Orthopoxvirus-Specific T-Cell Responses in Convalescent Mpox Patients

Abstract

Orthopoxvirus-specific T-cell responses were analyzed in 10 patients who had recovered from Mpox including 7 people with human immunodeficiency virus (PWH). Eight participants had detectable virus-specific T-cell responses, including a PWH who was not on antiretroviral therapy and a PWH on immunosuppressive therapy. These 2 participants had robust polyfunctional CD4⁺ T-cell responses to peptides from the 121L vaccinia virus (VACV) protein. T-cells from 4 of 5 HLA-A2–positive participants targeted at least 1 previously described HLA-A2–restricted VACV epitope, including an epitope targeted in 2 participants. These results advance our understanding of immunity in convalescent Mpox patients.

Orthopoxvirus-specific T-cell responses were characterized in 10 patients who recovered from mpox. Responses were present in 6 of 7 people with HIV and in 2 of 3 HIV-seronegative individuals.

The 2022 global mpox outbreak disproportionally affected men who have sex with men, and a large percentage of those affected in this group were people with HIV (PWH) [1]. In one study, PWH with uncontrolled human immunodeficiency virus (HIV) replication represented more than 80% of hospitalized patients with mpox [2]. The immune mechanisms behind the control of Mpox virus are unknown, but studies suggest that T-cell responses play a protective role against severe disease [3]. Prior studies have found comparable T-cell responses to vaccinia virus (VACV) in convalescent plasma from patients with Mpox and VACV vaccine recipients [4]. More recently, a study showed that T-cell responses to VACV peptide pools were seen within 8 to 10 days after onset of mpox symptoms [5]. However, there are little data on the exact epitopes T cells target in persons with Mpox. This study analyzed T-cell responses in convalescent mpox patients to orthopoxvirus peptides and previously described HLA-A2–restricted VACV epitopes [6–10]. Our results further our understanding of the immune response to the Mpox virus (MPXV).

METHODS

The study was approved by the Johns Hopkins Medicine Institutional Review Boards and informed consent was obtained from all study participants. Peripheral blood mononuclear cells (PBMCs) were collected from whole blood after Ficoll-Paque PLUS gradient centrifugation (GE Healthcare Life Sciences). For some experiments, CD8⁺ T cells were depleted using Miltenyi Biotec CD8⁺ T-Cell Positive Selection Kits.

An orthopoxvirus pool consisting of 127 peptides from selected proteins of VACV, MPXV, and variola virus, as well as peptide pools for the putative 49.8-kDa protein (O74R), the cell surface-binding protein (105L), and 2 subunits of the major core protein P4a (121L), were purchased from JPT Peptide Technology. The peptides were mostly 15 mers with 11 amino acid overlap. In addition, a panel of 34 previously described HLA-A2–restricted VACV peptides [6–8] were obtained from BEI resources. An additional 6 previously described HLA-A2 peptides were synthesized at Genscript. The peptides were VLSLELPEV from the D13L protein [9], ILMDNKGLGV (VACV) and ILMNNKGLGV (MPXV) from the F1L protein [9], SLSNLDFRL from the F11L protein [10], KLLLGELFFL from the J3R protein [10], and GLLDRLYDL from the O1L protein [10]. HIV Gag and Nef overlapping peptides were obtained from the NIH AIDS Reagent Program and used at a concentration of 10µg per peptide/mL. Overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human coronavirus NL63 (HCoV-NL63), HCoV-OC43, and HCoV-229e were obtained from the Biodefense and Emerging Infections Research Resources Repository and used at 10 µg per peptide/mL.

ELISpot Plus interferon-γ (IFN-γ) kits with precoated plates were purchased from Mabtech. The wells were plated with unfractionated PBMCs or CD8⁺ T cell–depleted PBMCs at 150,000 cells/well, and the cells were cultured for 20 hours with peptides at a concentration of 1 μg/mL. The plates were then processed according to the manufacturer's protocol and read by a blinded independent investigator using an automated reading system. Two replicates per pool were run; a peptide was only considered positive if both wells had values at least twice the average of the untreated wells and the average stimulation index was above 3, and more than 30 spot-forming units (SFU)/million cells were present. The stimulation index is the ratio of SFU for each peptide or peptide pool divided by the SFU in response to media alone.

Intracellular cytokine staining was performed after PBMCs had been cultured with peptide pools for 16 hours in the presence of Golgi Plug and Golgi Stop. The cells were stained with a live/dead dye and fluorescent antibodies to CD3, CD4, and CD8. They were then fixed, permeabilized, and stained with antibodies to IFN, tumor necrosis factor-α (TNF-α), and interleukin 2 (IL-2). Flow cytometry was performed with a LSRFortessa cytometer.

