A Penta-Component Mpox mRNA Vaccine Induced Protective Immunity in Naive and Simian Immunodeficiency Virus Infected Nonhuman Primates

doi:10.21203/rs.3.rs-4325129/v1

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A Penta-Component Mpox mRNA Vaccine Induced Protective Immunity in Naive and Simian Immunodeficiency Virus Infected Nonhuman Primates

https://doi.org/10.21203/rs.3.rs-4325129/v1

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The recent worldwide outbreaks of mpox (monkeypox) prioritize the development of a safe and effective mRNA vaccine. The contemporary mpox virus (MPXV) showed changing virological and epidemiological features, notably affecting populations already vulnerable to human immunodeficiency virus (HIV). Herein, we profiled the immunogenicity and protection of AR-MPXV5, a novel penta-component mRNA vaccine targeting five specific proteins (M1R, E8L, A29L, A35R, and B6R) from the representative contemporary MPXV clade II strain, in both naive and simian immunodeficiency virus (SIV)-infected nonhuman primates. Immunization with two doses of AR-MPXV5 to cynomolgus macaques resulted in robust antibody responses and cellular responses. Importantly, based on the challenge model with a contemporary MPXV clade II strain, AR-MPXV5 provided excellent protection in preventing skin lesions, eliminating viremia and reducing viral loads in multiple tissues including testis after challenge, thereby obviating the possibility of secondary sexual transmission. More importantly, AR-MPXV5 was well-tolerated in stable chronic SIV-infected rhesus monkeys, and comparable MPXV-specific humoral and cellular responses were elicited in both naive and SIV-infected monkeys. Together, these results support further clinical development of the AR-MPXV5 vaccine.

Biological sciences/Microbiology/Vaccines/RNA vaccines

Health sciences/Health care/Disease prevention

The re-emerging mpox (also known as monkeypox) is a zoonotic viral disease caused by the mpox virus (MPXV) which belongs to the Orthopoxvirus genus of Poxviridae family. The first documented human case of MPXV infection was reported in Democratic Republic of the Congo during 1970s ^1,2. Subsequently, MPXV infection has caused sporadic human cases and remained localized within countries across West and Central Africa ³. Since 2022, mpox suddenly emerged and spread into more than 110 countries, leading to the declaration of public health emergency of international concern ^4,5. MPXV is divided into two distinct clades, the Congo Basin (Central African) clade (clade I) and West African clade (clade II), while the contemporary circulating strains were classified as lineage B.1 of clade II ^6,7. The clinical symptoms and manifestations during the recent outbreak differ from those observed in previous endemics, as a reduced number of skin lesions were observed and the lesions were primarily localized in the genital and perianal regions^8–12. Besides, in the ongoing outbreak, most confirmed and suspected cases have no direct travel history to endemic areas ^4,13, and a large proportion of infected cases have been found in men who had sexual intercourse with men ^8,10. Of note, a significant number of cases have been detected among individuals living with human immunodeficiency virus (HIV) infection who may experience HIV-associated immunodeficiency^14–18, raising concerns regarding the potential risk of severe illness within this population. Although WHO declared the end of mpox emergency in May 2023, persistent transmission continues to occur in some regions, necessitating the sustained endeavor for long-term disease management¹⁹.

Vaccination has been well evidenced to be the most effective strategy to stop Orthopoxvirus infection. Most Orthopoxvirus members share highly conserved protein-coding sequences, including MPXV, vaccinia virus (VACV) and variola virus (VARV), and have been experimentally demonstrated to confer cross-protection against other orthopoxviruses^20–23. Currently, the U.S. Food and Drug Administration (FDA) has conditionally approved two smallpox vaccines for the prevention of mpox: JYNNEOS²⁴, a nonreplicating vaccine, and ACAM2000²⁵, a traditional live attenuated vaccine. However, recent clinical findings have unveiled that smallpox vaccine induced diminished levels of neutralizing antibodies against MPXV ^26,27.

Messenger RNA (mRNA)-based vaccine has been developed as a promising approach in combating emerging infectious diseases, making it an attractive strategy in addressing the mpox epidemic. Similar with other orthopoxvirus, MPXV exists two infectious forms: the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV). Experimental evidence has substantiated the necessity of combining antigens from both infectious forms to confer adequate protection against poxvirus infection^28–37. Previously, several IMV and EEV surface proteins of orthorpoxvirus were employed in the vaccine design as protective antigens. L1R, D8L and A27L of VACV (homologs of MPXV M1R, E8L, and A29L) are surface proteins of IMV that serve as targets for neutralizing antibodies and have been demonstrated to effectively induce protective antibodies^22,28,38. B5R (homologs of MPXV B6R) is an EEV surface protein that encompasses epitopes targeted by neutralizing antibodies in the presence of complement^39–41. A33R (homologs of MPXV A35R) is a target for antibody-dependent, complement-mediated cytolysis of infected cells ^21,41,42. The combination of these potential antigens holds great promise in conferring enhanced immunoprotection against poxvirus infection. However, the development of mRNA vaccines carrying multiple antigen-encoded mRNAs remains challenging, especially considering the cost and strict quality control requirement for large scale production.

Several MPXV mRNA vaccines have been reported in preclinical studies ^31–37. Base on the established mRNA-LNP platform ^43,44, we have shown that the penta-component vaccine candidate (AR-MPXV5) encoding five proteins of MPXV (M1R, E8L, A29L, A35R and B6R) elicited protective immunity in mice ³². In the current study, we sought to determine the safety, immunogenicity and protection efficacy in nonhuman primates, especially in the simian immunodeficiency virus (SIV)-infected animals.

AR-MPXV5 elicits humoral and cellular immunity in cynomolgus macaques

To induce a more balanced and potent immune response, our penta-component mRNA vaccine AR-MPXV5 were designed to encode five viral antigens of contemporary MPXV isolates, M1R, E8L and A29L from IMV, and A35R and B6R from EEV, respectively (Fig. 1a). The final mRNA-LNP formulation was prepared as previously described ⁴³. To investigate the immunogenicity of AR-MPXV5 in nonhuman primates, groups of cynomolgus macaques were intramuscularly immunized with 200 µg of AR-MPXV5 and boosted with the same dose 28 days later. Animals in sham group were immunized with placebo at the same procedure. Sera samples were subsequently collected on day 14, 28 and 42 post-initial immunization and subjected to antibody detection (Fig. 1b). Specifically, the total IgG antibody responses against MPXV specific antigens were determined by ELISA. The results indicated that a single dose vaccination elicited M1R, E8L, A29L, A35R and B6R-specific IgG antibodies, and a subsequent booster immunization obviously enhanced the antibody activity (Fig. 1c). Similarly, plaque reduction neutralization test (PRNT) showed that neutralizing antibodies against VACV were detected after the primary vaccination with a subsequent increase after the boost vaccination (Fig. 1d). The PRNT₅₀ titer against VACV reached ~ 1:1907 at 14 days after the second immunization. As expected, the IgG and neutralizing antibody titers were below the detection threshold in the sham animals.

Next, the antigen-specific cellular immune response in vaccinated macaques was assessed by flow cytometry 14 days after the second immunization (Fig. 2a, b). The stimulation of peripheral blood mononuclear cells (PBMCs) with corresponding antigen peptide pools resulted in an obvious increase of M1R-, E8L-, A29L-, A35R- and B6R-specific CD4⁺ T cells producing interleukin-2 (IL-2) in AR-MPXV5-immunized animals compared to the sham group (Fig. 2). Meanwhile, no significant differences were observed in the proportion of interferon γ (IFN-γ) or interleukin-4 (IL-4) secreting CD4⁺ T cells between the vaccine- and sham- immunized macaques (Fig. 2). Our results demonstrated the multicomponent mRNA vaccine activates a Th1-biased, antigen-specific CD4⁺ T cell response. Together, these results demonstrated that AR-MPXV5 elicits potent humoral and cellular immunity against poxvirus.

