Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine.

BACKGROUND: To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. METHODS: The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. RESULTS: Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. CONCLUSIONS: This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BACKGROUND: Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. CONCLUSIONS/SIGNIFICANCE: This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: Protective efficacy, immunogenicity, safety, and toxicity.

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.

Central Nervous System Inflammation and Infection during Early, Nonaccelerated Simian-Human Immunodeficiency Virus Infection in Rhesus Macaques.

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neurological AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this nonaccelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4+ T cell counts mirrored early human immunodeficiency virus (HIV) infection in humans. At 12 weeks postinfection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+ CD4- cellular infiltrate in the brain parenchyma without a concomitant increase in CD68/CD163+ monocytes, macrophages, and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAScope in situ hybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF are predominantly mediated by T cell-mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late-stage disease utilizing SIV. In the era of antiretroviral therapy, manifestations of late-stage HIV are less common. Furthermore, new interventions, such as monoclonal antibodies and therapeutic vaccinations, target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with SHIV expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans and that early neurologic inflammation is characterized by predominantly T cell-mediated inflammation accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end-stage disease.

Characterization of the rhesus macaque (Macaca mulatta) scrub typhus model: Susceptibility to intradermal challenge with the human pathogen Orientia tsutsugamushi Karp.

BACKGROUND: Scrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models. METHODOLOGY/PRINCIPLE FINDINGS: In this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7-21 post inoculation (pi); bacteremia was detected by qPCR on days 6-18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes. CONCLUSIONS/SIGNIFICANCE: These data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus.

Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver.

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites.

Tissue Distribution of Memory T and B Cells in Rhesus Monkeys following Influenza A Infection.

Studies of influenza-specific immune responses in humans have largely assessed systemic responses involving serum Ab and peripheral blood T cell responses. However, recent evidence indicates that tissue-resident memory T (TRM) cells play an important role in local murine intrapulmonary immunity. Rhesus monkeys were pulmonary exposed to 2009 pandemic H1N1 virus at days 0 and 28 and immune responses in different tissue compartments were measured. All animals were asymptomatic postinfection. Although only minimal memory immune responses were detected in peripheral blood, a high frequency of influenza nucleoprotein-specific memory T cells was detected in the lung at the "contraction phase," 49-58 d after second virus inoculation. A substantial proportion of lung nucleoprotein-specific memory CD8(+) T cells expressed CD103 and CD69, phenotypic markers of TRM cells. Lung CD103(+) and CD103(-) memory CD8(+) T cells expressed similar levels of IFN-γ and IL-2. Unlike memory T cells, spontaneous Ab secreting cells and memory B cells specific to influenza hemagglutinin were primarily observed in the mediastinal lymph nodes. Little difference in systemic and local immune responses against influenza was observed between young adult (6-8 y) and old animals (18-28 y). Using a nonhuman primate model, we revealed substantial induction of local T and B cell responses following 2009 pandemic H1N1 infection. Our study identified a subset of influenza-specific lung memory T cells characterized as TRM cells in rhesus monkeys. The rhesus monkey model may be useful to explore the role of TRM cells in local tissue protective immunity after rechallenge and vaccination.