Mpox-Specific Immune Responses Elicited by Vaccination or Infection in People With HIV

Abstract

In the recent mpox outbreak, people with human immunodeficiency virus (PWH) were at high risk both for contracting infection and for a more severe disease course. We studied cellular and humoral immune responses elicited by mpox infection (n = 5; n = 3 PWH) or smallpox vaccination (n = 17; all PWH) in a cohort of men who have sex with men. All PWH were successfully treated, with stable CD4 counts and undetectable HIV viral loads. Eleven of 17 vaccinated individuals had received childhood smallpox vaccination. In this group of individuals, both 2-dose modified vaccinia Ankara (MVA) vaccination and natural infection evoked mpox-specific immune responses mediated by B cells as well as CD4 and CD8 T cells. This study improves our understanding of smallpox vaccination-mediated cross-reactivity to other orthopox viruses, and long-lasting durability of childhood smallpox vaccination-mediated immune responses, including in PWH.

mpox virus, MVA vaccine, HIV, T cell response, antibody response

The mpox outbreak in previously nonendemic countries was declared a public health emergency of international concern by the World Health Organization (WHO) in 2022–2023 [1, 2]. This outbreak was mainly driven by human-to-human transmission through sexual contact [3–6], with people with HIV (PWH) disproportionately affected, accounting for 38%–54% of individuals diagnosed with mpox infection [6–9]. The severity of mpox disease is highly dependent on human immunodeficiency virus (HIV) infection status: while people with advanced and untreated HIV infection are at risk to experience fulminant clinical courses, patients with a well-controlled HIV infection show a similar clinical picture as individuals without HIV [7, 10, 11].

Several animal and human studies have detected significant cross-reactivity between smallpox- and mpox-specific immune responses, probably due to high sequence similarity between orthopox viruses (OPXV) and the wide breadth of evoked immune responses [3]. Thus, smallpox vaccination has a protective effect against mpox infection and the severity of symptoms [3, 5, 12]. Therefore, one preventive measure immediately implemented in 2022 was the pre- or postexposure immunization of people at risk with a vaccine derived from replication-deficient modified vaccinia virus Ankara (MVA), originally designed against smallpox [13–15]. This vaccine was approved for mpox vaccination by the Food and Drug Administration in 2019 and by the European Medicines Agency in 2022 [16, 17], and is both safe and immunogenic in PWH [18–21]. While the WHO and US health authorities recommend a 2-dose regimen for all vaccinees irrespective of childhood smallpox vaccination or immune status [13, 22], the German Standing Committee on Immunization recommends a 2-dose regimen for individuals with a history of smallpox vaccination only in the setting of immunosuppression [14, 15].

Here, we analyzed mpox-specific B and T-cell mediated immune responses elicited by MVA vaccination or natural infection longitudinally in a cohort of men who have sex with men, largely with concomitant HIV infection. This study expands our understanding of cross-reactivity to other OPXV viruses mediated by smallpox vaccination and the durability of immune responses elicited by childhood smallpox vaccination >40 years ago in PWH.

METHODS

Study Population

This double-site, observational study was approved by the Institutional Review Board of the Medical Faculty at Ludwig-Maximilians-Universität Munich (LMU; project number 19-0849), and was conducted in accordance with the ethical standards of the Helsinki Declaration. Patients recovered from mpox infection (n = 5) and individuals vaccinated subcutaneously with JYNNEOS (n = 17) were recruited at the outpatient Department of Infectious Diseases at LMU Hospital and Prinzmed practice (detailed cohort description in Supplementary Table 1). Fifteen of 17 vaccinated subjects received a 2-dose regimen, irrespective of historic smallpox vaccination. After obtaining informed written consent, clinical information and specimens were collected and pseudonymized. Samples from vaccinated individuals were collected before and 3 months after each vaccination. Convalescent participants were sampled shortly after clinical remission and 3 months later.

