Safety, pharmacokinetics, and potential neurological interactions of ivermectin, tafenoquine, and chloroquine in Rhesus macaques.

Ivermectin (IVM) could be used for malaria control as treated individuals are lethal to blood-feeding Anopheles, resulting in reduced transmission. Tafenoquine (TQ) is used to clear the liver reservoir of Plasmodium vivax and as a prophylactic treatment in high-risk populations. It has been suggested to use ivermectin and tafenoquine in combination, but the safety of these drugs in combination has not been evaluated. Early derivatives of 8-aminoquinolones (8-AQ) were neurotoxic, and ivermectin is an inhibitor of the P-glycoprotein (P-gp) blood brain barrier (BBB) transporter. Thus, there is concern that co-administration of these drugs could be neurotoxic. This study aimed to evaluate the safety and pharmacokinetic interaction of tafenoquine, ivermectin, and chloroquine (CQ) in Rhesus macaques. No clinical, biochemistry, or hematological outcomes of concern were observed. The Cambridge Neuropsychological Test Automated Battery (CANTAB) was employed to assess potential neurological deficits following drug administration. Some impairment was observed with tafenoquine alone and in the same monkeys with subsequent co-administrations. Co-administration of chloroquine and tafenoquine resulted in increased plasma exposure to tafenoquine. Urine concentrations of the 5,6 orthoquinone TQ metabolite were increased with co-administration of tafenoquine and ivermectin. There was an increase in ivermectin plasma exposure when co-administered with chloroquine. No interaction of tafenoquine on ivermectin was observed in vitro. Chloroquine and trace levels of ivermectin, but not tafenoquine, were observed in the cerebrospinal fluid. The 3''-O-demethyl ivermectin metabolite was observed in macaque plasma but not in urine or cerebrospinal fluid. Overall, the combination of ivermectin, tafenoquine, and chloroquine did not have clinical, neurological, or pharmacological interactions of concern in macaques; therefore, this combination could be considered for evaluation in human trials.

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.

Equex STM paste improves post-thawed epididymal sperm motility and acrosome integrity in rhesus monkey (Macaca mulatta).

Equex STM paste, a water-soluble detergent, exerts the protective effect of egg-yolk during sperm cryopreservation. This study aims to evaluate the post-thaw quality of rhesus monkeys' epididymal spermatozoa in the Tris-citric-glucose egg-yolk extender, supplemented with or without Equex STM paste (0.5%, v/v) (n = 6). Sperm motility, progressive motility, motion characteristics, viability, acrosome integrity and mitochondrial activity were compared immediately post-thaw. Equex STM paste supplementation significantly improved sperm motility (35.0 ± 4.5 vs. 23.7 ± 5.0%), progressive motility (15.4 ± 2.1 vs. 9.8 ± 2.7%) and percentage of sperm with intact acrosome (30.4 ± 4.5 vs. 26.3 ± 4.6%) compared to the controls, respectively. This is the first report applying Equex STM paste for monkey epididymal sperm cryopreservation and is expected to be beneficial as a model for endangered non-human primates.

Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 µM) and hypnozoites (IC50, 29.24 µM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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.

CD4+ Cell infiltration into subcutaneous adipose tissue is not indicative of productively infected cells during acute SHIV infection.

Limited longitudinal data exist on the effect of HIV on adipose tissue (AT). We found an increase in CD4+ cells and detectable SHIV-RNA in AT during acute SHIV infection. SHIV-RNA+ cells were rare, suggesting that AT is unlikely to be a major source of productively infected cells in SHIV infection.

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.

Efficacy of intravenous methylene blue, intravenous artesunate, and their combination in preclinical models of malaria.

BACKGROUND: Intravenous artesunate (IV AS) is the present treatment of choice for severe malaria, but development of artemisinin resistance indicates that a further agent will be needed. Methylene blue (MB) is an approved human agent for IV and oral use, and is already being investigated for oral treatment of uncomplicated malaria. To initiate investigation of IV MB for severe malaria, the efficacy of IV MB was compared to IV AS and to their combination in rat and non-human primate malaria models. METHODS: IV MB was compared to IV AS and to their combination in the Plasmodium berghei-infected rat, a self-curing model; the Plasmodium falciparum-infected Aotus monkey, a fatal model; and the Plasmodium cynomolgi-infected rhesus monkey, a fatal model. Key endpoints were clearance of all parasites from the blood and cure (clearance without recrudescence). RESULTS: In rats, the minimal dose of individual drugs and their combination that cleared parasites from all animals was 20 mg IV MB/kg/day, 60 mg IV AS/kg/day and 10 mg IV MB/kg/day plus 30 mg IV AS/kg/day. In Aotus, 8 mg IV MB/kg/day and 8 mg IV AS/kg/day each cured two of three monkeys by one day after therapy, and the third monkey in each group was cured two days later. The combination of both drugs did not result in superior efficacy. In rhesus, 8 mg IV MB/kg/day and 8 mg IV AS/kg/day performed comparably: parasite clearance occurred by day 3 of therapy, although only one of four animals in each dose group cured. Eight mg/kg/day of both drugs in combination was 100% successful: all four of four animals cured. CONCLUSIONS: In each of the three animal models, the efficacy of IV MB was approximately equal to that of standard of care IV AS. In the rat and rhesus models, the combination was more effective than either single agent. This preclinical data suggests that IV MB, alone or in combination with IV AS, is effective against Plasmodium spp. and can be evaluated in severe malaria models.