AR-MPXV5 protects non-human primates from MPXV infection

To further determine the protective efficacy of AR-MPXV5 in nonhuman primates, the infection model of MPXV was established with a contemporary circulating strain, MPXV-B.1-China-C-Tan-CQ01^45,46. Groups of cynomolgus macaques immunized with two doses of mRNA vaccine or placebo were challenged with 10⁷ TCID₅₀ of MPXV by the intravenous route (i.v.). All challenged macaques were then monitored for signs of disease, and skin lesion as well as viral shedding were recorded at 4,7 and 10 days post-infection (dpi) (Fig. 1b). No significant change was observed in body weight and body temperature between AR-MPXV5 and sham groups (Supplementary Fig. 1a, b). In the sham group, skin lesions were observed in all three infected animals, encompassing the regions of the head, face, arms, chest, abdomen and legs (Fig. 3a). The lesions progressed from a vesicular or pustular rash and ultimately developed into scabs, and lesion counts appeared at 7 dpi and peaked at 10 dpi in the sham group animals (Fig. 3b). In contrast, among the four animals immunized with AR-MPXV5, two didn't develop lesions after infection throughout the observation period; one animal developed a single small lesion and the other presented 4 small lesions at 7 dpi; all lesions faded away in the two AR-MPXV5-immunized animals at 10 dpi (Fig. 3b). The animals were then euthanized for histopathological analyses. In the sham group, the epidermis exhibited an increased thickness, accompanied by spinous layer hypertrophy and vacuolar degeneration of spinous cells. Cell necrosis was observed in both the epidermis and superficial dermis. Besides, disorganization of collagen fibers within the dermal layer can be observed, accompanied by infiltration of numerous granulocytes, lymphocytes, and macrophages (Fig. 3c). In contrast, the epidermis of the AR-MPXV5-vaccinated animals exhibited a normal and intact morphology, and collagen fibers arranged in a regular pattern within the dermal layer. These results clearly demonstrated that AR-MPXV5 is efficacious in delaying and alleviating skin lesions as well as pathological damage caused by MPXV infection in cynomolgus macaques.

Viremia has been well documented as a hallmark for MPXV infection, and the disappearance of viremia is recognized as a correlate for protection ^37,47–53. Upon challenge, all animals in the sham group developed rapid and sustained viremia within 10 dpi, with a peak titer of 4.17×10⁶ copies/mL at 7 dpi. Remarkably, all AR-MPXV5-immunized animals failed to develop any viremia throughout the observation period (Fig. 4a). Meanwhile, MPXV genomic DNA was detected in the urine samples at 10 dpi from the sham group animals, but viral DNA was absent in all vaccinated animals (Fig. 4b). Viral shedding in nasal and throat swabs was detected from 4 dpi and 7 dpi in the sham group, reaching peak titers at 10 dpi and 7 dpi, respectively; while no viral DNA was detected in nasal and throat swabs from the vaccinated animals (Fig. 4c, d). Our results clearly demonstrated that AR-MPXV5 not only eliminates viremia but also abolished viral shedding in MPXV-infected non-human primates.

To further determine the protective efficacy in various tissues of MPXV-infected macaques, animals were euthanized at 10 dpi. MPXV DNA was detected in all 11 tissues obtained from sham-immunized macaques, including the heart, liver, spleen, lung, kidney, skin, lymph node, muscle, ileum, colon and testis (Fig. 4e). The highest viral DNA load was detected in the skin, with a titer of ~ 2.44×10⁸ copies/µg. Meanwhile, substantial viral DNAs were determined in spleen, testis and lymph nodes. In contrast, an obvious reduction in viral loads was detected in all tissues of vaccinated animals, with MPXV genomic DNA remaining below the detection threshold in heart, liver, and lung. Additionally, a remarkable decrease with a fold change ranging from 32 to 1.4×10⁶ was observed in other tissues (Fig. 4e). The reduction of germ cells within the seminiferous tubules and loss of spermatogenic epithelial cells were observed in the histopathological analyses in a monkey from the sham group (Supplementary Fig. 2a), and the necrosis was also found in the epithelium and lymphoid tissue of tonsil in the same rhesus macaque (Supplementary Fig. 2b).