Specimen Collection and Processing

Within 2 hours after blood collection, plasma was extracted from EDTA blood and peripheral blood mononuclear cells (PBMC) were isolated and stored at −80°C or in liquid nitrogen, respectively. For anal swabs, a PAP-Cone (Menton Medizintechnik) was moistened either with H₂O or R10 media (RPMI-1640 [Sigma-Aldrich] supplemented with 10% fetal calf serum [FCS], 1% HEPES, 1% L-glutamine, 1% penicillin/streptomycin), inserted 2–3 cm into the anal canal, and rotated. The oral swab was obtained by wiping the PAP-Cone under and on the tongue and rotating on both sides of the jowl. The PAP-Cones were put into ThinPrep Preserve-Cyt solution (Hologic) for DNA extraction or in R10 media to obtain mucosal immune cells, and vortexed immediately. The PAP-Cone in R10 media was washed with 10 mL phosphate-buffered saline (PBS; Sigma-Aldrich) over a 100-µm cell strainer (Greiner). Cells in the media were pelleted, resuspended in PBS, filtered through the cell strainer, and cryopreserved in liquid nitrogen.

Activation-Induced Marker Assay

To analyze upregulation of activation-induced markers (AIM) on T cells, we developed an AIM assay based on previously published protocols with some modifications [23, 24]. In brief, PBMC were thawed and 1.5 × 10⁶ cells/well were plated on a 96-well plate (Sarstedt) and rested for 5 hours (37°C, 5% CO₂). Anti-CD40 monoclonal antibody (0.5 µg/mL; Miltenyi Biotec) was added to each well and incubated for 15 minutes (37°C, 5% CO₂). PBMC were stimulated with 1 µg/mL of mpox CD4 or HIV Gag (CD4 or CD8 [25]; Supplementary Table 2) peptide pools or 0.2 µg/mL of each of 5 different mpox CD8 peptide pools (MP1–5). The mpox peptide pools consisted of predicted CD4 or CD8 immunodominant epitopes as described elsewhere [24] (Supplementary Table 3). The unstimulated control was treated with the same volume of dimethyl sulfoxide as the stimulated samples. Staphylococcous Enterotoxin B (1 µg/mL; Sigma-Aldrich) served as a positive control. After 18 hours, the cell culture supernatant was harvested and frozen at −80°C for subsequent multiplex cytokine analysis. Cells were washed with PBS plus 1% FCS and stained for 15 minutes with a live/dead marker (ZombieNir Fixable Viability Kit; Biolegend) and for 30 minutes with the antibody master mix to detect CD8, CD4, CD19, CD14, CXCR5, CD3, CD137, CD69, and OX40 (Supplementary Table 4). After washing with PBS plus 1% FCS, cells were resuspended in Fix&Perm Medium A (Invitrogen) and incubated at room temperature for 15 minutes. Cells were washed and resuspended in PBS and acquired on a BD LSR Fortessa (BD Bioscience). Anti-mouse CompBeads (BD Bioscience) and rainbow beads (BD Bioscience) were used. Gating, as depicted in Supplementary Figure 1, was performed with FlowJo version 10.10. The depicted frequencies of activated CD4 (OX40⁺CD137⁺) and CD8 (CD69⁺CD137⁺) T cells were obtained after background subtraction (unspecific activation measured in the unstimulated control). Individuals, whose frequency of activated T cells exceeded > 1× SD of background were considered as responders.

Multiplex Cytokine Assay

The cell culture supernatant after stimulation (AIM) was used for multiplex cytokine analyses using the Luminex Performance Human XL Cytokine Panel (R&D System) according to the manufacturer's instructions. Beads were measured with a MAGPIX CCD Imager (Luminex) and detected CCL4, granzyme B, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin 2 (IL-2).

