were compared cross-sectionally between groups. Detailed follow-up time points were listed in Table S1. In addition, two control groups were included: four healthy controls (HC) and six antiretroviral therapy (ART)-treated, virologically suppressed PWH without MPXV infection, with all controls matched for age and gender. During the study period, no specific antiviral drugs for mpox had been approved in China; all patients received symptomatic supportive care according to the “Diagnosis and Treatment Guidelines for Monkeypox (2022 Edition)” issued by the National Health Commission of China [17]. Epidemiological, clinical, and laboratory data were systematically collected.

Mpox severity score System (mpox-SSS)

The Mpox-SSS, a validated tool for evaluating disease severity, includes seven parameters: number of active lesions, anatomic extent of lesion involvement, presence of confluent lesions, presence of bacterial superinfection, extent of mucosal areas affected, level of care, and analgesia requirement (Table S2). It is suitable for both clinical and research use and distinguishes severity among MPXV cases [18, 19].

Plasma HIV-1 viral load and CD4⁺ T cell count

Plasma HIV-1 RNA was quantified using the Amplicor HIV Monitor assay (Roche, detection limit: 40 copies/mL). The CD4⁺ T cell count was measured by flow cytometry using TruCOUNT tubes (BD Biosciences).

MPXV-specific IgG detection by ELISA

Plasma IgG against MPXV antigens A29L, E8L, and A35R was measured using ELISA kits (#KAV13402, #KAV13202, #KAV13102; Antibody System) based on a quantitative competitive enzyme immunoassay technique, following the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices).

Activation-induced cell marker (AIM) and intracellular cytokine staining (ICS) assay

Frozen PBMCs were thawed and recovered overnight. Cells were seeded into 96-well plates at 5 × 10⁵ cells/well for AIM and 1 × 10⁶ cells/well for ICS assays. Anti-CD40 monoclonal antibody (0.5 µg/mL; Miltenyi Biotec) was added 15 minutes before stimulation. Four stimulation groups were set up: DMSO (negative control), CD3/CD28 (5 µg/mL, positive control), orthopoxvirus peptide pool (0.4 µg/mL, JPT; 127 peptides derived from MPXV, vaccinia virus, and variola virus, of which 96% are fully conserved in MPXV), and HIV peptide pools (Gag, Nef, Pol, 0.2 µg/mL each, JPT). PWoH received the first three stimulations; PWH received all four. Anti-CD107a antibody (#563869, BD Biosciences) was added at the time of stimulation, and GolgiPlug (1:1000) was added 5 hours before harvest. After 24 hours, cells were stained for surface markers (CD3, CD4, CD8, CD45RA, CCR7, CD69, 4-1BB, CD40L, CXCR5, CCR4, CCR6, CCR10, CXCR3). Staining of intracellular markers: tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), Perforin, Granzyme B (GrzB) (Table S3). CD4⁺ T helper (Th) subsets were defined based on chemokine receptor expression patterns according to established criteria [20]: Tfh (CXCR5⁺CCR10^-), Th1 (CXCR5^-CCR6^-CCR4^-CXCR3⁺ CCR10^-), Th2 (CXCR5^-CCR6^-CCR4⁺CXCR3^-CCR10^-), Th9 (CXCR5–CCR6⁺CCR4^-CXCR3^-CCR10^-), ThGM (CCR6^-CCR4⁺CXCR3^-CCR10⁺), Th17 (CXCR5^-CCR6⁺CCR4⁺CXCR3^-CCR10^-), Th1/17 (CXCR5^-CCR6⁺CCR4^-CXCR3⁺CCR10^-) and Th22 (CXCR5^-CCR6⁺CCR4⁺CXCR3^-CCR10⁺). CD8⁺ cytotoxic T-cell (Tc) and Th subsets shared the same chemokine receptor expression signatures [21–23]. Cells were acquired on an LSRFortessa (BD), and data were analyzed with FlowJo software. The frequency of antigen-specific cells (AIM⁺ cells, ICS⁺ cells) was calculated by subtracting the frequency of the non-stimulation condition (DMSO) from each antigen-stimulated condition (orthopoxvirus peptide or HIV mixed peptides). CD4⁺ AIM⁺ T_m cells were defined as CD69⁺ CD40L⁺, and CD8⁺ AIM⁺ T_m cells as CD69⁺ 4-1BB⁺. ICS⁺ T_m cells were defined by cytokine and granule production (IFN-γ, TNF-α, GrzB, Perforin, CD107a) in CD40L⁺CD4⁺ or CD69⁺CD8⁺ T_m cells. The gating strategy was shown in Figure S1.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 10. Normality was assessed using the Kolmogorov-Smirnov test. For two-group comparisons, Student’s t-test was used for normally distributed data and the Mann-Whitney U test for non-normal data. For comparisons involving more than two groups, the Kruskal-Wallis test followed by Dunn’s multiple comparisons test was applied. Pearson correlation was used for normally distributed continuous variables; otherwise, Spearman’s rank correlation was used. Categorical variables were compared using the chi-squared or Fisher’s exact test. Dynamic changes in AIM⁺ and ICS⁺ T_m cell frequencies were analyzed using polynomial regression. Heatmaps and Pie charts were generated using R (version 4.4). p < 0.05 was considered statistically significant.

