Gregory S. Noland

Low Prevalence of Antibodies to Preerythrocytic but Not Blood-Stage Plasmodium falciparum Antigens in an Area of Unstable Malaria Transmission Compared to Prevalence in an Area of Stable Malaria Transmission

ABSTRACT In areas where levels of transmission of Plasmodium falciparum are high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.

Changes in B Cell Populations and Merozoite Surface Protein-1-Specific Memory B Cell Responses after Prolonged Absence of Detectable P. falciparum Infection

Clinical immunity to malaria declines in the absence of repeated parasite exposure. However, little is known about how B cell populations and antigen-specific memory B cells change in the absence of P. falciparum infection. A successful indoor residual insecticide spraying campaign in a highland area of western Kenya, led to an absence of blood-stage P. falciparum infection between March 2007 and April 2008. We assessed memory B cell responses in 45 adults at the beginning (April 2008) and end (April 2009) of a subsequent 12-month period during which none of the adults had evidence of asymptomatic parasitemia or clinical disease. Antibodies and memory B cells to the 42-kDa portion of the merozoite surface protein-1 (MSP-142) were measured using ELISA and ELISPOT assays, respectively. B cell populations were characterized by flow cytometry. From 2008 to 2009, the prevalence of MSP-142-specific memory B cells (45% vs. 55%, respectively, P = 0.32) or antibodies (91% vs. 82%, respectively, P = 0.32) did not differ significantly, although specific individuals did change from positive to negative and vice versa, particularly for memory B cells, suggesting possible low-level undetected parasitemia may have occurred in some individuals. The magnitude of MSP-142-specific memory B cells and levels of antibodies to MSP-142 also did not differ from 2008 to 2009 (P>0.10 for both). However, from 2008 to 2009 the proportions of both class-switched atypical (CD19+IgD-CD27-CD21-IgM-) and class-switched activated (CD19+IgD-CD27+CD21-IgM-) memory B cells decreased (both P<0.001). In contrast, class-switched resting classical memory B cells (CD19+IgD-CD27+CD21+IgM-) increased (P<0.001). In this area of seasonal malaria transmission, a one- year absence of detectable P. falciparum infection was not associated with changes in the prevalence or level of MSP-142 specific memory B cells, but was associated with major changes in overall memory B cell subsets.

A venue-based survey of malaria, anemia and mobility patterns among migrant farm workers in amhara region, Ethiopia

Background Mobile populations present unique challenges to malaria control and elimination efforts. Each year, a large number of individuals travel to northwest Amhara Region, Ethiopia to seek seasonal employment on large-scale farms. Agricultural areas typically report the heaviest malaria burden within Amhara thereby placing migrants at high risk of infection. Yet little is known about these seasonal migrants and their malaria-related risk factors. Methods and Findings In July 2013, a venue-based survey of 605 migrant laborers 18 years or older was conducted in two districts of North Gondar zone, Amhara. The study population was predominantly male (97.7%) and young (mean age 22.8 years). Plasmodium prevalence by rapid diagnostic test (RDT) was 12.0%; One quarter (28.3%) of individuals were anemic (hemoglobin <13 g/dl). Nearly all participants (95.6%) originated from within Amhara Region, with half (51.6%) coming from within North Gondar zone. Around half (51.2%) slept in temporary shelters, while 20.5% regularly slept outside. Only 11.9% of participants had access to a long lasting insecticidal net (LLIN). Reported net use the previous night was 8.8% overall but 74.6% among those with LLIN access. Nearly one-third (30.1%) reported having fever within the past two weeks, of whom 31.3% sought care. Cost and distance were the main reported barriers to seeking care. LLIN access (odds ratio [OR] = 0.30, P = 0.04) and malaria knowledge (OR = 0.50, P = 0.02) were significantly associated with reduced Plasmodium infection among migrants, with a similar but non-significant trend observed for reported net use the previous night (OR = 0.16, P = 0.14). Conclusions High prevalence of malaria and anemia were observed among a young population that originated from relatively proximate areas. Low access to care and low IRS and LLIN coverage likely place migrant workers at significant risk of malaria in this area and their return home may facilitate parasite transport to other areas. Strategies specifically tailored to migrant farm workers are needed to support malaria control and elimination activities in Ethiopia.

Determinants of Bed Net Use in Southeast Nigeria following Mass Distribution of LLINs: Implications for Social Behavior Change Interventions.

Millions of long-lasting insecticide treated nets (LLINs) have been distributed as part of the global malaria control strategy. LLIN ownership, however, does not necessarily guarantee use. Thus, even in the ideal setting in which universal coverage with LLINs has been achieved, maximal malaria protection will only be achieved if LLINs are used both correctly and consistently. This study investigated the factors associated with net use, independent of net ownership. Data were collected during a household survey conducted in Ebonyi State in southeastern Nigeria in November 2011 following a statewide mass LLIN distribution campaign and, in select locations, a community-based social behavior change (SBC) intervention. Logistic regression analyses, controlling for household bed net ownership, were conducted to examine the association between individual net use and various demographic, environmental, behavioral and social factors. The odds of net use increased among individuals who were exposed to tailored SBC in the context of a home visit (OR = 17.11; 95% CI 4.45–65.79) or who received greater degrees of social support from friends and family (ptrend < 0.001). Factors associated with decreased odds of net use included: increasing education level (ptrend = 0.020), increasing malaria knowledge level (ptrend = 0.022), and reporting any disadvantage of bed nets (OR = 0.39; 95% CI 0.23–0.78). The findings suggest that LLIN use is significantly influenced by social support and exposure to a malaria-related SBC home visit. The malaria community should thus further consider the importance of community outreach, interpersonal communication and social support on adoption of net use behaviors when designing future research and interventions.