Detection of Antibody Responses by ELISA and Neutralization Assay

The in-house enzyme-linked immunosorbent assay (ELISA) was performed as described before [26]. Briefly, binding of immunoglobulin G (IgG) and IgM antibodies to immobilized UV-inactivated RIPA lysate from vaccinia virus (VACV; New York City Department of Health Laboratories) infected or noninfected HEp2 cells was measured. Heparin was added to the plasma samples to a final concentration of 1% before they were inactivated with 0.5% Triton X-100% and 0.5% Tween-20 (56°C, 30 minutes). After centrifugation (1500g, 10 minutes) the supernatant was used for antibody detection. Each sample was measured in 1:100 and 1:1000 dilutions on VACV-infected and noninfected HEp2 cell lysate. Signals for binding to noninfected HEp2 lysate were subtracted from signals for binding to VACV-infected HEp2 lysates to account for unspecific binding, leading to Delta results. On each plate, a standard curve was included using a 1:4 dilution series of vaccinia immune globulin (polyclonal anti-vaccinia virus [immune globulin G, human], NR-2632; obtained through BEI Resources, National Institute of Allergy and Infectious Diseases). Delta results were quantified by interpolation to the standard curve by using a 4-parameter sigmoidal fit over log-transformed Vaccinia immune globulin concentrations using the statistical software R (version 4.1.2) and the drLumi package (version 0.1.2) [27]. Dilutions above or below the limit of quantification were excluded. For detection of neutralizing antibodies (nAb) a microneutralization test was performed as described previously [26]. Therefore, heparin was added to the plasma samples to a final concentration of 1%. After centrifugation (1500g, 10 minutes), samples were diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) to 6 two-fold dilution steps, resulting in dilutions of 1:10–1:320. A volume of 500 µL of virus stock (mpox virus [MPXV] clade IIb in-house isolate, titer of 1000 TCID₅₀/mL [50% tissue culture infectious dose/mL]) was added to 500 µL of each respective serum dilution and mixed. Following incubation (room temperature, 1 hour), 100 µL of each virus/serum dilution was added per well, in 8 replicates, to 96-well plates containing Vero E6 cells seeded the previous day (1.5 × 10⁴ cells/well in 100 µL of medium) and incubated (37°C, 5% CO₂). The virus stock was titrated as a control: 1 mL of the virus dilution was mixed with 1 mL of medium and incubated (room temperature, 1 hour). Decimal dilutions were prepared in medium, resulting in dilutions of 1:10¹–1:10⁹, then 100 µL of each virus dilution was added to cells as described above. As a negative control, 100 µL of medium/well was added to 8 wells. After 7 days, the cells were inspected by microscopy for cytopathic effects (CPE). The titer of the virus stock was calculated according to the following formula: TCID₅₀/mL = 10^((n/8 + 0.5))/0.5, where n = number of wells with CPE. The titer of the plasma samples was calculated according to the following formula: titer (1:x) = 10 × 2^((n/8 + 0.5)), where n = number of wells without CPE.

Phenotyping of Mucosal Immune Cells

Cells isolated from anal swabs were analyzed by flow cytomentry. After thawing, cells were washed and stained with a live/dead marker and antibodies as described above. Following antigens were detected: CCR5, CD8, CD3, HLA-DR, PD-1, CD4, CD45RO, CD69, CD45, and CD103 (Supplementary Table 5). After washing with a PBS-based buffer supplemented with 1 mM EDTA and 1% bovine serum albumin, cells were fixated with Fix&Perm Medium A. Briefly before acquiring the sample, cells were filtered through a 35-µm cell strainer (Falcon). Flow cytometry data were analyzed with FlowJo version 10.10 and the detailed gating strategy is depicted in Supplementary Figure 2. Only samples with sufficient cells were included (>50 CD3⁺CD45⁺, whole analysis; < 50 CD3⁺CD45⁺ and >10 CD4⁺ or CD8⁺ cells, CD4⁺/CD8⁺ ratio).

DNA Isolation and Mpox PCR

DNA isolated from swabs with QIAamp DNA Mini Kit (Qiagen) was analyzed with the Novaplex MPXV Assay (Seegene), both following the manufacturer's protocol.

