Burden of Submicroscopic Plasmodium Infections and Detection of kelch13 Mutant Parasites in Military and Civilian Populations in Papua New Guinea.

Malaria remains a major public health problem in Papua New Guinea (PNG) and an important force health protection issue for both PNG and Australian Defence Forces. To investigate the malaria burden in the military and civilians residing on military bases, a cross-sectional survey was conducted in April 2019 at three military bases in Wewak, Manus Island, and Vanimo, PNG. A total of 1,041 participants were enrolled; 235 military personnel from three bases and 806 civilians from Wewak and Vanimo. Polymerase chain reaction (PCR) revealed an overall high prevalence of Plasmodium infection in both the military and civilians. Among the military, the infection prevalence was significantly higher in Wewak (35.5%) and Vanimo (33.3%) bases than on Manus Island (11.8%). Among civilians, children (<16 years old) had significantly higher odds of being PCR positive than adults (≥16 years old). At Wewak and Vanimo, Plasmodium vivax accounted for 85.4%, 78.2%, and 66.2% of infections in military, children, and adult populations. Overall, 87.3%, 41.3%, and 61.3% of Plasmodium infections in the military, children, and adults, respectively, were detected only by PCR, not by microscopy (submicroscopic [SM] infections). Children had a significantly lower proportion of SM infections than adults and Papua New Guinea Defence Force personnel. Infection status was not associated with hemoglobin levels in these populations at the time of the survey. Mutant kelch13 (C580Y) parasites were identified in 5/68 Plasmodium falciparum-infected individuals. The survey results indicate extensive malaria transmission on these bases, especially in Wewak and Vanimo. More intensified interventions are required to reduce malaria transmission on PNG military bases.

A Health Survey Revealing Prevalence of Vector-Borne Diseases and Tuberculosis in Papua New Guinea Defence Force Personnel and Families.

The Papua New Guinean Defence Force (PNGDF) and the Australian Defence Force (ADF) work and train closely. Infectious diseases pose a health threat to both forces, but recent knowledge about the risk at military bases in Papua New Guinea is limited. To improve understanding, a collaborative cross-sectional survey was conducted (March-April 2019) at Lombrum Naval base (Manus province) and Moem Army barracks (Wewak, East Sepik province) plus its Vanimo outpost (Sandaun province). Clinical data, venous blood, and sputum were collected from PNGDF personnel (DF) from the three sites, with point-of-care testing conducted for malaria (microscopy and rapid diagnostic test [RDT]), lymphatic filariasis (RDT), glucose-6-phosphate-dehydrogenase (G6PD) deficiency (RDT), tuberculosis (GeneXpert), and hemoglobin level. Finger prick blood collected from family members residing at the Wewak base was tested for malaria and hemoglobin level. Overall, 235 DF and 793 family members completed the survey. Microscopy revealed malaria prevalence as 0.4% Plasmodium falciparum and 3.1% Plasmodium vivax among DF and 3.5% P. falciparum, 14.3% P. vivax, and 0.3% mixed P. falciparum/P. vivax among family members. Among DF, 3.9% were G6PD deficient and none tested positive for tuberculosis or for lymphatic filariasis antigen. Anemia was present in 6.5% of DF and 47.3% of family members, predominantly females. Results suggest ongoing exposure to malaria, particularly P. vivax, at study sites, whereas infections of lymphatic filariasis and tuberculosis were not detected. Survey results will inform the PNGDF and the ADF regarding vector-borne disease risk for future sustainable health and disease control interventions.

Prevalence of Barmah Forest Virus, Chikungunya Virus and Ross River Virus Antibodies among Papua New Guinea Military Personnel before 2019.

Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Ross River virus (RRV) belong to the Alphavirus genus of the family Togaviridae. All three virus infections have been reported in Papua New Guinea (PNG) previously, but the exact prevalence and distribution of these three alphaviruses in PNG has not been established. Sera collected from 204 PNG Military Personnel (PNGMP) study participants in April 2019 was tested for the presence of anti-BFV, anti-CHIKV and anti-RRV immunoglobulin G (IgG) antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) IgG detection kits, as well as for specific neutralizing antibodies (NAb) against individual viruses. Overall, sero-positivity of the sera was anti-BFV IgG 12.3% (25/204), anti-BFV NAb 8.3% (17/204); anti-CHIKV IgG 47.1% (96/204), anti-CHIKV NAb 34.8% (71/204); and anti-RRV IgG 93.1% (190/204), anti-RRV NAb 56.4% (115/204), respectively. Of the 137/204 participants that were Nab-positive for at least one virus, we identified 4 BFV, 40 CHIKV and 73 RRV single infections, and 9 RRV+CHIKV and 11 BFV+RRV double infections. The lower proportion of NAb sero-positive compared to the ELISA IgG sero-positive assay samples suggests that the currently available commercial ELISA detection kits for these three alphaviruses may not be suitable for diagnostic/surveillance purposes in endemic areas such as PNG, due to serological cross-reactivity among these three alphaviruses. Laboratory testing using known positive control sera indicated no cross-neutralization between BFV and RRV; however, some RRV or BFV single infection human sera demonstrated low-level cross-neutralization against CHIKV (the ratio of RRV/CHIKV NAb titers or BFV/CHIKV ≥ 4). Our preliminary results indicate that the majority of PNGMP have previously been exposed to RRV, with mild exposure to CHIKV and low-level exposure to BFV, suggesting that multiple alphaviruses have been circulating among PNGMP. The transmission landscapes of these three alphaviruses across PNG should be prioritized for further investigation, including identification of specific vectors and hosts that mediate human spillover in order to mitigate future outbreaks. Ongoing education regarding precautionary and protective measures are needed to better protect individuals who travel to PNG.