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 µg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Correction: 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.

[This corrects the article DOI: 10.1371/journal.pntd.0010611.].

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.

Chimeric Virus-like Particle-Based COVID-19 Vaccine Confers Strong Protection against SARS-CoV-2 Viremia in K18-hACE2 Mice.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

Establishment of a Rhesus Macaque Model for Scrub Typhus Transmission: Pilot Study to Evaluate the Minimal Orientia tsutsugamushi Transmission Time by Leptotrombidium chiangraiensis Chiggers.

Recently, an intradermal inoculation of the rhesus macaque model of scrub typhus has been characterized at our institution. The current project was to establish a rhesus macaque model of scrub typhus using the naturally infected chigger challenge method that faithfully mimics the natural route of pathogen transmission to fully understand the host-pathogen-vector interactions influencing pathogen transmission. Unlike the needle-based inoculation route, Orientia tsutsugamushi-infected chiggers introduce both pathogen and chigger saliva into the host epidermis at the bite site. However, information on the interaction or influence of chigger saliva on pathogenesis and immunity of host has been limited, consequently hindering vaccine development and transmission-blocking studies. To characterize chigger inoculated O. tsutsugamushi in rhesus macaques, we determined the minimum chigger attachment time required to efficiently transmit O. tsutsugamushi to the immunocompetent hosts and preliminary assessed clinical parameters, course of bacterial infection, and host's immunological response to identifying potential factors influencing pathogen infection. Chigger infestation on hosts resulted in: (i) Rapid transmission of O. tsutsugamushi within 1 h and (ii) antigen-specific type I and II T-cell responses were markedly increased during the acute phase of infection, suggesting that both systems play critical roles in response to the pathogen control during the primary infection. In summary, we demonstrate that O. tsutsugamushi infection in rhesus macaques via chigger challenge recapitulates the time of disease onset and bacteremia observed in scrub typhus patients. Levels of proinflammatory cytokines and chemokines were positively correlated with bacteremia.

Virological and Immunological Outcomes in Rhesus Monkeys after Exposure to Dengue Virus-Infected Aedes aegypti Mosquitoes.

This study describes the natural history of dengue virus (DENV) infection in rhesus monkeys exposed to the bites of DENV-infected Aedes aegypti mosquitoes. Dengue virus-infected mosquitoes were generated by either intrathoracic inoculation or by oral feeding on viremic blood meals. Each of the six rhesus monkeys that were fed upon by intrathoracically infected mosquitoes developed non-structural protein 1 (NS1) antigenemia and an IgM response; viremia was detected in 4/6 individuals. No virological or immunological evidence of DENV infection was detected in the three monkeys exposed to mosquitoes that had been orally infected with DENV. These results demonstrate the utility of mosquito-borne challenge of rhesus monkeys with DENV.

Impact of analytical treatment interruption on the central nervous system in a simian-HIV model.

OBJECTIVE(S): Analytical treatment interruption (ATI) studies are often used to evaluate potential HIV cure strategies. This study was conducted to determine the impact of ATI on simian-HIV (SHIV) infection in the central nervous system. DESIGN: Animal study. METHODS: Nine rhesus macaques were inoculated with SHIV-1157ipd3N4. Antiretroviral therapy (ART) was administered from week 2 to 18. At week 18, four animals were euthanized (no-ATI-group) and five underwent ATI (ATI-group) and were euthanized at 12 weeks post viral rebound. Plasma and cerebrospinal fluid (CSF) SHIV-RNA, markers of inflammation and brain CD3+, CD68+/CD163+ and RNA+ cells were measured. RESULTS: All nine animals were SHIV-infected, with median pre-ART plasma and CSF SHIV-RNA of 6.2 and 3.6 log10copies/ml. Plasma and CSF IL-15, monocyte chemoattractant protein-1, IFN-γ-induced protein-10 and neopterin increased postinfection. ART initiation was associated with rapid and complete suppression of plasma viremia and reductions in plasma and CSF IL-15, IFN-γ-induced protein-10, neopterin and CSF monocyte chemoattractant protein-1. Median time to plasma viral rebound was 21 days post-ATI. At 12 weeks postrebound, CSF SHIV-RNA was undetectable and no increases in plasma and CSF markers of inflammation were found. Higher numbers of CD3+ and CD68+/CD163+ cells were seen in the brains of 3/5 and 1/5 animals, respectively, in the ATI-group when compared with no-ATI-group. SHIV-RNA+ cells were not identified in the brain in either group post-ATI. CONCLUSION: ATI in macaques that initiated ART during early SHIV-1157ipd3N4 infection was associated with mild, localized T-cell infiltrate in the brain without detectable SHIV-RNA in the brain or CSF, or elevation in CSF soluble markers of inflammation.