In addition, the cytokine storm has been reported in human mpox cases, and is likely to be associated with disease exacerbation and unfavorable clinical outcomes^54,55. In our study, MPXV challenge led to readily production of cytokines and chemokines in the sham-immunized macaques (Fig. 5a). Notably, the up-regulation of sera cytokines were detected at 1 dpi, peaked at 4 or 7 dpi, and faded at 10 dpi in all sham animals (Fig. 5a). Of all up-regulated cytokines, IFN-γ, SDF-1α and VEGF-A showed a remarkable increase of 365.8-, 46.5- and 24.9-fold, respectively (Fig. 5b). Strikingly, all AR-MPXV5-vaccinated macaques showed a milder cytokine production profile upon MPXV challenge, and the levels of all selected cytokines were significantly lower than that from the sham group animals (Fig. 5a, b). These results clearly demonstrated that AR-MPXV5 vaccination has hindered the upregulation of cytokines caused by MPXV challenge in nonhuman primates.

More importantly, the neutralizing antibody titers against VACV were determined and compared pre- and post-infection. A significantly elevated neutralizing antibody response was detected in the sham-immunized macaques at 10 dpi, while all animals immunized with AR-MPXV5 exhibited no significant increase in neutralizing antibody titers after challenge (Fig. 5c), indicating sterile immunity against MPXV was induced.

AR-MPXV5 induces protective immune responses in SIV-infected rhesus monkeys

To further investigate whether AR-MPXV5 induces a protective immune response in immunodeficient individuals, groups of naive or stable chronic SIV-infected rhesus monkeys were intramuscularly immunized with 200 µg of AR-MPXV5 and boosted with the same dose 28 days later. CD4⁺ T cell counts of naive or SIV-infected monkeys were analyzed before immunization. The result indicated an obvious reduction of CD4⁺ T cells in chronic SIV-infected monkeys compared with their uninfected counterparts (Fig. 6a), and the plasma SIV loads were detected prior to immunization, with ~ 8.71×10⁴ copies/mL of viral RNAs detected in the SIV-infected monkeys (Fig. 6b). Next, the neutralizing antibodies against VACV were determined after vaccination. The administration of two doses of AR-MPXV5 effectively induced robust antibody responses in both of the naive and SIV-infected monkeys, with the PRNT₅₀ titers reaching ~ 1: 1607 and ~ 1: 634 on day 14 after the booster immunization, respectively (Fig. 6c). No significant difference was detected between the two groups.

Besides, the antigen-specific cellular response was assessed 14 days after the second immunization by flow cytometry (Fig. 6d, e). An obvious increase of M1R-, E8L-, A35R- and B6R-specific CD4⁺ T cells producing IL-2 was detected in both of the naive and SIV-infected groups compared to pre-immunization levels (Fig. 6d, e). The naive animals also exhibited an elevation of M1R- and E8L-specific CD4⁺ T cells producing IFN-γ (Fig. 6d). Together, our results demonstrate that MPXV mRNA vaccine AR-MPXV5 effectively elicits both humoral and cellular immune responses in stable chronic SIV-infected nonhuman primates.