Statistical Analysis

Statistical analyses were performed with GraphPad Prism version 10.1.2 (GraphPad Software) and P values <.05 were considered significant. Statistic was performed with 2-tailed Mann-Whitney test for longitudinal comparison and 2-tailed Wilcoxon matched-pairs signed rank test to compare different groups.

RESULTS

Description of the Study Cohort

All participants were male. The median age was higher in the vaccinated than in the convalescent group (Table 1; detailed cohort description in Supplementary Table 1). Except for 2 convalescent participants all individuals were PWH on antiretroviral therapy with stable CD4 counts. Eleven of 17 vaccinated individuals reported childhood smallpox vaccination. One of the convalescent participants was vaccinated 5 days prior symptom onset; however, it is unclear whether the infection was acquired before or after vaccination. All convalescent participants had a mild, self-limiting clinical course without requiring hospitalization. All except 1 reported to have acquired mpox in Germany. Diagnosis was made median 5 days (interquartile range, 4–6 days) and patients were released from isolation 27 days after symptom onset. Mpox-specific skin lesions were detected at multiple sites: anus 2 of 4, penis 2 of 4, face and neck 2 of 4, back 2 of 4, hand 2 of 4, legs 1 of 4, and 1 patient with missing documentation. Although patients were recruited after complete clinical remission, mpox DNA was still detectable in 2 participants. One patient was orally positive (cycle threshold [Ct] value, 39.31) and another anally (Ct value, 34.35; cut-off, 45).

Table 1.

Basic Cohort Characteristics

Characteristic	MVA-Vaccinated Individuals	Individuals Recovered From Mpox
n	17	5
Age, y, median (IQR)	57 (47–63)	51 (49–52)
Ethnicity	15 Caucasian 2 Asian	4 Caucasian 1 Hispanic
PWH	17 of 17, all on ART	3 of 5, all on ART
Plasma VL, copies/mL	all <50	all <50
CD4 cell count, cells/µL, median (IQR)	628 (594–689)	581 (574–689)
CD4 nadir, cells/µL, median (IQR)	236 (174–409)	139 (94.5–350)
Time between immunization and sampling, median (IQR)	1st dose 11 (6–13) wk 2nd dose 13 (11–13) wk	27 (23–27) d

For detailed information see Supplementary Table 1.

Abbreviations: ART, antiretroviral therapy; IQR, interquartile range; MVA, modified vaccinia Ankara; PWH, people with HIV; VL, viral load.

High OPXV-Specific IgG Titers After 2-Dose Vaccination and Mpox Remission

As OPXV-specific antibodies are thought to correlate with protection mediated by human smallpox vaccination [12, 28], we first analyzed IgM and IgG responses evoked by vaccination or mpox infection. Due to high cross-reactivity [3], IgG and IgM titers against VACV-infected cells served as correlate for mpox-specific antibody response and were assessed by ELISA.

While none of the participants had an OPXV-specific IgM response at the analyzed time points (data not shown), a robust IgG response was detected in both groups (Figure 1A). Already at the prevaccination sampling time point, 59% of the vaccinated cohort showed OPXV-specific IgG responses, albeit at a low level, reflecting childhood smallpox vaccination (Figure 1A). The proportion of individuals responding with IgG titers above the cutoff increased to 93% after the first dose and to 100% after the second dose. Simultaneously, the magnitude of IgG responses increased significantly compared to the prevaccination time point (Figure 1A). Mpox infection evoked a strong IgG response in all convalescent individuals (Figure 1A). The IgG responses of the 2 mpox convalescent individuals without HIV infection were within the range of convalescent PWH. Interestingly, there was a significant difference in magnitude of IgG responses between individuals with and without childhood smallpox vaccination at every sampling time point (Figure 1B) and 1 vaccination was sufficient to elicit detectable IgG responses in all childhood smallpox-vaccinated individuals.