Results

MPXV-specific antibody responses were weaker and delayed in PWH than in PWoH

All 41 MPXV-infected participants (28 PWH, 13 PWoH) were male, aged 23–40 years, and smallpox-unvaccinated. Sexual contact was the most likely transmission route in both groups (PWH: 89.3%, PWoH: 84.6%). Median CD4⁺ T cell count was 521.5 cells/μL (IQR 377.5–777.75) in PWoH and 494 cells/μL (IQR 104.25–694.5) in PWH, with 19 PWH (67.9%) achieving viral suppression on ART. None of the participants received antiviral treatment for mpox, and three PWH died during hospitalization. The control group included four healthy individuals (age range: 21–27) and six ART-treated PWH without MPXV infection (age range: 22–40), all unvaccinated against smallpox. Demographic and clinical characteristics were shown in Table 1; individual-level detailed information was provided in Table S1. Mpox-SSS assessments at 1–2 weeks post-MPXV symptom onset were shown in Table S4.

Table 1.

Demographic and clinical characteristics of study participants

	PWoH (n = 13)	PWH (n = 28)
Age, years
Median age	29 (27.5–32.25)	32 (26–36.5)
43 years or older	0 (0%)	0 (0%)
Sex
Male	13 (100%)	28 (100%)
Famale	0 (0%)	0 (0%)
Route of possible exposure†
MSM	11 (84.6%)	25 (89.3%)
Other	2 (15.4%)	3 (10.7%)
Days from last exposure to onset	7 (5–8)	8 (3–15)
Smallpox vaccination history	0 (0%)	0 (0%)
HIV-positive	-	28 (100%)
Regular ART	-	19 (67.9%)
Regular ART duration, years	-	4 (2–6)
CD4+ T cell count, cells/μL	521.5 (377.5–777.75)	494 (104.25–694.5)
Undetectable HIV RNA	-	19 (67.9%)
Coexisting conditions
Syphilis	1 (7.7%)	15 (53.6%)
Hepatitis B	0 (0%)	2 (7.1%)
Hepatitis C	0 (0%)	2 (7.1%)
Mode of admission
Inpatient	10 (76.9%)	25 (89.3%)
outpatient	3 (23.1%)	3 (10.7%)
Days from symptom onset to mpox diagnostic test, days	6 (5–7.5)	6 (5.5–10)
Length of hospital stay, days	8 (3.5–10)	7 (5–18.5)
Systemic symptoms
Rash or skin lesions	13 (100%)	28 (100%)
Fever	10 (76.9%)	16 (57.1%)
Lymphadenopathy	8 (61.5%)	18 (64.3%)
Perianal pain	0 (0%)	6 (21.4%)
Antiviral treatment for mpox	0 (0%)	0 (0%)
Death	0 (0%)	3 (10.7%)
PCR positive
Throat swab	8/10 (80.0%)	15/19 (78.9%)
Skin lesions	11/11 (100%)	20/21 (95.2%)
Perianal swab	0/1 (0%)	4/4 (100%)
Serum	1/5 (20%)	6/10 (60%)

Notes: Data are expressed as median (lQR) of n (%)

†The last possible exposure before symptom onset

Abbreviations: PWoH, people without HIV; PWH, people with HIV; HIV, human immunodeficiency virus; MSM, men who have sex with men; ART, antiretroviral therapy; PCR, Polymerase Chain Reaction