AR-MPXV5 is well tolerated in naive and SIV-infected nonhuman primates

In our experiment, the administration of two doses of AR-MPXV5 was totally tolerated in cynomolgus macaques and rhesus monkeys. All naive and SIV-infected animals did not exhibit any significant clinical events after vaccination. The body weight changes as well as plasma viremia of SIV-infected animals were monitored for 42 days following the initial immunization (Supplementary Fig. 3a, b). Only a transient and minimal weight loss was observed during the administration (Supplementary Fig. 3a). No obvious increase in SIV RNA loads was detected throughout the immunization process, indicating the absence of SIV activation following vaccination (Supplementary Fig. 3b). Meanwhile, there was no significant decline in CD4⁺ T cell counts after immunization, while a transient increase was observed following the second immunization (Supplementary Fig. 3c). In addition, hematological parameters were recorded and analyzed in naive and SIV-infected monkeys after immunization (Supplementary Table 1, 2 and Supplementary Fig. 4). The white blood cell (WBC) count, neutrophil (Neu) count, and monocyte (Mon) count exhibited a transient elevation on day 1 after the first and second immunization, and return to normal ranges on day 3 (Supplementary Fig. 4). Meanwhile, a slight and transient decrease in lymphocyte (Lym) count was observed on day 1 post-immunization and subsequently returned to normal on day 3. The red blood cell (RBC) count and hemoglobin (HGB) remained within the normal range throughout the vaccination process without any significant alterations (Supplementary Fig. 4). No significant differences were observed in the alteration of hematological parameters between the naive and SIV-infected groups. Our results demonstrated that AR-MPXV5 was safe and well-tolerated in naive and SIV-infected nonhuman primates.

A safe and effective vaccine is indispensable for the prevention and control of MPXV infection and transmission. In the present study, the immune profile and protective efficacy of a penta-component mRNA vaccine (AR-MPXV5) were well elucidated in a MPXV-infected nonhuman primate model, which have simulated and restated the natural course and clinical manifestations of MPXV infection^{48–51,53,56,57}. Two administrations of AR-MPXV5 elicited robust levels of antigen-specific IgG antibodies as well as neutralizing antibodies with a peak titer of approximately 1:1907, exhibiting a superior or comparable protective antibody response compared to that induced by attenuated vaccine^50,53,56,58, subunit vaccine⁵⁹, virus-like replicon particles (VRP)⁶⁰, DNA vaccine^21,28,29 as well as another mRNA vaccine³⁷ as recently reported. Besides, our results revealed that AR-MPXV5 induces an elevation of MPXV antigen specific, Th1 cytokine-secreting CD4⁺ T cells in cynomolgus macaques, suggesting the potent immunogenicity of the multiple antigens in combination.

The MPXV responsible for the global outbreak in 2022 belongs to clades IIb of West African clade⁶. Recently, another MPXV mRNA vaccine (BNT166) encoding four surface proteins (A35, B6, M1 and H3) was assessed in a cynomolgus macaques model challenged with a lethal clade I MPXV strain³⁷. Interestingly, BNT166 vaccination failed to eliminate the viremia caused by MPXV in cynomolgus macaques ³⁷. As the clinical manifestations and transmission characteristics of the recent mpox differ from those observed in previous studies, we established a nonhuman primate model infected with a circulating MPXV clade II strain to evaluate the protective efficacy of AR-MPXV5. In contrast to the clade I MPXV-infected lethal model, this macaque model represents a non-lethal infection model characterized by substantial viremia and obvious skin lesions. AR-MPXV5 effectively prevents the development of skin lesions and absolutely eliminates viremia, suggesting that the inclusion of additional antigen components may potentially confer enhanced protection against poxvirus infection. In addition, the sham-immunized animals exhibited substantial viral DNA loads in multiple tissues, whereas a significant reduction in viral loads was observed in AR-MPXV5-immunized animals. Notably, high levels of viral DNA were detected in the testis from animals in the sham group, while an obviously decreased viral load was observed in the testis from vaccinated macaques, suggesting the potent efficacy of AR-MPXV5 in preventing sexual transmission⁶¹. Moreover, the elevation of multiple sera cytokine and chemokine expressions have been reported in MPXV-infected human or nonhuman primates, indicating a strong proinflammatory response associated with MPXV infection^52,54,55,62, while the administration of AR-MPXV5 significantly attenuated the cytokine storm following infection.

