IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

BACKGROUND: The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. METHODS: We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. RESULTS: IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). CONCLUSIONS: In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies.

BACKGROUND: When rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed. METHODS: Several species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented. RESULTS: Anopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner. CONCLUSIONS: Anopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.

Remotely-sensed, nocturnal, dew point correlates with malaria transmission in Southern Province, Zambia: a time-series study.

BACKGROUND: Plasmodium falciparum transmission has decreased significantly in Zambia in the last decade. The malaria transmission is influenced by environmental variables. Incorporation of environmental variables in models of malaria transmission likely improves model fit and predicts probable trends in malaria disease. This work is based on the hypothesis that remotely-sensed environmental factors, including nocturnal dew point, are associated with malaria transmission and sustain foci of transmission during the low transmission season in the Southern Province of Zambia. METHODS: Thirty-eight rural health centres in Southern Province, Zambia were divided into three zones based on transmission patterns. Correlations between weekly malaria cases and remotely-sensed nocturnal dew point, nocturnal land surface temperature as well as vegetation indices and rainfall were evaluated in time-series analyses from 2012 week 19 to 2013 week 36. Zonal as well as clinic-based, multivariate, autoregressive, integrated, moving average (ARIMAX) models implementing environmental variables were developed to model transmission in 2011 week 19 to 2012 week 18 and forecast transmission in 2013 week 37 to week 41. RESULTS: During the dry, low transmission season significantly higher vegetation indices, nocturnal land surface temperature and nocturnal dew point were associated with the areas of higher transmission. Environmental variables improved ARIMAX models. Dew point and normalized differentiated vegetation index were significant predictors and improved all zonal transmission models. In the high-transmission zone, this was also seen for land surface temperature. Clinic models were improved by adding dew point and land surface temperature as well as normalized differentiated vegetation index. The mean average error of prediction for ARIMAX models ranged from 0.7 to 33.5%. Forecasts of malaria incidence were valid for three out of five rural health centres; however, with poor results at the zonal level. CONCLUSIONS: In this study, the fit of ARIMAX models improves when environmental variables are included. There is a significant association of remotely-sensed nocturnal dew point with malaria transmission. Interestingly, dew point might be one of the factors sustaining malaria transmission in areas of general aridity during the dry season.

Prevalence of ataxia in children: a systematic review.

OBJECTIVE: To estimate the prevalence of childhood ataxia resulting from both genetic and acquired causes. METHODS: A systematic review was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) statement. Five databases were searched for articles reporting a frequency measure (e.g., prevalence, incidence) of ataxia in children. Included articles were first grouped according to the World Health Organization (WHO) regions and subsequently classified according to etiology (genetic, acquired, or mixed). Each article was assessed for its risk of bias on the domains of sampling, measurement, and analysis. Incidence values were converted to prevalence estimates whenever possible. European prevalence estimates for different etiologies of ataxia were summed to gauge the overall prevalence of childhood ataxia. RESULTS: One hundred fifteen articles were included in the review. More than 50% of the data originated from the Europe WHO region. Data from this region also showed the least susceptibility to bias. Little data were available for Africa and Southeast Asia. The prevalence of acquired ataxias was found to vary more greatly across regions than the genetic ataxias. Ataxic cerebral palsy was found to be a significant contributor to the overall prevalence of childhood ataxia across WHO regions. The prevalence of childhood ataxias in Europe was estimated to be ∼26/100,000 children and likely reflects a minimum prevalence worldwide. CONCLUSIONS: The findings show that ataxia is a common childhood motor disorder with a higher prevalence than previously assumed. More research concerning the epidemiology, assessment, and treatment of childhood ataxia is warranted.

Measuring malaria by passive case detection: a new perspective based on Zambian experience.

Most measurements of malaria are based on cross-sectional data and do not reflect the dynamic nature of transmission, particularly when interventions require timely data for planning strategies. Such data can be collected from local rural health centres (RHCs) where the infrastructure is sufficiently developed and where rapid diagnostics are in use. Because in rural areas, the population served by RHC is reasonably static, the regular use of malaria rapid diagnosis in RHCs can provide data to assess local weekly incidence rates, and such data are easily dispersed by cell phones. Essentially each RHC is a potential sentinel site that can deliver critical information to programme planners. Data collected during this process of passive case detection over a five-year period in the Macha area of Zambia show the importance of ecological zones and refugia in the seasonal fluctuation of malaria cases. If this process is implemented nationally it can assist in planning efficient use of resources and may contribute to local management and even elimination of malaria in this region.

Full-length Plasmodium falciparum circumsporozoite protein administered with long-chain poly(I·C) or the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion elicits potent antibody and CD4+ T cell immunity and protection in mice.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I · C) [poly(I · C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4(+) T cell responses. Moreover, E. coli-derived CS proteins in combination with poly(I · C)LC induced potent multifunctional (interleukin 2-positive [IL-2(+)], tumor necrosis factor alpha-positive [TNF-α(+)], gamma interferon-positive [IFN-γ(+)]) CD4(+) effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ~50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4(+) T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4(+) T cell immunity that was significantly less potent than that with poly(I · C)LC. Overall, these data suggest that full-length CS proteins and poly(I · C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development.

The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5' end of YF17D, in frame and upstream of the polyprotein (YF-5'/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8(+) IFN-gamma T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4(+) T cells expressing IFN-gamma and IL-2. A balanced CD4(+) and CD8(+) T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development.

Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii.

The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of naïve mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8(+) T-cells. A single immunization of naïve mice with recombinant YF17D resulted in robust production of IFN-gamma by CD8(+) T-cells and IFN-gamma and IL-2 by CD4(+) T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo.