Pan‐pox‐specific T‐cell responses in HIV‐1‐infected individuals after JYNNEOS vaccination

People living with human immunodeficiency virus (HIV) are the individuals most affected by the current Monkeypox virus outbreak that was first announced in May 2022. Here we report Pan‐pox‐specific T‐cell responses in a cohort of HIV‐1‐infected individuals after receiving the nonreplicative, attenuated smallpox vaccine JYNNEOS from Bavarian Nordic. Intradermal (i.d.) and subcutaneous (s.c.) vaccination was safe without major side effects. Dose‐sparing i.d. vaccination was superior to s.c. vaccination and promoted T‐cell polyfunctionality, and the expression of the gut‐homing marker α4β7 integrin on lymphocytes. HIV‐1‐infected individuals with CD4 T‐cell counts ≤500/mm3 blood required at least a booster vaccination to exhibit efficient virus‐specific T‐cell responses. The magnitude of the Th1 response after this booster directly correlated with the CD4 T‐cell count of the vaccinees. Further studies with a larger number of participants are warranted to confirm and expand our observations.


| INTRODUCTION
Monkeypox (mpox) is a zoonotic virus infectious disease that can spread autochthonously between humans via direct contacts and respiratory routes. 1 It is caused by the mpox virus (MPXV), a member of the genus Orthopoxvirus of the family of Poxviridae, and is associated with fever, headache, muscle pain, swollen lymph nodes, rash, respiratory and rectal symptoms, exhaustion, and others. 2pending on the MPXV strain, and the age and immunocompetence of the host, disease symptoms can be very severe and may lead to death in few cases. 3MPXV is antigenically related to other Orthopoxviruses including variola virus, the cause of smallpox. 4This antigenic relatedness is the reason why smallpox vaccines provide >85% protection against MPXV infection in humans. 5th the worldwide eradication of smallpox in 1978 and the cessation of the mass smallpox vaccination campaigns, the incidence of mpox has continuously increased. 6Mpox usually appeared as individual outbreaks that were geographically limited to endemic areas in Central and West Africa. 7This situation dramatically changed in spring 2022 when a worldwide outbreak beyond the endemic regions was reported, 8 and human-to-human MPXV transmission has been estimated to occur since at least 2016.The cases were mainly among men having sex with men, many of which being persons living with human immunodeficiency virus (HIV) infection. 8,9Within this latter group, individuals with high HIV loads and/or low CD4 T-cell counts were especially susceptible to severe mpox disease. 9To protect this highly vulnerable group of individuals against MPXV infection and reduce virus spreading, vaccination would be the preferred option.However, a specific MPXV vaccine does not exist and, thus, a vaccination campaign would rely on the protective crossreactivity of smallpox vaccines.From the available ones, the nonreplicative JYNNEOS vaccine, approved in 2019, seems the best option. 10It induces negligible antibody responses but robust T-cell responses 11,12 that play a critical role against poxvirus infections. 13rthermore, it has less side effects than vaccines such as ACAM2000 TM , LC16 or Dryvax that are based on replicative vaccinia viruses and are contraindicated for immuno-compromised individuals. 14Importantly, American and European health authorities proposed a dose sparing intradermal (i.d.) route of JYNNEOS administration instead of the usual subcutaneous route (s.c), which extends the available vaccine doses by a factor of 5 and thus better covers increasing vaccine demands without compromising vaccine efficacy. 14,15However, clinical trials that evaluate JYNNEOS vaccination responses and compare different administration routes in HIV-1-infected individuals are lacking.Here we analyzed JYNNEOS vaccination-induced pan-pox-specific T-cell responses from HIV-1infected individuals whose virus load was controlled by antiretroviral therapy and whose CD4 T-cell counts were either ≤500/mm 3 blood (loCD4-group) or ≥500/mm 3 blood (hiCD4-group).The vaccine responses were compared with those from vaccinated healthy control (HC) individuals.We demonstrate that the dose-sparing i.d.vaccination route is preferable over the s.c.route, and that loCD4 individuals may require at least one vaccine booster to generate an efficient pan-pox-specific T-cell response.

| Sample collection and participant characteristics
This is an observational study that prospectively collected data and blood samples from HIV-1-infected individuals and HCs who were vaccinated with JYNNEOS, a modified vaccinia Ankara attenuated vaccine developed by Bavarian Nordic.The study groups consisted of (i) HIV-1-infected individuals with CD4 T-cell counts ≤ 500/mm 3 blood (n = 10; loCD4), (ii) HIV-1-infected individuals with CD4 counts ≥ 500/mm 3 (hiCD4) vaccinated s.c (n = 7), (iii) HIV-1-infected individuals with CD4 counts ≥ 500/mm 3 (hiCD4) vaccinated i.d (n = 7), (iv) HC vaccinated i.d (n = 7) as positive controls, and (v) unvaccinated healthy individuals (n = 7) serving as negative controls for the T-cell assays.All groups received a single JYNNEOS dose (0.5 mL; s.c vaccinees or 0.1 mL; i.d.vaccinees), except the loCD4 individuals who received a booster after 28 days due to their immunocompromised condition.Blood samples were collected before (Day 0) (pre-vaccine) and after first vaccination (28 days after vaccination) (1st vacc), with an additional collection 28 days after the booster vaccination for the loCD4 group (2nd vacc).Additionally, from 24 immunized participants, serum samples were taken 4-12 months post vaccination.Participant characteristics of the 24 HIV-1-infected individuals are outlined in Table 1.The median age of the HIV-1-infected individuals was 34 years (interquartile range [IQR]: 23-44), all were male, and only one had a coinfection.All were on highly active antiretroviral therapy, with 95.83% (23/24) having an undetectable HIV-1 viral load.The vaccinated HC individuals had a median age of 43 years (IQR: 27-59) and were all male.Unvaccinated HC individuals had a median age of 47 years (IQR: 27-67), with 28.56% (2/7) being male.All participants provided written informed consent and the study protocol was approved by the institutional review board of the Hospital del Mar, Barcelona.

| Quantification of pan-pox-specific immune responses
Specific T-cell responses were assessed from peripheral blood mononuclear cells (PBMCs) as previously described using enzymelinked immunospot (ELISpot) 16 and intracellular cytokine staining (ICS) 17 with adaptations as specified in the Supporting Information Binding antibodies against the MPXV surface proteins A29 (Sino Biologicals, SIB-40891-V08E-100) and A35 (Sino Biologicals, SIB-40886-V08H-100) were determined by enzyme-linked immunosorbent assays, 16 whereas neutralizing antibodies were determined by reduction of MPXV infectivity of Vero E6 cells. 18An extended Method description is given in the Supporting Information S8.

| RESULTS AND DISCUSSION
JYNNEOS vaccination has been shown to reduce the incidence of mpox and provide some protection against MPXV infection. 19To potentially protect highly vulnerable HIV-1-infected individuals during the recent MPXV outbreak and to evaluate their poxvirusspecific T-cell responses after vaccination, 24 HIV-1-infected individuals, all outpatients of the Infectiology Unit of the Hospital del Mar, Barcelona, participated in this study (see Table 1 for details).To cover the increased vaccine demands for protecting against mpox, the dose sparing i.d.route of JYNNEOS administration was proposed by the health authorities in the US and Europe. 14,15cordingly, we adapted our vaccine administration route so that study participants after August 2022 were given 0.1 mL i.d.instead of 0.5 mL s.c.JYNNEOS shots.This enabled us to compare both administration routes in the hiCD4 group of HIV-1-infected individuals (Figure 3A-C).Importantly, all HIV-1-infected individuals generated significant pan-pox-specific T-cell responses after i.d.vaccination, which was not the case after s.c.vaccination.Furthermore, IFN-γ ELISpot levels were significantly higher after i.d.than after s.c.vaccine administration.This strongly supports the proposed dose-sparing vaccination route also for the protection of immuno- Abbreviations: HHV8, human herpesvirus-8; HIV, human immunodeficiency virus; i.d., intradermally; uLD: undetectable HIV-1 viral load; HAART, highly active antiretroviral therapy; s.c., subcutaneously.
They were grouped according to their CD4+ T-cell counts as loCD4 or hiCD4, and received either the standard s.c.JYNNEOS vaccination (before August 2022) or the emergency dose-sparing i.d.vaccination after its approval in August 2022.14Seven HC individuals that work in the hospital and are exposed to MPXV-infected individuals also received the JYNNEOS vaccine as a prophylaxis.They were all i.d.vaccinated and served as positive controls for the induced T-cell responses.PBMCs from seven unvaccinated HCs served as negative controls.None of the participating individuals had been infected with MPXV.There were no changes in CD4+ T-cell counts or viral loads nor major side effects due to vaccination.The binding and neutralizing antibody titers from the available sera of 24 study participants (see Supporting Information S2: Figure S2) were low as previously described. 11,12To first evaluate JYNNEOS vaccine-induced T-cell responses in HIV-1-infected individuals (n = 24) and HCs (n = 7), PBMCs were isolated from all participants before and 28 days after the first vaccination time point, stimulated with a peptide pool of 127 panpox-specific HLA-I and HLA-II epitopes, and subjected to IFN-γ ELISpot and ICS assays.Results for the latter are given as composite Th1 responses in Figures 1-3, whereas the individual cytokine responses (IFN-γ, TNF-α, and IL-2) are given in Supporting Information S3-S5: Figures S3-S5.Strong and specific Th1 responses were observed for both groups of immunized participants (Figure 1A,B), emphasizing the high immunogenicity of the vaccine even after a single immunization.However, although all HCs responded well to the vaccine, nearly 30% (six of 22) of the HIV-1-infected individuals did not adequately respond (Figure 1A).All six low responders were vaccinated s.c.Three were from the loCD4-and three from the hiCD4 group.As all HC individuals were vaccinated i.d., it remains uncertain whether the low response was due to a lower immunogenicity of a single s.c.vaccination or to a lower vaccine response due to HIV-1 infection.Nonetheless, our previous results in immunizing HIV-1-infected individuals with the potent T-cell response-inducing messenger RNA-based coronavirus 2019 vaccine BNT162b2 argue to strongly consider a low immune responsiveness of this patient group. 16The CD4+ T-cell count in HIV-1-infected individuals is inversely related to their immunodeficiency.To test whether and how the CD4+ T-cell count relates to JYNNEOS vaccine responsiveness, pan-pox-specific T-cell responses of the loCD4 group were analyzed 28 days after the first (1st vacc) and 28 days after the second s.c.vaccination (2nd vacc) (Figure 2).Albeit not all vaccinees responded, the second vaccination increased median ELISpot (1st vacc: 78 [0-156]; 2nd vacc: 130 [6-254]) and Th1 CD4 (1st vacc: 0.455 [0.07-0.84];2nd vacc: 0.815 [0-1.63]) and CD8 T-cell responses (1st vacc: 0.340 [0-0.68];2nd vacc: 0.480 [0-0.96])(Figure 2A-D).There was a significant linear relation between CD4+ T-cell counts and the vaccine-induced IFN-γ ELISpot response after the second vaccination (Figure 2B).Thus, the standard two-dose s.c.JYNNEOS vaccination efficiently induces pan-pox-specific T-cell responses in HIV-1-infected individuals in a CD4+ T-cell-count-dependent manner.
compromised individuals.Figures combining all vaccination responses(Supporting Information S6: FigureS6) and T-cell polyfunctionalities (Supporting Information S7: FigureS7) are provided to enable simple head-to head comparisons.The integrin α4β7 plays a crucial role in the trafficking of T cells to the gut-associated lymphoid tissue.There, the cells downregulate α4β7 and differentiate into long-lived resident memory cells that participate in tissue protection against intestinal pathogens.20As sexual MPXV transmission was considered a dominant infection route in the current MPXV outbreak,8,9 it is conceivable that mucosal immune responses will have an important impact in protection from infection.To test whether JYNNEOS vaccine-induced T-cell responses might have mucosal-homing potential, we evaluated the expression of the α4β7 gut-homing integrin on pan-pox-specific T cells via ICS (Figure3D,E).Interestingly, the i.d.immunized groups of HCs (7/7 individuals for CD4 T cells and 6/7 individuals for CD8 T F I G U R E 1 Pan-pox-specific T-cell responses in healthy controls (HCs) and human immunodeficiency virus-1 (HIV-1)-infected individuals after JYNNEOS vaccination.(A) Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) values (SFU per 10 6 peripheral blood mononuclear cells [PBMCs]) for HCs and HIV-1-infected individuals before vaccination and 28 days after the first vaccination.Percentages (%) of pan-poxspecific Th1 CD4+ (B) or CD8+ (C) responses determined by intracellular cytokine staining (ICS) for HC and HIV-1-infected individuals at prevaccination and after the first vaccination.Red triangles in the HIV-1-infected individuals group refer to the HIV-1 loCD4 group, whereas black triangles refer to the HIV-1 hiCD4 group.Statistically significant differences between the groups were calculated using a one-way analysis of variance test for parametric data or a Kruskal-Wallis test for nonparametric data.Nonsignificant differences were indicated as "ns" and differences with p values < 0.05 were considered significant and were indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.Donut charts under figures A, B, and C show the number "n" of individuals analyzed and percentages of responders (gray-colored in chart) and non-responders (white-colored in chart).