Plasma from responders was subsequently analyzed for the neutralization capacity against mpox virus. Trends observed for overall IgG response were strengthened for nAb (Figure 1C). Significantly higher and preexisting nAb titers were detectable in childhood-vaccinated individuals compared to their counterparts without childhood smallpox vaccination. Nevertheless, plasma from mpox recovered individuals showed by far the highest neutralization capacity.

Figure 1.

Humoral immune responses in MVA-vaccinated and mpox recovered individuals. A, Longitudinal IgG levels before and after vaccination(s) (circles) and after natural infection (triangles). Dark brown triangles indicate patients without HIV infection. Green segments of pie charts depict the proportion of individuals with IgG titers above the cutoff (responders; cutoff, >62.98 µg/mL). B, Differences in IgG titers between vaccinees with (born before 1976; dark purple circles) and without (born after 1976; light purple circles) preceding childhood smallpox vaccination before and after each of the MVA vaccination doses. C, Neutralizing capacity against mpox detected in responders as defined in (A). A–C, Median and interquartile range are depicted. P values were calculated with 2-tailed Mann-Whitney test for longitudinal comparison or 2-tailed Wilcoxon matched-pairs signed rank test to compare different groups. A and B, Plasma IgG levels were analyzed by ELISA. Abbreviations: ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IgG, immunoglobulin G; MVA, modified vaccinia Ankara; nAb, neutralizing antibody; prevac, prevaccination.

Mpox-Specific T-Cell Responses Elicited by Vaccination and Infection

Next, we investigated mpox-specific T-cell responses to address immunological breadth of recalled memory immune responses. T-cell responses were assessed by AIM assay coupled with a multiplex bead-based immunoassay of cytokines released into cell supernatant after stimulation. The first dose of vaccination led to a 4-fold, but not significant, increase of activated CD4 T cells, an effect not profoundly altered by the second vaccination (Figure 2A). This trend reflected the number of individuals responding with an mpox-specific CD4 response: the first dose raised the frequency of responders from 47% before vaccination to 73% after first vaccination. After the second dose, 80% of vaccinated individuals showed a detectable response. The magnitude of mpox-specific CD4 T cells overall was low and fell within the range of HIV-specific CD4 T cells in our cohort of PWH with suppressed viral loads. The convalescent participants responded with a high interindividual range. Consistent with the nonsignificant increase of overall activated CD4 T cells, the levels of secreted IFN-γ (not significant) and IL-2 (P = .0353) were elevated after the second vaccination, with comparable magnitude of responses observed in convalescent individuals (Figure 2B and 2C). The frequency of activated CD4 T cells positively correlated with the amount of IFN-γ (Spearman r = 0.4636; P = .0099) and IL-2 (Spearman r = 0.4927; P = .0057) measured in cell supernatant after stimulation in vaccinated individuals (Supplementary Figure 3A). Regarding CCL4, we found a significant difference in magnitude based on childhood smallpox vaccination status, which was reduced following the second dose of vaccination (Figure 2D). This was the only detectable difference between patients with and without childhood smallpox vaccination regarding CD4 T-cell responses.

Figure 2.

