Monkeypox Virus Infection of Rhesus Macaques Induces Massive Expansion of Natural Killer Cells but Suppresses Natural Killer Cell Functions

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques

Development of a Flow Cytometry Assay to Study Monkeypox Virus Infection and Immune Response Regulation in Nonhuman Primates

Monkeypox virus (MPXV) is divided into two clades, Congo Basin and West African. Congo Basin MPXV infection is associated with severe symptoms and fatality of up to 10 percent in humans; whereas, West African MPXV infection is associated with less severe symptoms. To study infection and differential immune response regulation among virus isolates from Congo Basin and West African clades in nonhuman primates, a flow cytometry assay was developed. Annexin V, a protein used to detect apoptosis previously did not permit fixation and permeabilization for intracellular measurements, such as pathogen-infected cells. The method developed permits measurements of Annexin V apoptosis combined with intracellular staining. Reagents compatible with Annexin V were identified for fixation/permeabilization, and the need for calcium in intracellular assay steps was determined. The combined Annexin V/intracellular method described can be used to study disease pathogenesis of many BSL-3 or BSL-4 pathogens in nonhuman primates

Poxvirus antigen staining of immune cells as a biomarker to predict disease outcome in monkeypox and cowpox virus infection in non-human primates.

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1-2×10(7) PFU) or CPXV (>10(2) PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection

Characterization of Yellow Fever Virus Infection of Human and Non-human Primate Antigen Presenting Cells and Their Interaction with CD4⁺ T Cells

<div><p>Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4⁺ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4⁺ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4⁺ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.</p></div

Gating strategy for phenotyping of MDM and MoDC.

<p>Following differentiation and maturation, MoDC and MDM were characterized by flow cytometry. The gating strategy included initial gating on monocyte populations from the FSC-A vs SSC-A plot, followed by exclusion of doublets and then gating on viable cells. Within the viable cell population, CD14 was plotted against all other markers for MDM gating and CD11c was plotted against all other markers for MoDC gating. An unstained control was used to set the gating for all plots. Shown here is an example of typical populations determined in the phenotyping analysis.</p

Cytokine response in CD4⁺ T cells: Vaccinated vs. unvaccinated.

<p>IFN-γ and IL-2 production by human CD4⁺ T cells in re-stimulation assays. Each data point represents the response from an individual donor (n = 6) with the horizontal bar indicating the mean of the six values. Red circles indicate cells isolated from vaccinated donors and green squares indicate cells isolated from unvaccinated donors. Yellow triangles indicate mock-treated (N+N) control cells. Closed data points indicate cells infected with YFV 17D while open data points indicate cells infected with YFV Asibi. (L) indicates treatment with live virus, (D) indicates treatment with gamma-irradiated inactivated virus and (N) indicates mock-treated MoDC prior to co-culturing with CD4⁺ T cells (See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004709#pntd.0004709.g007" target="_blank">Fig 7</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004709#sec012" target="_blank">Materials and Methods</a>). (*) indicates points of significant (p<0.05) difference between the indicated datasets (bracket). A non-parametric multi-T test was used to determine statistical significance.</p

Cytokine and chemokine response in human MoDC.

<p>Cytokine and chemokine response in human MoDC infected with either wild-type Asibi virus or the vaccine strain 17D virus (yellow) in relation to mock (black) infected cells. (*) indicates points of significant (p<0.05) difference between virus and mock infected cells. Repeated measures ANOVA run in SAS was used to determine statistical significance.</p

Cytokine response in CD4⁺ T cells: Wild-type Asibi virus vs. vaccine 17D virus infection.

<p>IFN-γ and IL-2 production by human CD4⁺ T cells in re-stimulation assays. Each data point represents the response from an individual donor (n = 6) with the horizontal bar indicating the mean of the six values. Red data points indicate 17D YFV-treated cells, green squares indicate Asibi YFV-treated cells and yellow triangles indicate mock-treated cells. Closed data points indicate cells from unvaccinated donors while open data points indicate cells from vaccinated donors. (L) indicates treatment with live virus, (D) indicates treatment with gamma-irradiated inactivated virus and (N) indicates mock-treated MoDC prior to co-culturing with CD4⁺ T cells (See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004709#pntd.0004709.g007" target="_blank">Fig 7</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004709#sec012" target="_blank">Materials and Methods</a>). (*) indicates points of significant (p<0.05) difference between the indicated datasets (bracket). A non-parametric multi-T test was used to determine statistical significance.</p

YFV kinetics in MDM and MoDC.

<p>Yellow fever propagation kinetics in NHP and human derived (A) MDM and (B) MoDC. Each line represents an individual donor and values in the legend provide the donor reference number. Titrations were performed in triplicate with data points representing the mean of the triplicate values.</p

Cytokine and chemokine response in NHP MDM and MoDC.

<p>Cytokine and chemokine response in NHP (A) MDM or (B) MoDC infected with either wild-type Asibi virus or the vaccine strain 17D virus (yellow) in relation to mock (black) infected cells. (*) indicates points of significant (p<0.05) difference between virus and mock infected cells. Repeated measures ANOVA run in SAS was used to determine statistical significance.</p