F I G U R E 2
Pan-pox-specific T-cell responses in the human immunodeficiency virus-1 (HIV-1) loCD4 group after one or two JYNNEOS vaccinations.(A) Interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) values (SFU per 10 6 peripheral blood mononuclear cells [PBMCs]) for the HIV-1 loCD4 at three defined time points: prevaccination (pre), after first vaccination (1st vacc; 28 days post vaccination), and after second vaccination (2nd vacc; 28 days post 2nd vaccination).(B) Correlation plots of CD4 counts/mm 3 blood (x axis) and IFN-γ values (y axis) for each individual as shown under (A) after the first and the second vaccination.Red point indicates a HIV-1-infected individual that was considered an outlier by Grubb's test and excluded from analyses.Percentages (%) of pan-pox-specific Th1 CD4+ (C) or CD8+ (D) T-cell responses determined by intracellular cytokine staining (ICS) for the HIV-1 loCD4 group at prevaccination and after the first and second vaccination.Statistically significant differences between the groups were calculated using a one-way analysis of variance test for parametric data or a Kruskal-Wallis test for nonparametric data.Nonsignificant differences were indicated as "ns" and differences with p values < 0.05 were considered significant and were indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; p < 0.0001.Donut charts under figures A, C and D show the number "n" of individuals analyzed, and percentages of responders (gray-colored in chart) and nonresponders (white-colored in chart).cells) and HIV-1-infected individuals (7/8 individuals for CD4 T cells and 8/8 individuals for CD8 T cells) showed higher integrin expression than the s.c immunized groups suggesting an additional potential benefit of i.d.vaccination over the standard s.c.JYNNEOS vaccination.This hypothesis awaits direct demonstration of pan-poxspecific T cells in mucosal tissue.In summary, this study provides direct evidence for a superior immunogenicity of the dose-sparing i.d.JYNNEOS vaccination in HIV-1-infected individuals that are particularly susceptible to MPXV infection and pathogenicity.It emphasizes the need to specifically control vaccine responses in HIV-1-infected individuals with low CD4 T-cell counts as their vaccination responses may be severely F I G U R E 3 Comparison of subcutaneous (s.c.) and intradermal (i.d.) JYNNEOS vaccination routes.Pan-pox-specific interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) (A), Th1 CD4+ (B), and Th1 CD8+ (C) T-cell responses after i.d. or s.c.JYNNEOS vaccination of human immunodeficiency virus-1 (HIV-1) hiCD4 group individuals.Pan-pox-specific α4β7+ CD4+ (D) or CD8+ (E) T-cell responses for HC and HIV-1-infected individuals after s.c. or i.d.JYNNEOS vaccination.Given are the differences in percentages to the prevaccination time point.Individual values for IFN-γ ELISpots and intracellular cytokine staining (ICS) assays are shown.Statistically significant differences between the groups were calculated using a one-way analysis of variance test for parametric data or a Kruskal-Wallis test for nonparametric data.Nonsignificant differences were indicated as "ns" and differences with p values < 0.05 were considered significant and were indicated with asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.Donut charts under figures A-E show the number "n" of individuals analyzed and percentages of responders (gray-colored in chart) and nonresponders (white-colored in chart).