To assess the MPXV-specific antibody response in PWoH and PWH, we quantified plasma IgG levels against the MPXV antigens A29L, E8L, and A35R. At 1 month, PWH exhibited significantly lower IgG levels than PWoH for all three antigens (p = 0.0280 for A29L, E8L, and A35R). Although IgG levels decreased over time in both groups, PWH continued to show lower levels at 18 months for A29L (p = 0.0556) and E8L (p = 0.0792), although these differences did not reach statistical significance. Furthermore, distinct kinetic patterns were observed between the groups. In PWoH, IgG responses peaked at 1 month and subsequently declined. In PWH, anti-A29L IgG showed a similar early peak, while anti-E8L IgG responses rose more gradually and peaked around 3–6 months. Conversely, anti-A35R IgG responses remained low without a clear peak. Overall, IgG levels in PWH remained consistently lower than those in PWoH across all time points (Fig. 1A–C).

Fig. 1.

Kinetics of MPXV-specific IgG responses. (A-B) levels of anti-A29L, E8L, and A35R IgG antibody in PWoH (n = 7 at 1–2 weeks; n = 3 at 1 month; n = 2 at 3 months; n = 2 at 6 months; n = 3 at 12 months; n = 5 at 18 months) and PWH (n = 7, 10, 3, 5, 7, and 5 at corresponding time points). Bar plots represented group-wise comparisons at each time point; line graphs illustrated temporal dynamics. Data were shown as geometric means ± geometric standard deviation. Statistical comparisons used the mann-whitney U test; P values were indicated when significant. Abbreviations: w, weeks; m, months. (C) dynamic changes of anti-A29L, E8L, and A35R IgG antibodies in each participant after the onset of symptoms (PWoH: n = 13, samples = 26; PWH: n = 28, samples = 52)

PWH exhibited decreased poxvirus-specific T_m cell responses compared to PWoH

Given the high sequence homology between MPXV and other orthopoxviruses [10, 24] and the fact that all participants were smallpox vaccine-naïve, we evaluated the overall MPXV memory T cell response by stimulating PBMCs with an orthopoxvirus peptide pool [25] and conducting AIM and ICS assays across multiple time points. Furthermore, the analyses focused on memory T cells, particularly the central memory and effector memory subsets, which represent the major populations responsible for long-term immune surveillance and effector functions. Compared to HCs, MPXV-infected participants exhibited stronger poxvirus-specific T_m cell responses, regardless of HIV status. Comparison across time points revealed that differences between PWoH and PWH were most pronounced at 1–6 months. At this time point, PWoH showed a higher responder rate of AIM⁺ CD4⁺ T_m cells than PWH (100% vs 71%) with significantly elevated frequencies (p = 0.0107; Fig. 2A). A similar trend was observed in CD8⁺ T_m cells at 1–6 months (100% vs 53%; p = 0.048; Fig. 2B). No significant group differences were observed at 1–2 weeks and 12–18 months. ICS⁺ CD4⁺ and CD8⁺ T_m cell responses remained generally comparable between groups across most time points. Notably, a trend toward increased ICS⁺ CD8⁺ T_m cell responses in PWoH at 12–18 months (p = 0.0594) may reflect sustained cytotoxic potential (Fig. 2C–D). To assess temporal dynamics, AIM⁺ and ICS⁺ T_m cell frequencies were plotted over time since symptom onset. Over time, AIM⁺ T_m cell frequencies declined in PWoH but remained stable in PWH, resulting in a reduced difference between groups at later time points. In contrast, ICS⁺ T_m cell responses remained relatively stable across time points, with no significant group differences (Fig. 2E–F). Poxvirus-specific T_m cell responses persisted in most participants at 12–18 months, suggesting sustained cellular immunity following MPXV infection.

Fig. 2.

Poxvirus-specific T_m cell responses at different time points in PWoH and PWH. (A-D) frequencies of poxvirus-specific AIM⁺ CD4⁺ T_m (A), AIM⁺ CD8⁺ T_m (B), ICS⁺ CD4⁺ T_m (C), and ICS⁺ CD8⁺ T_m (D) cells at three time points: 1–2 weeks (PWoH: 7 participants, 7 samples; PWH: 6 participants, 6 samples), 1–6 months (PWoH: 3 participants, 7 samples; PWH: 12 participants, 17 samples), and 12–18 months (PWoH: 8 participants, 8 samples; PWH: 14 participants, 14 samples). Dashed line identified the median of poxvirus-specific T_m cells in healthy controls (4 participants, 4 samples). Blue, red, and gray circles represented PWoH responders, PWH responders, and non-responders, respectively. Data were shown as geometric means ± geometric standard deviation. The mann-whitney U tests were used for group comparisons; P values were displayed in the plots. Pie charts below each panel represented responder frequencies, with P values from Chi-squared or Fisher’s exact tests shown beneath each chart. (E-F) temporal dynamics of AIM⁺ (E) and ICS⁺ (F) CD4⁺ and CD8⁺ T_m cell frequencies. The solid lines represented the fitted curve obtained using curve fitting polynomial regression, and the shaded areas represented 95% confidence intervals