Considering the crucial role of CD4⁺ T cells in antibody production and their necessity for effective protection against poxvirus infection, there are concerns that people living with HIV may be at a heightened risk of severe MPXV infection, particularly those with low CD4⁺ T cell counts^18,63–65. Recent studies revealed that severe MPXV infections were observed in HIV-infected individuals, especially those with advanced HIV infection or acquired immunodeficiency syndrome (AIDS) characterized by significantly diminished CD4⁺ T cell counts ^16,65–69. In our study, two immunizations of the multicomponent mRNA vaccine successfully induced a potent and comparable neutralizing antibody response in both of the naive and stable chronic SIV-infected monkeys. In addition, a Th1-biased, antigen-specific CD4⁺ T cell response was also detected in SIV-infected monkeys. Overall, our results suggested that AR-MPXV5 was potent to induce protective immune response in immunodeficiency virus infected as well as immunocompetent individuals.

Cells and Viruses

BS-C-1 (ATCC) cells were cultured in Minimum Essential Medium (MEM, Procell, China) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (PS, Gibco, USA) at 37 ℃ with 5% CO₂. VACV Western Reserve strain was propagated and titrated in BS-C-1 cells before use. Cells were collected in phosphate buffered saline (PBS, Solarbio, China) at 2 dpi, and lysed by repeated freezing and thawing. Cell fragments were removed by centrifugation and virus stocks were obtained. Virus titers were determined by standard plaque assay on BS-C-1 cells. Low-passage stocks of MPXV (MPXV-B.1-China-C-Tan-CQ01) was originally isolated from a German imported case of Chinese nationality, which belongs to B.1 branch of the West African linage⁴⁶. MPXV was cultivated and titrated in Vero cells as described previously⁴⁶, which were performed in the BSL-3 containment at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

AR-MPXV5 formulation

Lipid-nanoparticle (LNP) formulations were prepared in the same way as previously described ⁴³. Briefly, we dissolved lipids in ethanol containing an ionizable lipid, 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and PEG-lipid (with molar ratios of 50: 10: 38.5: 1.5). The lipid mixture was then mixed with 20 mM citrate buffer (pH 4.0) containing mRNA through a T-mixer in a ratio of 1:2. Formulations were permeated into a 10-fold volume of PBS with a 100 KD tangential flow filtration membrane, and filtered with a 0.22 µm filter to obtain the final LNP formulations. LNP formulations were stored at 2–8 ℃. All formulations were tested for particle size, distribution, RNA concentration and encapsulation.

Vaccination and challenge experiments

All animal procedures were reviewed and approved by the Animal Experiment Committee of Laboratory Animal Center, AMMS (Approval Number. IACUC-DWZX-2022-055) and the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Approval No. XJ23004). Animal experiments with infectious MPXV were conducted in animal biosafety level 3 (ABSL-3) facilities in Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. MPXV-seronegative cynomolgus macaques (3–4 years old) were intramuscularly immunized with 200 µg of AR-MPXV5 (n = 4) or empty LNPs (n = 3) and boosted with an equal dose 28 days later. Empty LNPs were utilized as sham treatment. Plasma samples were collected prior to immunization and 14, 28 and 42 days post-initial immunization and subjected to IgG and neutralizing antibody detection. Whole blood samples were collected and peripheral blood mononuclear cells (PBMCs) were separated 14 days after the boost immunization. On day 52 after initial immunization, AR-MPXV5 or sham-immunized cynomolgus macaques were intravenously challenged with 10⁷ TCID₅₀ of MPXV CQ202209. Weight changes and body temperature were monitored for 10 days after infection. Skin lesions were observed and counted at 0, 4, 7 and 10 dpi. MPXV DNA loads in plasma, urine, nasal swab and throat swab were determined at 0, 4, 7 and 10 dpi. Neutralizing antibody titers of infected macaques were determined at 10 dpi. All infected animals were euthanized at 10 dpi for tissue harvest and virological analyses.