To determine HLA-A2 status, PBMCs were stained with a monoclonal antibody to HLA-A2 (clone BB7.2) from Biolegend.

Statistical comparisons were performed using GraphPad Prism 9.2.0. A 2-tailed P test with Welch correction was used to compare responses in Mpox participants and healthy donors.

RESULTS

Study Participants

Details of the participants demographics are shown in the Supplementary Table. We enrolled 10 participants based on positive orthopoxvirus polymerase chain reaction (PCR) results in the Johns Hopkins Health System. The median time between the positive test and study enrollment was 104 days (range 55 to 160 days). Participants who had received a VACV vaccine were excluded from the study. The participants included 7 non-Hispanic black cisgender men, 2 Hispanic white cisgender men, and 1 non-Hispanic black transgender woman, and the median age was 33 years (range 18–41 years). Of the 10 participants, 7 were PWH and 3 were HIV negative. Of the PWH, the median CD4 count was 554 cells/ul (range 360−1025). All the PWH participants were on suppressive antiretroviral therapy (ART) regimens except for MP4 who was recently diagnosed with HIV and had a viral load of 2160 copies/mL off antiretroviral treatment. Two of the PWH were admitted with proctitis (MP2, MP9), the remaining 8 patients had mild disease. Five PWH received the antiviral drug tecovirimat, whereas none of the 3 HIV-seronegative patients received tecovirimat. We also enrolled 10 healthy donors as controls. None of the 20 participants in the study had ever received a VACV vaccine.

T Cell Responses to Orthopoxvirus Peptides

T-cell responses to orthopoxvirus protein peptide pools were identified in 8 of 10 participants. Six of the 7 PHW had positive responses to the orthopoxvirus peptide pool, including MP4, who had recently been diagnosed with HIV and was not on ART, and MP9, who was on mycophenolate mofetil and tacrolimus for a solid organ transplant (Figure 1A). Two of the 3 seronegative patients also had detectable responses to this peptide. In contrast, no responses were seen in any of the 10 healthy donor controls and there was a significant difference in responses between the 10 convalescent mpox patients and the healthy donors (Supplementary Figure 1A). There was no obvious relationship between having received tecovirimat and the magnitude of the immune response and no obvious differences between PWH and HIV-seronegative participants, although this analysis is limited by the small number of patients in each category. We depleted CD8⁺ T cells in samples from 4 participants and saw a marked decline in the ELISpot responses, suggesting that the responses to the orthopoxvirus pool were generated by CD8⁺ T-cells (Supplementary Figure 1B). The T-cell responses of 5 PWH to the orthopoxvirus pool was comparable to their responses to HIV Gag and Nef peptides (Supplementary Figure 1C). The 2 participants who did not have significant responses to orthopoxviruses (MP7 and MP8) had relatively mild disease. They both had detectable T-cell responses to spike proteins from at least 1 common cold coronavirus (Supplementary Figure 1D), suggesting that they could make responses to other viral antigens.

Figure 1.

T-cell recognition of orthopoxvirus peptides. T-cell responses to the orthopoxvirus peptide pool (A), and the vaccinia virus 121L subunit 2 peptide pool (B) are shown. Each column represents responses for an individual HD or convalescent MP. Each dot represents an individual replicate. People with HIV are shown in red, and HIV-seronegative patients are shown in blue. MP1, MP2, MP3, MP7, and MP9 were treated with tecovirimat. The dashed line represents the cutoff for a positive response. C, Pie charts showing the proportion of polyfunctional cytokine expression for MP4 and MP9 CD4⁺ T cells. Pie arcs show the proportion making each cytokine as annotated. Abbreviations: HD, healthy donor; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IL-2, interleukin 2; MP, mpox patient; SFU, spot-forming unit; TNF, tumor necrosis factor.

There were no significant differences between the healthy donor and Mpox patient responses generated to peptide pools from the 2 subunits of the 121L protein and proteins 074R and 105L (Supplementary Figure 2). However, MP4 and MP9 made robust T-cell responses to peptides from the second subunit of the 121L protein (Figure 1B). Flow cytometry showed that the response was generated by polyfunctional CD4⁺ T cells that secreted IFN-γ, TNF-α, and IL-2 (Figure 1C and Supplementary Figure 3). In comparison, the CD8⁺ T-cell cytokine response to the orthopoxvirus peptide pool was lower in magnitude when analyzed by flow cytometry, and thus we could not determine whether the cells were polyfunctional.