Mpox- and HIV-specific CD4 T-cell responses in MVA-vaccinated and mpox convalescent individuals. PBMC were stimulated with CD4 T-cell–specific mpox or HIV peptide pools. A, Cells were analyzed by flow cytometry-based AIM and (B–D) cytokines were quantified from the cell culture supernatants by a multiplex bead-based immunoassay. A, Frequency of activated (OX40⁺CD137⁺) CD4 T cells after stimulation with HIV (first bar) or mpox-specific (bars 2–5) peptide pools at different time points. The unspecific activation measured in the unstimulated control (background) was substracted from all data. The frequency of responders is depicted as green segments of the pie charts (% of activated CD4 T cells > 1× SD of background). B, IFN-γ and (C) IL-2 expression after mpox stimulation in vaccinated (prevaccination and after the second dose) and recovered individuals. D, Differences in CCL4 levels in cell supernatant following mpox CD4 peptide pool stimulation in mpox vaccinees depending on the childhood smallpox vaccination status. A–D, Median and interquartile range are depicted. Circles indicate vaccinated individuals (n = 17) and triangles indicate convalescent individuals (recovered, n = 5). Dark purple circles indicate patients with childhood smallpox vaccination, dark brown triangles indicate patients without HIV infection. ULOQ and LLOQ within the range of the graph are depicted as dotted lines. Statistics were performed with 2-tailed Mann-Whitney test for longitudinal comparison and 2-tailed Wilcoxon matched-pairs signed rank test to compare different groups. Abbreviations: AIM, activation induced marker assay; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IL-2, interleukin 2; LLOQ, lower limit of quantification; MVA, modified vaccinia Ankara; PBMC, peripheral blood mononuclear cell; prevac, prevaccination; ULOQ, upper lower limit of quantification.

Slightly different results were observed regarding the dynamics of mpox-specific CD8 T-cell responses in our cohort. Here, the first vaccination dose increased the level of activated CD8 T cells above the level of positivity only at an individual basis and did not change the proportion of individuals with detectable mpox-specific response (Figure 3A). After the second dose, however, the frequency of activated CD8 T cells increased significantly. Here, the frequency of responders was unaltered at 40% after the first vaccination and increased to 73% after the second vaccination. Of note, only convalescent individuals without concomitant HIV infection showed a measurable surface activation of CD8 T cells following stimulation. Conversely, vaccination led to a significant increase in cytokine secretion following mpox-specific stimulation to comparable levels as in the group of convalescent individuals (Figure 3B and 3C). Here, the increase of overall activated CD8 T cells significantly correlated with the increase of IFN-γ (Spearman r = 0.5272; P = .0028), but not with IL-2 secretion (Spearman r = 0.3348; not significant) in vaccinated individuals (Supplementary Figure 3B). Although the highest responses of CD8 T cells was observed in patients with childhood smallpox vaccination, the differences were not significant when compared to patients without childhood smallpox vaccination. However, as observed for the CD4 T-cell response, CCL4 levels in cell supernatant were significantly elevated in childhood-vaccinated individuals, an effect that faded after the second vaccination (Figure 3D).

Figure 3.

Mpox- and HIV-specific CD8 T-cell responses in MVA-vaccinated and mpox convalescent individuals. After stimulation with CD8 T-cell specific mpox or HIV peptide pools, PBMC and the cell culture supernatant were analyzed as described in Figure 2. A, Frequency of activated (CD69⁺CD137⁺) CD8 T cells after stimulation. Data is background subtracted. The frequency of responders is depicted as green segments of the pie charts as in Figure 2A. B, IFN-γ and (C) IL-2 expression after mpox stimulation in vaccinated (before vaccination and after the second dose) and recovered individuals. D, Differences in CCL4 levels in cell supernatant following mpox CD8 peptide pool stimulation in mpox vaccinees depending on the childhood smallpox vaccination status. A–D, Median and interquartile range are depicted. Purple circles indicate vaccinated individuals (n = 17) and brown triangles indicate convalescent individuals (recovered, n = 5). Dark purple circles indicate patients with childhood smallpox vaccination, dark brown triangles indicate patients without HIV infection. ULOQ and LLOQ within the range of the graph are depicted as dotted lines. Statistic was performed with 2-tailed Mann-Whitney test for longitudinal comparison and 2-tailed Wilcoxon matched-pairs signed rank test to compare different groups. Abbreviations: AIM, activation induced marker assay; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IL-2, interleukin 2; LLOQ, lower limit of quantification; MVA, modified vaccinia Ankara; PBMC, peripheral blood mononuclear cell; prevac, prevaccination; ULOQ, upper lower limit of quantification.