Naive and chronic SIV-infected rhesus monkeys (4–9 years old, stable infected with SIV for 27, 43 and 49 months) were intramuscularly immunized with 200 µg of AR-MPXV5 (n = 3) and boosted with an equal dose 28 days later. CD4⁺ T cell counts were determined by flow cytometry at 60 days prior to immunization and at 0, 7, 14, 28, 35 and 42 days post-initial immunization. Viral RNA loads in plasma of SIV-infected monkeys were determined at 60 days prior to immunization and at 0, 14 and 42 days post-initial immunization. The body weight changes of SIV-infected monkeys were monitored for 42 days post-initial immunization. Plasma samples were collected 14 days following the second immunization and subsequently subjected to antibody detection. Whole blood samples were collected at 0, 1, 3, 5, 7 and 14 days after the prime and boost vaccination for routine blood analyses by using hematologic analyzer (Mindray, China). PBMCs were separated 14 days after the boost immunization.

Enzyme-linked immunosorbent assay (ELISA)

The IgG antibody titers in serum samples were determined by ELISA. Serum samples were heated at 56 ℃ for 30 min before use. MPXV antigens diluted with 1 × coating buffer (Solarbio, China) were coated on ELISA plates at 4 ℃ overnight, respectively. Serum samples were twofold-diluted and incubated in blocked ELISA plates for 2 h at 37 ℃. Plates were washed with PBS-T (Gene-Protein Link, China) for three times and then incubated with goat anti-monkey IgG-HRP at 37℃ for 1 h. ELISA plates were washed three times with PBS-T after incubation. Soluble TMB (Cwbio, China) was added to plates and incubated for 15 min at room temperature, followed by subsequent termination through the addition of stop solution (Solarbio, China). Synergy H1 hybrid multimode microplate reader (BioTek, USA) was used to read the absorbance. The endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance exceeding background values by more than 2-fold.

Plaque reduction neutralization test (PRNT)

50% plaque reduction neutralization test (PRNT₅₀) was used to detect neutralizing antibody titers in serum samples. BS-C-1 cells were seeded in 12-well plates and incubated at 37 ℃ and 5% CO₂. Serum samples were heated at 56 ℃ for 30 min before use. The 3-fold diluted sera were mixed with an equal volume of virus at 37 ℃ for 90 min. The mixture was added to BS-C-1 cells and incubated at 37 ℃ for 90 min. Then the mixture was removed, and cells were covered by 0.5% methylcellulose in Dulbecco’s minimal essential medium (DMEM, Gibco, USA) with 2.5% FBS. All FBS were inactivated before use. Cells were incubated at 37 ℃ and 5% CO₂ for 2 days. The PRNT₅₀ titers were calculated by the method of Spearman-Karber.

Flow cytometry analyses

Flow cytometry analyses was used to evaluate MPXV antigen-specific T cell responses in nonhuman primates. Briefly, a total of 10⁶ PBMCs were co-stimulated with anti-monkey CD28 antibody, CD49d antibody (Biolegend, USA) and the overlapping antigen peptide pools (GeneScript, China) of M1R, E8L, A29L, A35R and B6R at 37 ℃ and 5% CO₂ for 1 h. PBMCs were incubated with Protein Transport Inhibitor (BD Biosciences, USA) at 37 ℃ and 5% CO₂ for 8 h. Cells were washed with PBS containing 1% FBS, blocked with anti-CD16/CD32 antibody (Biolegend, USA) and then incubated with anti-monkey CD3 antibody (Biolegend, USA), CD4 antibody (BD Biosciences, USA), and CD8 antibody (Biolegend, USA) at 4 ℃ for 30 min in the dark. Cells were fixed, washed by PBS containing 1%FBS and incubated with fluorescently conjugated antibodies to interferon-γ (IFN-γ, Biolegend, USA), interleukin-2 (IL-2, Biolegend, USA), and interleukin-4 (IL-4, Biolegend, USA) for 30 min at 4°C in the dark. PBMCs were resuspended with PBS containing 1%FBS, and analyzed by using the FACSVerse Flow Cytometer (BD Biosciences, USA). Data are analyzed with Flow Jo software.