T-Cell Responses to HLA-A2–Restricted Peptides

MPXV has more than 200 open reading frames making the mapping of epitopes challenging. We thus analyzed the responses to previously described VACV epitopes. Five participants were HLA-A2 positive. We analyzed their responses to a total of 40 HLA-A2–restricted peptides (Table 1). These participants targeted a median of 1 epitope (range 0 to 5 epitopes). Only 1 epitope, SLSNLDFRL from the F11L protein [10], was targeted by more than 1 individual. Antigen-induced CD8⁺ T-cell proliferation is a correlate of immunity and thus we tested the capacity of MP2 cells to proliferate in response to the peptide ILDDNLYKV. As shown in Supplementary Figure 4, there was a marked expansion in the number of reactive T cells after the cells were first incubated with the peptide for 10 days.

Table 1.

HLA-A2–Restricted VACV Peptides Targeted by 5 HLA-A2⁺ Convalescent Mpox Patients

Participant	Peptide Sequence	Protein	SFU/106 PBMCs	SI	Reference
MP2	ILDDNLYKV	VACWR082	110	11	7
MP2	HVDGKILFV	B14R	33	3	7
MP3	FLVIAINAM	D3R	58	115	8
MP3	MILVPLITV	A36R	55	110	8
MP3	RTLLGLILFV	A17L	110	220	7
MP3	IIIPFIAYFV	M1L	40	80	7
MP3	SLSNLDFRL	F11L	43	85	10
MP7	VLPFDIKKL	VETFsm	33	3	6
MP11	SLSNLDFRL	F11L	63	16	10
MP8	None	NA	NA	NA	NA

VACV peptides that were recognized out of a 40-peptide pool by each HLA-A2–positive mpox patient. The SFU per million PBMCs are shown, as is the SI.

Abbreviations: NA, not applicable; PBMC, peripheral blood mononuclear cell; SFU, spot-forming unit; SI, stimulation index; VACV, vaccinia virus. The reference refers to the manuscript describing the epitope.

DISCUSSION

Cellular immunity may play a role in the control of MPXV [3], but the human T-cell response to this virus has not been fully described. Agrati et al recently demonstrated that virus-specific T cells were present as early as 8 days after symptom onset in mpox patients [5]. We extend these results by showing that convalescent patients have robust T-cell responses. We also show potent polyfunctional CD4⁺ T-cell responses to the 121L protein in a PWH on immunosuppressive therapy and in a PWH who was not on antiretroviral therapy. This is particularly interesting as a polyfunctional response, where multiple cytokines and/or other effector proteins are secreted by the same cell, is a correlate of immunity against other viral infections [11]. It is notable that few of the previously described HLA-A2–restricted epitopes were targeted by our patients and only 1 peptide was targeted by more than 1 individual. However, this is consistent with prior studies that showed that allele-specific immunodominant responses, where the same epitopes are targeted by multiple individuals, were not commonly seen in VACV vaccine recipients [12, 13].

We did not observe potent responses to the 105L and O74R peptide pools, but VACV and MPXV both have more than 200 open reading frames (ORFs), and a study showed that VACV vaccine recipients made CD4⁺ T-cell responses to an average of 39% of 180 studied VACV ORFs [14]. Thus, it is possible that the commercially available proteins we studied were not immunodominant in the 10 participants with Mpox in our study and we would have observed better responses had we been able to study more proteins.

Our study is limited by the relatively small number of participants and the fact that we analyzed responses to VACV rather than MPXV proteins. However, a recent study demonstrated that 94% of CD4⁺ T-cell epitopes and 82% of CD8⁺ T-cell epitopes are conserved between VACV and MPXV, which is consistent with the high degree of sequence homology between the 2 viruses [15]. Thus, we expect the responses to VACV to be a decent reflection of MPXV-specific T-cell responses.

In summary, we show robust responses to orthopoxvirus peptides in samples from participants with convalescent Mpox, including polyfunctional CD4⁺ T-cell responses in a transplant PWH on immunosuppressive medication. We only identified a subset of epitopes targeted and it will be important to perform nonbiased mapping of the entire proteome in order to get a better picture of the breadth of the T-cell response. However, our identification of targeted HLA-A2–restricted epitopes in these patients will allow for the synthesis of tetramers and the detailed analysis of unstimulated antigen-specific T-cells. Further studies with larger cohorts will be needed to determine if there are quantitative or qualitative differences in these responses that might explain the high prevalence of Mpox in PWH.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases (grant number R21AI167705); the Clinical Characterization Protocol for Severe Emerging Infections, Johns Hopkins University; the Centers for AIDS Research, Johns Hopkins University; and the Infectious Diseases Precision Medicine Center of Excellence, Johns Hopkins University.