Serological evidence of possible high levels of undetected transmission of Zika virus among Papua New Guinea military personnel, 2019.

Objectives: The Papua New Guinea (PNG) Health Department retrospectively reported six cases of Zika virus (ZIKV) from a cohort of febrile patients during outbreaks of dengue and malaria in 2016. However, the transmission of ZIKV remains unclear due to lack of testing capability. This study aimed to determine the level of immunity to ZIKV among PNG military personnel (PNGMP) in 2019. Methods: Sera of 208 PNGMP recruited in April 2019 was tested for the presence of anti-ZIKV immunoglobulin G (IgG) and M (IgM) antibodies using Euroimmun IgG/IgM detection kits, and anti-ZIKV neutralizing antibody (Nab) against a ZIKV African strain on all anti-ZIKV-IgG/IgM+ samples. Results: Anti-ZIKV seropositivity of these sera was as follows: IgG, 67%; IgM, 9%; and Nab, 65%. Five of 19 anti-ZIKV-IgM+ samples had anti-ZIKV-Nab titres ≥20, as well as an anti-ZIKV-Nab titre ratio ≥4 compared with the Nab titres of four anti-dengue serotypes, so met the criteria of the World Health Organization (WHO) for confirmed ZIKV infection. Conclusions: The prevalence of anti-ZIKV-Nab of 65% suggests that there are high levels of ZIKV exposure among PNGMP. Five of the 19 anti-ZIKV-IgM+ samples met the WHO criteria for confirmed ZIKV infection, suggesting a recent undetected outbreak in PNGMP. These results provide better understanding of the current ZIKV epidemic status in PNGMP.

Seroprevalence of chikungunya virus among military personnel in Papua New Guinea, 2019.

Objectives: The first outbreak of chikungunya virus (CHIKV) was reported in West Sepik, Papua New Guinea (PNG) in June 2012, and spread rapidly throughout PNG. CHIKV imported from PNG to Queensland has been reported occasionally, but transmission of CHIKV in PNG remains unclear due to the lack of testing capability. This study investigated the degree of CHIKV exposure among PNG military personnel (PNGMP) in 2019, 7 years after its first emergence. Methods: Sera of 204 PNGMP recruited in April 2019 was tested for the presence of anti-CHIKV immunoglobulin G (IgG) antibodies using a commercially available IgG detection kit, and anti-CHIKV neutralizing antibodies against a CHIKV Reunion strain using a neutralizing assay. Results: Anti-CHIKV seropositivity of the sera was 47% and 35%, respectively, using the enzyme-linked immunosorbent assay (ELISA) and neutralizing assay. Five percent (n=11) of samples were found to be IgG negative or borderline, but neutralizing antibody positive. Conclusions: The prevalence of anti-CHIKV neutralizing antibody of 35% suggests that CHIKV infection has become endemic among PNGMP. Current commercially available CHIKV ELISA detection kits may not be suitable for diagnostic purposes in multiple alphavirus endemic areas such as PNG, due to serological cross-reactivity among alphaviruses. Re-emergence of CHIKV in PNGMP is possible.

Circulation of 2 Barmah Forest Virus Lineages in Military Training Areas, Australia.

During 2017-2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3' untranslated region suggest that 2 lineages are circulating in eastern Australia.

In Vivo Efficacy and Pharmacokinetics of the 2-Aminomethylphenol Antimalarial JPC-3210 in the Aotus Monkey-Human Malaria Model.

Nonimmune Aotus monkeys infected with Plasmodium falciparum and Plasmodium vivax were cured of their infections when treated with a single oral dose of 5 mg/kg and 10 mg/kg of the 2-aminomethylphenol, JPC-3210, respectively. Corresponding mean blood elimination half-lives of JPC-3210 were lengthy at 19.1 days and 20.5 days, respectively. This in vivo potency and lengthy half-life supports the further development of JPC-3210 as a promising, long-acting blood schizontocidal antimalarial for malaria treatment and prevention.

Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance.

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance.

Persistence and immunogenicity of chemically attenuated blood stage Plasmodium falciparum in Aotus monkeys.

Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5-0.7million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2days following inoculation as determined by thick blood smears, and for up to 8days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria.

Liar, liar, working memory on fire: Investigating the role of working memory in childhood verbal deception.

The aim of the current study was to investigate the role of working memory in verbal deception in children. We presented 6- and 7-year-olds with a temptation resistance paradigm; they played a trivia game and were then given an opportunity to peek at the final answers on the back of a card. Measures of both verbal and visuospatial working memory were included. The good liars performed better on the verbal working memory test in both processing and recall compared with the bad liars. However, there was no difference in visuospatial working scores between good liars and bad liars. This pattern suggests that verbal working memory plays a role in processing and manipulating the multiple pieces of information involved in lie-telling.

Opsonic phagocytosis of Plasmodium falciparum merozoites: mechanism in human immunity and a correlate of protection against malaria.

BACKGROUND: An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development. METHODS: Using a validated assay to measure opsonic phagocytosis of Plasmodium falciparum merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection. RESULTS: Antibodies promoting opsonic phagocytosis of merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of merozoites, providing a functional link between these antigen-specific responses and protection for the first time. CONCLUSIONS: Opsonic phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.

Antibodies to polymorphic invasion-inhibitory and non-Inhibitory epitopes of Plasmodium falciparum apical membrane antigen 1 in human malaria.

BACKGROUND: Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1. METHODOLOGY/FINDINGS: We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies. CONCLUSIONS/SIGNIFICANCE: Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1.

Erythrocyte-binding antigens of Plasmodium falciparum are targets of human inhibitory antibodies and function to evade naturally acquired immunity.

Abs that inhibit Plasmodium falciparum invasion of erythrocytes form an important component of human immunity against malaria, but key target Ags are largely unknown. Phenotypic variation by P. falciparum mediates the evasion of inhibitory Abs, contributing to the capacity of P. falciparum to cause repeat and chronic infections. However, Ags involved in mediating immune evasion have not been defined, and studies of the function of human Abs are limited. In this study, we used novel approaches to determine the importance of P. falciparum erythrocyte-binding Ags (EBAs), which are important invasion ligands, as targets of human invasion-inhibitory Abs and define their role in contributing to immune evasion through variation in function. We evaluated the invasion-inhibitory activity of acquired Abs from malaria-exposed children and adults from Kenya, using P. falciparum with disruption of genes encoding EBA140, EBA175, and EBA181, either individually or combined as EBA140/EBA175 or EBA175/EBA181 double knockouts. Our findings provide important new evidence that variation in the expression and function of the EBAs plays an important role in evasion of acquired Abs and that a substantial amount of phenotypic diversity results from variation in expression of different EBAs that contributes to immune evasion by P. falciparum. All three EBAs were identified as important targets of naturally acquired inhibitory Abs demonstrated by differential inhibition of parental parasites greater than EBA knockout lines. This knowledge will help to advance malaria vaccine development and suggests that multiple invasion ligands need to be targeted to overcome the capacity of P. falciparum for immune evasion.

Immunoglobulin G subclass-specific responses against Plasmodium falciparum merozoite antigens are associated with control of parasitemia and protection from symptomatic illness.

Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum.

BACKGROUND: Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood. METHODS: We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42). RESULTS: Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied. CONCLUSIONS: Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.

Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies.

Antibodies that inhibit Plasmodium falciparum invasion of erythrocytes are believed to be an important component of immunity against malaria. During blood-stage infection, P. falciparum can use different pathways for erythrocyte invasion by varying the expression and/or utilization of members of 2 invasion ligand families: the erythrocyte-binding antigens (EBAs) and reticulocyte-binding homologs (PfRhs). Invasion pathways can be broadly classified into 2 groups based on the use of sialic acid (SA) on the erythrocyte surface by parasite ligands. We found that inhibitory antibodies are acquired by malaria-exposed Kenyan children and adults against ligands of SA-dependent and SA-independent invasion pathways, and the ability of antibodies to inhibit erythrocyte invasion depended on the pathway used by P. falciparum isolates. Differential inhibition of P. falciparum lines that varied in their use of specific EBA and PfRh proteins pointed to these ligand families as major targets of inhibitory antibodies. Antibodies against recombinant EBA and PfRh proteins were acquired in an age-associated manner, and inhibitory antibodies against EBA175 appeared prominent among some individuals. These findings suggest that variation in invasion phenotype might have evolved as a mechanism that facilitates immune evasion by P. falciparum and that a broad inhibitory response against multiple ligands may be required for effective immunity.