Quantification of viral RNA and DNA loads

MPXV genomic DNA was determined by real-time quantitative PCR (qPCR). Total DNA was extracted by using DNeasy Blood & Tissue Kit (QIAGEN, Germany). The following primer-probe set was used: F3L.forward (5’-CATCTATTATAGCATCAGCATCAGA-3’), F3L.reverse (5’-GATACTCCTCCTCGTTGGTCTAC-3’), and F3L.probe: (5’-TGTAGGCCGTGTATCAGCATCCATT-3’) ⁴⁷. qPCR was conducted in a LightCycler® 480 Instrument (Roche Diagnostics Ltd). SIV RNA loads in plasma were determined by qPCR assay following a previously described method ⁷⁰. qPCR was carried out on an ABI 7500 Real-time PCR system (Applied Biosystems, USA) using the same protocol and primers as previously⁷¹.

Histopathological analysis

For histopathology, skin, testis and tonsil from infected macaques were fixed in 4% neutral-buffered formaldehyde, embedded in paraffin, sectioned, and stained with H&E. Images were captured using Olympus BX51 microscope equipped with a DP72 camera.

Cytokine and chemokines analysis

Cytokine and chemokine expressions in plasma samples pre- and post-infection were measured using a Cytokine/Chemokine/Growth Factor 37-Plex NHP ProcartaPlex Panel (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The data were collected on Luminex 200 and analyzed by Luminex PONENT (Thermo Fisher Scientific, USA).

Statistical analysis

Data were analyzed using GraphPad Prism 10 (GraphPad Software). The values shown in the graphs are presented as the mean ± SEM. Statistical differences between groups were analyzed using two-tailed unpaired t tests or two-way ANOVA statistical tests, and P < 0.05 was considered statistically significant.

Data Availability

The data that support the findings of this study are available from the corresponding author, Prof. Cheng-Feng Qin ([email protected]).

Competing Interests

C.F.Q., Q.Y., R.R.Z. and Z.J.W. have filed a patent related to the vaccine reported in the study.

Acknowledgements

This work was supported by the National Key Research and Development Project of China (2021YFC2302400, 2023YFC2309000, 2022YFC2304101), and the National Natural Science Foundation of China (82241069, 82241068). C.-F.Q. was supported by the National Science Fund for Distinguished Young Scholars (81925025), and the Innovation Fund for Medical Sciences (2019-I2M-5-049) from the Chinese Academy of Medical Sciences. J.X. was supported by the National Science Fund for Excellent Young Scholars (82222041).

Author Contributions:

C.F.Q., J.X. and Q.Y. designed the experiments and wrote the manuscript. Q.Y., D.Z., R.R.Z., Q.X., X.Y.H., M.X.S. performed the majority of the experiments. Z.C., L.Z., J.R.M., N.L., J.J.Z., T.C., J.H.L., Y.Z.H. performed the animal experiments and sample collection. X.C., H.T.L., C.Z., M.W., Z.J.W., J.Y., Y.F.Q., B.Y contributed specific experiments and data analysis. Y.B.H. and W.T. contributed the MPXV strain. All authors read and approved the contents of the manuscript.

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Yes there is potential Competing Interest. C.F.Q., Q.Y., R.R.Z. and Z.J.W. have filed a patent related to the vaccine reported in the study.

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A Penta-Component Mpox mRNA Vaccine Induced Protective Immunity in Naive and Simian Immunodeficiency Virus Infected Nonhuman Primates

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Abstract

Figures

Introduction

Results

AR-MPXV5 elicits humoral and cellular immunity in cynomolgus macaques

AR-MPXV5 protects non-human primates from MPXV infection

AR-MPXV5 induces protective immune responses in SIV-infected rhesus monkeys

AR-MPXV5 is well tolerated in naive and SIV-infected nonhuman primates

Discussion

Materials and Methods

Cells and Viruses

AR-MPXV5 formulation

Vaccination and challenge experiments

Enzyme-linked immunosorbent assay (ELISA)

Plaque reduction neutralization test (PRNT)

Flow cytometry analyses

Quantification of viral RNA and DNA loads

Histopathological analysis

Cytokine and chemokines analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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