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.

Polytopic vaccination with a live-attenuated dengue vaccine enhances B-cell and T-cell activation, but not neutralizing antibodies.

Dengue, caused by dengue viruses (DENVs), is the most common arboviral disease of humans. Several dengue vaccine candidates are at different stages of clinical development and one has been licensed. Inoculation with live-attenuated DENV constructs is an approach that has been used by vaccine developers. Unfortunately, the simultaneous injection of all four attenuated DENV serotypes (DENV1-4) into a single injection site (monotopic vaccination) has been postulated to result in interference in the replication of some serotypes in favor of others, an important obstacle in obtaining a balanced immune response against all serotypes. Here, we demonstrate the virus replicative and immunostimulatory effects of polytopic monovalent dengue vaccination (PV) in which, each of the four components of the tetravalent vaccine is simultaneously delivered to four different sites versus the more traditional monotopic tetravalent vaccination (MV) in a non-human primate (NHP) model. With the exception of DENV-2, there was no significant difference in detectable viral RNA levels between PV and MV inoculation. Interestingly, longer periods of detection and higher viral RNA levels were seen in the lymph nodes of NHPs inoculated PV compared to MV. Induction of lymph node dendritic cell maturation and of blood T- and B-cell activation showed different kinetics in PV inoculated NHPs compared to MV. The MV inoculated group showed earlier maturation of dendritic cells and activation of B and T cells compared to PV inoculated NHPs. A similar kinetic difference was also observed in the cytokine response: MV induced earlier cytokine responses compared to PV. However, similar levels of DENV neutralizing antibodies were observed in PV and MV NHPs. These findings indicate that cellular immune response after vaccination may be affected by the location of inoculation. Design of vaccine delivery may need to take into account the effects of locations of vaccine delivery of multiples serotype live viral vaccine on the induction of immune response.

Strong interferon-gamma mediated cellular immunity to scrub typhus demonstrated using a novel whole cell antigen ELISpot assay in rhesus macaques and humans.

Scrub typhus is a febrile infection caused by the obligate intracellular bacterium Orientia tsutsugamushi, which causes significant morbidity and mortality across the Asia-Pacific region. The control of this vector-borne disease is challenging due to humans being dead-end hosts, vertical maintenance of the pathogen in the vector itself, and a potentially large rodent reservoir of unclear significance, coupled with a lack of accurate diagnostic tests. Development of an effective vaccine is highly desirable. This however requires better characterization of the natural immune response of this neglected but important disease. Here we implement a novel IFN-γ ELISpot assay as a tool for studying O. tsutsugamushi induced cellular immune responses in an experimental scrub typhus rhesus macaque model and human populations. Whole cell antigen for O. tsutsugamushi (OT-WCA) was prepared by heat inactivation of Karp-strain bacteria. Rhesus macaques were infected intradermally with O. tsutsugamushi. Freshly isolated peripheral blood mononuclear cells (PBMC) from infected (n = 10) and uninfected animals (n = 5) were stimulated with OT-WCA, and IFN-γ secreting cells quantitated by ELISpot assay at five time points over 28 days. PBMC were then assayed from people in a scrub typhus-endemic region of Thailand (n = 105) and responses compared to those from a partially exposed population in a non-endemic region (n = 14), and to a naïve population in UK (n = 12). Mean results at Day 0 prior to O. tsutsugamushi infection were 12 (95% CI 0-25) and 15 (2-27) spot-forming cells (SFC)/106 PBMC for infected and control macaques respectively. Strong O. tsutsugamushi-specific IFN-γ responses were seen post infection, with ELISpot responses 20-fold higher than baseline at Day 7 (mean 235, 95% CI 200-270 SFC/106 PBMC), 105-fold higher at Day 14 (mean 1261, 95% CI 1,097-1,425 SFC/106 PBMC), 125-fold higher at Day 21 (mean 1,498, 95% CI 1,496-1,500 SFC/106 PBMC) and 118-fold higher at Day 28 (mean 1,416, 95% CI 1,306-1,527 SFC/106 PBMC). No significant change was found in the control group at any time point compared to baseline. Humans from a scrub typhus endemic region of Thailand had mean responses of 189 (95% CI 88-290) SFC/106 PBMC compared to mean responses of 40 (95% CI 9-71) SFC/106 PBMC in people from a non-endemic region and 3 (95% CI 0-7) SFC/106 PBMC in naïve controls. In summary, this highly sensitive assay will enable field immunogenicity studies and further characterization of the host response to O. tsutsugamushi, and provides a link between human and animal models to accelerate vaccine development.