We examined the polyfunctionality of poxvirus-specific T_m cells by profiling the co-expression of IFN-γ, TNF-α, GrzB, Perforin, and CD107a. At 1–2 weeks, CD4⁺ T_m cells from PWoH displayed broader polyfunctionality, with subsets expressing combinations of IFN-γ⁺ TNF-α⁺, IFN-γ⁺ Perforin⁺, and IFN-γ⁺ TNF-α⁺ GrzB⁺. In contrast, CD4⁺ T_m cells from PWH were skewed toward Perforin⁺ and GrzB⁺ single- or dual-positive subsets with minimal IFN-γ expression and limited cytokine co-expression. Over time, CD4⁺ T_m cells in PWH increased functional diversity, progressively resembling the pattern observed in PWoH. CD8⁺ T_m cells in both groups were dominated by GrzB⁺ Perforin⁺, or single-positive subsets, with stable profiles across time points (Fig. 3A). We further compared the frequencies of functional subsets between PWoH and PWH. The results showed that PWH had significantly elevated proportions of GrzB⁺ CD4⁺ T_m cells at 1–2 weeks (p = 0.014). At 1–6 months, PWoH exhibited higher frequencies of IFN-γ⁺ GrzB⁺ CD4⁺ T_m cells (p = 0.0169) and Perforin⁺ CD8⁺ T_m cells than PWH (p = 0.0296). By 12–18 months, TNF-α⁺ Perforin⁺ CD4⁺ T_m cells were increased in PWoH (p = 0.0044), along with elevated frequencies of TNF-α⁺ Perforin⁺ CD107a⁺, Perforin⁺ CD107a⁺, and Perforin⁺ CD8⁺ T_m cells (p = 0.0096, 0.0018, and 0.0419, respectively; Fig. 3B–D).

Fig. 3.

Polyfunctional CD4⁺ and CD8⁺ T_m cell responses in PWoH and PWH post-MPXV infection. (A) polyfunctionality profiles of poxvirus-specific CD4⁺ T_m and CD8⁺ T_m cells at three time points: 1–2 weeks (PWoH: 7 participants, 7 samples; PWH: 6 participants, 6 samples), 1–6 months (PWoH: 3 participants, 7 samples; PWH: 12 participants, 17 samples), and 12–18 months (PWoH: 8 participants, 8 samples; PWH: 14 participants, 14 samples). Pie charts illustrated the proportion of cells producing 5 (dark orange), 4 (light orange), 3 (pale orange), 2 (dark gray), or 1 (light gray) effector molecules. (B-D) frequency comparisons of polyfunctional CD4⁺ T_m (left) and CD8⁺ T_m (right) cells between PWoH and PWH. Data were shown as geometric means ± geometric standard deviation. The mann-whitney U test was applied to each graph; significant P values were shown. Abbreviations: IFN-γ = interferon-γ. TNF-α = tumour necrosis factor-α. GrzB = Granzyme B

PWH and PWoH displayed differential polarization of T cell subsets following MPXV infection

Considering the distinct roles of various T cell subsets in antiviral immunity and immune regulation, we compared the distribution of Th and Tc subsets between MPXV-infected PWH and PWoH (Figure S2A-C). At 1–2 weeks, no significant differences were observed between groups. At 1–6 months, PWH exhibited decreased frequencies of T follicular helper (Tfh) cells (p = 0.0409), along with lower proportions of Th1 and Th1/17 subsets and increased Th2 cells (p = 0.0021, 0.0053, and 0.0408, respectively). In the CD8 compartment, T follicular cytotoxic (Tfc), Tc1/17, and TcGM subsets were significantly decreased in PWH (p = 0.0472, 0.0160, and 0.0005, respectively), while Tc9 cells were also lower but the difference was not statistically significant (p = 0.0553). At 12–18 months, Th22 and Tc22 cell frequencies were lower in PWH compared to PWoH (p = 0.0338 and 0.002, respectively). These data indicated that T cell polarization was affected by HIV infection. Furthermore, we assessed chemokine receptor expression within AIM⁺ CD4⁺ and CD8⁺ T_m cells (Figure S3A-C). The analysis revealed an elevated proportion of CCR4⁺ AIM⁺CD4⁺ T_m cells in PWH at 1–2 weeks (p = 0.0103). In contrast, CXCR3⁺ AIM⁺CD4⁺ T_m cells were significantly more frequent in PWoH at 1–6 months (p = 0.0186). By 12–18 months, CCR6⁺ CCR4⁺ AIM⁺CD4⁺ T_m cells were higher in PWH than in PWoH (p = 0.0361). No significant differences in chemokine receptor expression were found among poxvirus-specific CD8⁺ T_m cells.