Mucosal Immune Status After Mpox Infection

Anal mucosa is a relevant infection site for mpox virus in the recent outbreak, with 50% of individuals in our cohort presenting with anal lesions. Therefore, we investigated anal T-cell phenotypes after mpox infection. Patients recovered from mpox showed comparable frequencies of anal CD4 and CD8 T cells to the control group of vaccinated individuals without mpox infection. Nevertheless, a trend towards higher frequencies of tissue-resident and activated CD4 T cells was observed after mpox infection independent of the sampling time point. No difference was detected for memory CD4 and for CD8 T-cell subpopulations. Remarkably, the patient with anally detectable mpox DNA after complete clinical remission showed the highest level of memory, tissue-resident, and activated CD4 T cells as well as the highest level of memory and tissue-resident CD8 T cells (Figure 4).

Figure 4.

Phenotyping of anal mucosal (A) CD4 and (B) CD8 T cells after mpox infection compared to samples from noninfected individuals. Cells isolated from an anal swab were analyzed by fluorescence-activated cell sorting. Triangles indicate convalescent individuals and circles noninfected individuals (vaccinees, control). The red triangle highlights the patient with detectable anal mpox DNA by polymerase chain reaction (PCR). Only samples with sufficient cells were included (> 50 CD3⁺CD45⁺, whole analysis; < 50 CD3⁺CD45⁺ and > 10 CD4⁺ or CD8⁺ cells, CD4⁺/CD8⁺ ratio). Median and interquartile range are depicted.

DISCUSSION

During the global mpox outbreak in 2022–2023, PWH were disproportionally at risk for contracting mpox infection [6–9] and potentially for suffering more severe disease courses due to HIV-associated immune deficiency [9, 11, 29–31]. In the presented study we aimed to broaden the understanding of mpox-specific immune responses elicited by MVA vaccination or natural infection, focusing on PWH. While others report only a moderate antibody response, including neutralization capacity, elicited by the MVA-based vaccine [32–34], we detected strong humoral immune responses. This discrepancy might be explained by the broader antigen spectra used in our assay, the reported delayed kinetics (peak 42 days after first immunization) regarding antibody production following MVA vaccination [35], and the high percentage of individuals reporting childhood smallpox vaccination in our study. The absence of IgM responses can be explained by the large time interval of 12 weeks between immunization and sampling. Cellular immunity is believed to be important for controlling poxvirus infection [3, 36], and previous studies have analyzed T-cell immunity in recovered [37–39] and MVA-vaccinated individuals [32–34, 40]. In an animal model of mpox infection, macaques with <300 CD4 T cells/mm³ prior to immunization had increased infection severity after virus challenge [41]. In line with this, patients with HIV-related CD4 T-cell deficiency might experience a fulminant course after infection [7, 12, 29, 30], and more frequent breakthrough infections after vaccination due to insufficient immune responses [42, 43]. In our cohort of PWH with stable disease, 2-dose vaccination led to detectable, albeit low-frequency, mpox-specific CD4 and CD8 T-cell responses. However, the increase of total mpox-specific CD4 T cells in response to MVA vaccination was not significant and peaked within the range of low-frequency HIV-specific responses in aviremic individuals. While the second dose did not profoundly alter mpox-specific CD4 T-cell responses, this did not hold true for CD8 T-cell immunity. Here, only the second dose increased the number of individuals responding and the magnitude of mpox-specific immune responses compared to a single-dose regimen. We detected higher levels of CD8 T-cell responses compared to others [24, 32], which might be explained by the composition of the cohort or the stimulation conditions.

