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.

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.

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

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.

Evaluation of the safety and immunogenicity in rhesus monkeys of a recombinant malaria vaccine for Plasmodium vivax with a synthetic Toll-like receptor 4 agonist formulated in an emulsion.

Plasmodium vivax is the major cause of malaria outside sub-Saharan Africa and inflicts debilitating morbidity and consequent economic impacts in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of a novel chimeric recombinant protein, VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Very few adjuvant formulations are currently available for human use. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study rhesus monkeys were immunized intramuscularly three times with VMP001 in combination with a stable emulsion (SE) or a synthetic Toll-like receptor 4 (TLR4) agonist (glucopyranosyl lipid A [GLA]) in SE (GLA-SE). Sera and peripheral blood mononuclear cells (PBMCs) were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of monkeys generated high titers of anti-P. vivax IgG antibodies, as detected by enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. In addition, all groups generated a cellular immune response characterized by antigen-specific CD4(+) T cells secreting predominantly interleukin-2 (IL-2) and lesser amounts of tumor necrosis factor (TNF). We conclude that the combination of VMP001 and GLA-SE is safe and immunogenic in monkeys and may serve as a potential second-generation vaccine candidate against P. vivax malaria.

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10.

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-alpha mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-alpha led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-alpha and IFN-alpha also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-alpha and TNF-alpha may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.

Randomized, controlled, double-blind trial of daily oral azithromycin in adults for the prophylaxis of Plasmodium vivax malaria in Western Thailand.

We assessed the prophylactic efficacy of azithromycin (250 mg/day) against malaria in 276 adults in western Thailand in a randomized, double-blind, placebo-controlled trial. After antimalarial suppressive treatment, volunteers were randomized in a 2:1 ratio to either the azithromycin or placebo, respectively. Study medication was given for an average of 74 days. The azithromycin group (n = 179) had five endpoint parasitemias (1 Plasmodium vivax and 4 P. falciparum), and the placebo group (n = 97) had 28 endpoint parasitemias (21 P. vivax, 5 P. falciparum, and 2 mixed infections). Adverse events and compliance and withdrawal rates were similar in both groups. The protective efficacy (PE) of azithromycin was 98% for P. vivax (95% confidence interval [CI] = 88-100%). There were too few cases to reliably estimate the efficacy of azithromycin for P. falciparum (PE =71%, 95% C =-14-94%). We conclude that daily azithromycin was safe, well-tolerated, and had a high efficacy for the prevention of P. vivax malaria.

Characterization of circulating monocytes expressing HLA-DR or CD71 and related soluble factors for 2 weeks after severe, non-thermal injury.

BACKGROUND: Severe injury is associated with changes in monocytes that may contribute to poor outcomes. Longitudinal characterization of monocyte response patterns after trauma may provide added insight into these immunological alterations. METHODS: Venous blood obtained seven times during post-injury days 1 through 13 from 61 patients with an injury severity score >20 was assessed by flow cytometry for monocytes (CD14+) expressing HLA-DR or CD71 (transferrin receptor) and for circulating levels of interleukin (IL) 1alpha, IL-1beta, IL-6, soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and endotoxin. Urine neopterin was measured by high-pressure liquid chromatography, expressed as a neopterin-creatinine ratio. RESULTS: Trauma patients had leucocytosis days 1 through 13, monocytosis days 5 through 13, reduced proportions of CD14+HLA-DR+ cells days 2 through 5, and elevated proportions of CD14+CD71+ cells days 1 through 13. Neopterin was elevated all days, peaking on day 10. sCD14 was elevated days 2 through 13, and there were sporadic elevations of IL-1alpha, IL-1beta, IL-6, TNF-alpha, PGE(2), TXB(2), and endotoxin. Sepsis syndrome patients (n = 6) had larger and more prolonged reductions in CD14+HLA-DR+ cells and higher neopterin values, in comparison with uneventful patient outcomes. CONCLUSIONS: Altered proportions of monocytes expressing HLA-DR and CD71 and elevated sCD14 and urine neopterin levels, for up to 2 weeks after severe injury, underscores an extended period of profound immunological effects. Additional studies to more fully assess temporal monocyte response patterns after severe injury, including activation, may be warranted.

Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a Toll-like receptor 9-dependent pathway.

A common feature of severe Plasmodium falciparum infection is the increased systemic release of proinflammatory cytokines that contributes to the pathogenesis of malaria. Using human blood, we found that blood stage schizonts or soluble schizont extracts activated plasmacytoid dendritic cells (PDCs) to up-regulate CD86 expression and produce IFN-alpha. IFN-alpha production was also detected in malaria-infected patients, but the levels of circulating PDCs were markedly reduced, possibly because of schizont-stimulated up-regulation of CCR7, which is critical for PDC migration. The schizont-stimulated PDCs elicited a poor T cell response, but promoted gamma delta T cell proliferation and IFN-gamma production. The schizont immune stimulatory effects could be reproduced using murine DCs and required the Toll-like receptor 9 (TLR9)-MyD88 signaling pathway. Although the only known TLR9 ligand is CpG motifs in pathogen DNA, the activity of the soluble schizont extract was far greater than that of schizont DNA, and it was heat labile and precipitable with ammonium sulfate, unlike the activity of bacterial DNA. These results demonstrate that schizont extracts contain a novel and previously unknown ligand for TLR9 and suggest that the stimulatory effects of this ligand on PDCs may play a key role in immunoregulation and immunopathogenesis of human falciparum malaria.