Protective immune responses were associated with mpox disease severity in PWoH and with CD4⁺ T cell count in PWH

To assess the association between immune responses and mpox disease severity, we correlated humoral and cellular parameters with the disease severity score at 1–2 weeks post-symptom onset. In PWoH, higher frequencies of ICS⁺ CD4⁺ T_m cells (r = −0.8365, p = 0.027) and Perforin⁺ CD69⁺ CD8⁺ T_m cells (r = −0.8729, p = 0.019) were associated with lower disease severity score, suggesting a protective role of polyfunctional T cells. No such associations were observed in PWH, further highlighting their impaired immune response (Figure S4A-B).

The lack of correlation between protective immune responses and disease severity in PWH suggested a potential role of underlying immune impairment. Therefore, we assessed whether CD4⁺ T cell count was associated with the magnitude of immune responses against MPXV in PWH. At 1 month post-infection, PWH were stratified by CD4⁺ T cell count into two groups: ≥200 cells/μL and <200 cells/μL. PWH with a high CD4 count exhibited a higher responder rate of AIM⁺ CD4⁺ T_m cells (p = 0.048), although overall frequencies did not differ significantly (Figure S5A). The response rates and frequencies of AIM⁺ CD8⁺ T_m cells, ICS⁺ CD4⁺ T_m cells, and ICS⁺ CD8⁺ T_m cells were comparable between groups (Figure S5B-D). Polyfunctionality profiling revealed a significantly higher proportion of IFN-γ⁺ TNF-α⁺ GrzB⁺ CD4⁺ T_m cells in PWH with high CD4 count (p = 0.0333), suggesting enhanced polyfunctionality. No differences in polyfunctional subsets were observed in CD8⁺ T_m cells between groups (Figure S5E). In addition, plasma anti-A29L, E8L, and A35R IgG levels were slightly higher but non-significant in PWH with high CD4 count (Figure S5F). Furthermore, we performed correlation analyses between CD4⁺ T cell count and the frequencies of poxvirus-specific T_m cell subsets among PWH. Significant positive correlations were observed between CD4⁺ T cell count and the proportions of ICS⁺ CD40L⁺ (r = 0.7716, p = 0.0135), IFN-γ⁺ CD40L⁺ (r = 0.8128, p = 0.0028), TNF-α⁺ CD40L⁺ (r = 0.7169, p = 0.0246), GrzB⁺ CD40L⁺ (r = 0.8128, p = 0.0028), and Perforin⁺ CD40L⁺ (r = 0.8125, p = 0.0067) CD4⁺ T_m cells (Figure S5G). These results suggested that stronger poxvirus-specific CD4⁺ T_m cell responses were associated with a more favorable immune status.

PWH exhibited impaired coordination between humoral and cellular immune responses

To assess immune coordination, correlation analyses were performed across all measured immune parameters within 18 months post-symptom onset (Figure S6). In PWoH, anti-E8L IgG levels positively correlated with the frequencies of Tfh cells (r = 0.4315, p = 0.0449), Tfc cells (r = 0.4869, p = 0.0216), and CXCR5⁺ AIM⁺ CD4⁺ T_m cells (r = 0.4249, p = 0.0487). Anti-A35R IgG levels also showed significant correlations with Tfh (r = 0.4349, p = 0.0431) and Tfc cells (r = 0.4620, p = 0.0304). Moreover, IgG levels against different MPXV antigens demonstrated strong concordance: A29L and E8L (r = 0.8639, p < 0.0001), A29L and A35R (r = 0.9378, p < 0.0001), and E8L and A35R (r = 0.905, p < 0.0001). These results highlighted the coordination of humoral responses and their association with T cells (Fig. 4A). In PWoH, functional coordination between CD4⁺ and CD8⁺ T_m cell subsets was also evident, as frequencies of ICS⁺ CD4⁺ and ICS⁺ CD8⁺ T_m cells positively correlated (r = 0.5991, p = 0.0032). Similar coordination was observed across other functional T cell subsets (Fig. 4B). In contrast, these associations were attenuated or lost in PWH, who exhibited weaker humoral coordination and markedly disrupted T cell subset interactions (Fig. 4A–B).