One conceivable reason for the increasing number of mpox outbreaks in the past decades is a worldwide decline of OPXV immunity due to the decreasing number of people who received a smallpox vaccination before smallpox eradication in the 1980s [3, 5, 44, 45]. This supports the hypothesis that historic smallpox vaccination mediates a cross-protective effect against other OPXV infections, like mpox [45, 46]. Regarding T-cell immunity, however, the only difference we observed between childhood smallpox-vaccinated individuals and others was CCL4 expression in the prevaccination state. This marker has been associated with memory formation [47, 48]; however, as the sole differing T-cell–associated factor in our experiments, no conclusion regarding T-cell–mediated protection can be drawn from this observation. We, like others [49, 50], detected a stronger association of childhood smallpox vaccination with the magnitude of humoral than of cellular responses. Indeed, antibody-mediated immunity to OPXV is very long lasting, even in PWH [21], including our cohort of people infected with HIV for a median of 10 years. We observed significant differences between patients with and without childhood smallpox vaccination regarding the magnitude of IgG responses and neutralization capacity of the elicited antibodies. Individuals with detectable IgG and nAb before MVA vaccination due to childhood smallpox vaccination only moderately profited serologically from the second dose in our cohort. Mazzotta et al [33] reported that a single-dose regimen in prevaccinated PWH without detectable humoral immune response before MVA vaccination led to lower rates of seroconversion compared to healthy controls. Because measurement of antibody titers to guide the vaccination schedule is not always readily available in clinical practice, a pragmatic way to handle these findings in PWH is the recommendation of a 2-dose regimen irrespective of childhood smallpox vaccination, as currently done by most health authorities [13, 14, 22].

The 5 mpox convalescent individuals showed high interindividual ranges in the magnitude of immune responses, but the responses did not significantly differ from vaccinated individuals, except for high nAb titers. In this study, we also analyzed anal immune responses in mpox-convalescent individuals, but the small number of 4 participants per group with evaluable mucosal immune cells due to sample processing difficulties prohibits general conclusions. However, it is remarkable that it is the individual with anally detectable mpox DNA despite clinical cure at the time of sampling that drives the increase of tissue-resident and activated CD4 T cells in the convalescent group. General conclusions cannot be drawn from our small study, and larger studies are required to strengthen the findings, especially regarding the recovered group. Reflecting the advanced age of PWH attending our outpatient clinic, a history of childhood smallpox vaccination was unevenly distributed among our vaccinated group.

In conclusion, this study shows that 2-dose MVA vaccination as well as natural infection evoked detectable mpox-specific immune responses mediated by B cells as well as CD4 and CD8 T cells in PWH. Moreover, it improves our understanding of smallpox vaccination-mediated cross-reactivity to other OPXV viruses and the durability of childhood smallpox vaccination-mediated immune responses.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. E. G. conducted all experiments except for the serological assays. M. G. and D. S. performed the serological assays. V. O. established the AIM assay in our laboratory. T. M. E. supported the multiplex cytokine assay. G. I., N. P., and U. S. recruited participants and collected samples. G. R. and R. S. processed the samples. R. C. and C. D. provided technical equipment. A. G. and A. S. provided the mpox and HIV peptide pools. J. R. obtained funding for this study. E. G., U. S., and J. R. conceptualized the study. E. G., J. B., U. S., and J. R. wrote and revised the manuscript. All authors had full access to the data used in the study, interpreted the data, provided critical input, and revised the manuscript.

Acknowledgment. We thank all study participants and physicians who recruited participants.

Financial support. This work was supported by the Else-Kröner-Fresenius-Stiftung (grant number 2021_EKEA25 to J. R.).

Author notes

Presented in part: Kongress für Infektionskrankheiten und Tropenmedizin, 16 June 2023, Leipzig, Germany, talk; and German Center for Infection Research Annual Meeting, 25 September 2023, Hannover, Germany, poster presentation, abstract No. P-2-63.

Potential conflicts of interest. A. S. is a consultant for Gritstone Bio, Flow Pharma, Moderna, AstraZeneca, Qiagen, Fortress, Gilead, Sanofi, Merck, RiverVest, MedaCorp, Turnstone, NA Vaccine Institute, Emervax, Gerson Lehrman Group, and Guggenheim. A. G. is a consultant for Pfizer and Sanofi. L. J. I. has filed for patent protection for various aspects of T-cell epitope and vaccine design work. All other authors report no potential conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.