Fig. 4.

Immune coordination within 18 months post-MPXV symptom onset in both PWH and PWoH. (A) correlations between MPXV-specific IgG levels (A29L, E8L, A35R) and T cell subsets in PWoH (n = 13) and PWH (n = 28). (B) correlation between CD4⁺ T_m and CD8⁺ T_m cell immune response in PWoH (n = 13) and PWH (n = 28). Analyses were conducted using Pearson’s or Spearman’s tests, depending on distribution. Only significant correlations (p < 0.05) were shown. IgG values were log₁₀-transformed

MPXV coinfection did not alter HIV-specific T_m cell immunity in ART-treated individuals

To determine whether MPXV coinfection alters HIV-specific T_m cell responses, we evaluated immune responses in ART-treated PWH with or without prior MPXV infection. These participants were then stratified by time since MPXV infection, and six ART-treated PWH without MPXV exposure were included as PWH controls. HIV-specific responses were measured by stimulating PBMCs with pooled HIV peptide mixes (Gag, Nef, Pol). The proportions of HIV-specific AIM⁺ T_m cells were comparable across groups (Fig. 5A–B). Similarly, the frequencies of HIV-specific ICS⁺ T_m cells showed no significant differences (Fig. 5C–D). Polyfunctional profiling revealed no differences between groups (Fig. 5E–F). HIV-specific CD8⁺ T_m cells were predominantly Perforin⁺ and GrzB⁺ subsets, while CD4⁺ T_m cells exhibited broader functional diversity (Fig. 5G). These findings suggested that MPXV coinfection does not markedly alter HIV-specific T_m cell responses in individuals on ART.

Fig. 5.

HIV-specific immune response at different time points in PWH. (A-F) frequencies of HIV-specific AIM⁺ CD4⁺ T_m (A), AIM⁺ CD8⁺ T_m (B), ICS⁺ CD4⁺ T_m (C), ICS⁺ CD8⁺ T_m (D), polyfunctional CD4⁺ T_m (E) and polyfunctional CD8⁺ T_m (F) cells in ART-treated PWH at 1–2 weeks (6 participants, 6 samples), 1–6 months (7 participants, 11 samples), and 12–18 months post-event (14 participants, 14 samples), and compared to ART-treated MPXV-naïve PWH controls (6 participants, 6 samples). Dark blue, light blue, pink, orange, and gray circles represented: (1) ART-treated PWH responders at 1–2 weeks post-MPXV infection, (2) 1–6 months, (3) 12–18 months, (4) ART-treated MPXV-naïve PWH responders, and (5) non-responders, respectively. Data were shown as geometric means ± geometric standard deviation. The Kruskal-Wallis tests and Dunn’s multiple comparisons test were used for group comparisons. (G) polyfunctionality profiles of HIV-specific CD4⁺ T_m and CD8⁺ T_m cells in PWH at different time points after MPXV infection and in PWH who have never been infected with MPXV

Discussion

Since the global mpox outbreak in 2022, PWH have been recognized as a highly vulnerable population due to immune dysregulation [26]. While previous studies have characterized MPXV-specific immune responses in PWH, data on the durability of antibody and T cell responses, as well as coordination between humoral and cellular immunity, remain limited [15, 16]. Here, we conducted a comprehensive, multidimensional assessment of immune responses at multiple time points. Our findings revealed that HIV infection impacts immune responses against MPXV through multiple mechanisms: it decreased antibody levels, reduced proportion and polyfunctionality of antigen-specific T_m cells, and impaired immune coordination.

A key finding of this study was that PWH exhibited significantly lower frequencies and polyfunctionality of poxvirus-specific T_m cells, compared to PWoH at 1–6 months after MPXV infection. Of note, these deficiencies persisted at 12–18 months. Previous studies did not observe such differences at any time points, likely due to methodological variations. Earlier studies primarily assessed total T cell responses without distinguishing memory subsets. For instance, an Italian study employed IFN-γ ELISpot assays, which preclude the analysis of memory subsets, while a Spanish study, despite utilizing AIM and ICS assays, focused solely on total T cell populations [15, 16]. Given that HIV infection depletes naive T cells and expands memory T cells, and that this imbalance often persists even after ART [27], evaluating total T cell responses may obscure differences in memory functionality. Our study addressed this limitation by profiling poxvirus-specific T_m cells, enabling accurate functional comparison between PWH and PWoH. More importantly, previous studies might have overlooked these distinctions due to incomplete T cell functional characterization [16]. In contrast, our study incorporated cytotoxic markers (GrzB, Perforin, and CD107a) and effector cytokine markers (IFN-γ and TNF-α) to comprehensively evaluate T cell effector functions. Consequently, we revealed that several cytotoxic T cell subsets were significantly more abundant in PWoH than in PWH at both 1–6 and 12–18 months post-infection, further supporting functional impairments in PWH. These findings aligned with reports of diminished smallpox vaccination-induced T cells in PWH compared to PWoH, suggesting that HIV infection might cause long-lasting damage to poxvirus-specific immune memory [28]. Similar impairments have also been reported in other viral infections, with PWH exhibiting attenuated SARS-CoV-2-specific T cell responses compared to PWoH following both natural infection and vaccination [29, 30]. Collectively, these findings suggested that HIV infection could weaken antigen-specific T cell responses to acute viral infections, potentially affecting clinical outcomes.

Consistent with previous reports [15, 16, 31], our study also revealed that PWH exhibited lower antibody levels than PWoH, suggesting impaired humoral response. More importantly, anti-A29L and E8L IgG levels remained reduced in PWH even 18 months post-infection, demonstrating persistent defects in long-term humoral immunity. This impairment may be linked to decreased frequencies of Tfh, Tfc, and Tc9 cells in PWH. Tfh and Tfc cells assist B cells via CD40L and IL-21 signals, supporting robust antibody production, while Tc9 cells promote functional B cell memory via the IL-9-ZBTB18 axis [32, 33]. The depletion of these helper populations could compromise B cell function, resulting in suboptimal antibody quality and durability. Moreover, HIV infection induces irreversible B cell exhaustion and impairs antigen responses despite virologic control [34], contributing to defective memory B cell development and long-lasting antibody responses against MPXV in PWH.

Another important finding was disrupted immune coordination in PWH after MPXV infection. Successful immune responses require precise coordination across immune components for effective antigen recognition, immune activation, and memory formation. While previous mpox studies have investigated immune coordination, most have focused on associations within T cell subsets or antibody profiles [15, 35–37]. Only one Spanish study analyzed cellular-humoral interplay, but it was limited to total T cells without examining the interactions of specific subsets and antigen-specific antibody responses [16]. In contrast, our study systematically evaluated three key aspects of immune coordination: the functional synergy among poxvirus-specific T_m cell subsets, the relationships between T cell populations and IgG antibodies, and the concordance across various antibody responses. This multidimensional analysis revealed striking differences between PWoH and PWH. PWoH demonstrated robust immune integration, characterized by strong correlations between functional T cell subsets, coupling between Tfh/Tfc cells and MPXV-specific IgG antibodies, and highly consistent IgG profiles across different MPXV antigens. Conversely, PWH exhibited significant disruptions in immune coordination that extended beyond simple quantitative deficiencies. This dysregulation likely resulted from the dual pathological effects of HIV infection: impaired CD4⁺ T cell helper functions compromising both cytotoxic T cell and B cell responses, combined with chronic immune activation that disrupts normal intercellular communication [38]. These findings suggested that achieving protective immunity in PWH might require not only enhancing response magnitude but also restoring proper coordination within the immune system. Thus, future vaccine development should strengthen T-B cell interactions and functional synergy among T cell subsets to establish long-lasting immunity in this vulnerable population.

The relationship between immune status and immune responses against MPXV in PWH warrants further investigation. PWH with high CD4⁺ T cell count typically developed mild and self-limiting illness comparable to that observed in PWoH, whereas those with low CD4⁺ T cell count were at increased risk of severe disease, hospitalization, and death [7, 13, 39]. However, comprehensive immunological correlates underlying these clinical observations remain limited. Our study provides important mechanistic insights, revealing that PWH with CD4⁺ T cell count above 200 cells/μL mounted significantly stronger poxvirus-specific immune responses. Notably, we identified a positive correlation between CD4⁺ T cell count and poxvirus-specific T_m cell response. These findings highlighted the clinical importance of early HIV diagnosis and prompt ART initiation in high-risk populations. Timely immune reconstitution not only enhances protection against opportunistic pathogens, such as MPXV, but also reduces risks of severe morbidity and mortality.

In addition to examining the impact of HIV infection on MPXV-specific immunity, we further investigated potential reciprocal effects of MPXV infection on HIV-specific immune responses. Previous research documented stable levels of both integrated and defective HIV proviral DNA in a virologically suppressed PWH before and after MPXV infection, indicating no significant alteration in the latent reservoir size [40]. Our results corroborated this virologic observation, demonstrating preserved frequencies and polyfunctional capacity of HIV-specific T_m cells in ART-treated PWH throughout MPXV infection, with no discernible differences compared to MPXV-naïve controls. These results provided compelling evidence that MPXV infection does not substantially compromise HIV-specific T_m cell populations. The conclusion aligned with prior research showing that COVID-19 mRNA vaccination in ART-treated PWH neither expanded the intact HIV reservoir in peripheral CD4⁺ T cells nor modified HIV-Nef- or Gag-specific T cell frequencies [41, 42]. These findings had important clinical implications, supporting the safety of vaccination protocols and the management of acute infections in virologically suppressed PWH receiving ART.

This study has several limitations. First, humoral immunity was assessed only through MPXV-specific IgG levels, excluding IgA responses and neutralizing activity. Second, the exclusive analysis of peripheral blood limited insights into tissue-resident immunity. Third, we did not explore the potential impact of MPXV infection on the HIV-1 latent reservoir during ART, limiting our understanding of reservoir dynamics. Lastly, the single-center design and modest sample size may restrict the external validity of our findings. Future studies should incorporate multicenter cohorts with larger sample sizes, more comprehensive immune profiling, and longitudinal tracking of latent reservoir dynamics to fully elucidate the immunological features of MPXV/HIV coinfection.

In summary, this study demonstrated that MPXV-specific IgG antibodies and poxvirus-specific T_m cell responses were durably maintained for up to 18 months in both PWH and PWoH. However, PWH exhibited significantly attenuated immunological memory compared to PWoH, as evidenced by lower MPXV-specific IgG levels, reduced frequencies and polyfunctionality of poxvirus-specific T_m cells, and impaired immune network coordination. While these disparities showed partial attenuation over time, they persisted throughout the observation period, indicating that HIV infection compromises the durability of immunological memory. Notably, ART-treated PWH maintained stable HIV-specific T_m cell responses despite MPXV infection. These findings underscored the necessity for customized immunotherapeutic approaches in PWH to optimize long-term protection against emerging viral threats.

Electronic supplementary material

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Abbreviations

MPXV

Monkeypox virus

OPXV

Orthopoxvirus

MSM

Men who have sex with men

HIV

human immunodeficiency virus

PWH

People with HIV

PWoH

People without HIV

T_m

Memory T

HC

Healthy controls

ART

Antiretroviral therapy

AIM

Activation-induced marker

ICS

Intracellular cytokine staining

TNF-α

Tumour necrosis factor-α

IFN-γ

Interferon-γ

GrzB

Granzyme B

Th

T helper

Tc

Cytotoxic T

Tfh

T follicular helper

Tfc

T follicular cytotoxic

Author contributions

LX, XW, and MJ performed the experiments, analyzed the data, and wrote the manuscript. HY, XZ, JL, JX, and YL contributed to recruitment and clinical data collection. CS and QP analyzed the data. RJ reviewed the manuscript. YK, HZ, and XW designed the study, accessed and verified the data, and revised the manuscript. All authors have read and approved the article.

Funding

This work was supported by the Beijing Hospitals Authority’s Ascent Plan [DFL20241802], Beijing Natural Science Foundation [L254074], Beijing Natural Science Foundation [L222069], National Natural Science Foundation of China [82171548], Capital’s Funds for Health Improvement and Research [CFH2024-2-2175], and the science foundation of Beijing Ditan Hospital, Capital Medical University [DTYM-202408].

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Beijing Ditan Hospital (No. DTEC-YJ2023-001–02), and was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent.

Consent for publication

Not applicable.

Competing interests

We declare no competing interests.