Effect of transmission intensity and age on subclass antibody responses to Plasmodium falciparum pre-erythrocytic and blood-stage antigens

Cytophilic immunoglobulin (IgG) subclass responses (IgG1 and IgG3) to Plasmodium falciparum antigens have been associated with protection from malaria, yet the relative importance of transmission intensity and age in generation of subclass responses to pre-erythrocytic and blood-stage antigens have not been clearly defined. We analyzed IgG subclass responses to the pre-erythrocytic antigens CSP, LSA-1, and TRAP and the blood-stage antigens AMA-1, EBA-175, and MSP-1 in asymptomatic residents age 2 years or older in stable (n=116) and unstable (n=96) transmission areas in Western Kenya. In the area of stable malaria transmission, a high prevalence of cytophilic (IgG1 and IgG3) antibodies to each antigen was seen in all age groups. Prevalence and levels of cytophilic antibodies to pre-erythrocytic and blood-stage P. falciparum antigens increased with age in the unstable transmission area, yet IgG1 and IgG3 responses to most antigens for all ages in the unstable transmission area were less prevalent and lower in magnitude than even the youngest age group from the stable transmission area. The dominance of cytophilic responses over non-cytophilic (IgG2 and IgG4) was more pronounced in the stable transmission area, and the ratio of IgG3 over IgG1 generally increased with age. In the unstable transmission area, the ratio of cytophilic to non-cytophilic antibodies did not increase with age, and tended to be IgG3-biased for pre-erythrocytic antigens yet IgG1-biased for blood-stage antigens. The differences between areas could not be attributed to active parasitemia status, as there were minimal differences in antibody responses between those positive and negative for Plasmodium infection by microscopy in the stable transmission area. Individuals in areas of unstable transmission have low cytophilic to non-cytophilic IgG subclass ratios and low IgG3:IgG1 ratios to P. falciparum antigens. These imbalances could contribute to the persistent risk of clinical malaria in these areas and serve as population-level, age-specific biomarkers of transmission.

Decreased Prevalence of Anemia in Highland Areas of Low Malaria Transmission After a 1-Year Interruption of Transmission

BACKGROUND Malaria control campaigns have reduced malaria transmission to very low levels in many areas of Africa. Yet the extent to which malaria interruption or elimination might decrease the prevalence of anemia in areas of low malaria transmission is unknown. METHODS Kapsisiywa and Kipsamoite, highland areas of Kenya with low, unstable malaria transmission, experienced a 12-month interruption in malaria transmission from April 2007 to May 2008, following high-level coverage (>70% of households) with indoor residual insecticide spraying in 2007. Hemoglobin levels were tested in 1697 randomly selected asymptomatic residents of Kapsisiywa (n = 910) and Kipsamoite (n = 787) at the beginning of a 12-month period of interrupted transmission (in May 2007) and 14 months later (in July 2008). RESULTS From May 2007 to July 2008, only 1 of 1697 study cohort members developed clinical malaria. In this period, the prevalence of anemia decreased in Kapsisiywa in all age groups (from 57.5% to 37.9% in children aged <5 years [P < .001], from 21.7% to 10.5% in children aged 5-14 years [P < .001], and from 22.7% to 16.6% in individuals aged ≥ 15 years [P = .004]). The prevalence of anemia in Kipsamoite also decreased in children aged <5 years (from 47.2% to 31.3%; P = .001) but was unchanged in children aged 5-14 years and in individuals aged ≥15 years. Among children <5 years, anemia prevalence was reduced by 34% in both Kapsisiywa (95% confidence interval [CI], 21%-45%) and Kipsamoite (95% CI, 16%-48%). CONCLUSIONS Successful malaria elimination or interruption may lead to substantial reductions in anemia prevalence even in areas of very low transmission.

Criteria to Stop Mass Drug Administration for Lymphatic Filariasis Have Been Achieved Throughout Plateau and Nasarawa States, Nigeria

Nigeria has the largest population at risk for lymphatic filariasis (LF) in Africa. This study used a transmission assessment survey (TAS) to determine whether mass drug administration (MDA) for LF could stop in 21 districts, divided into four evaluation units (EUs), of Plateau and Nasarawa States, Nigeria, after 8-12 years of annual albendazole-ivermectin treatment. A total of 7,131 first- and second-year primary school children (approximately 6-7 years old) were tested for LF antigen by immunochromatographic test (ICT) from May to June 2012. The target sample size of 1,692 was exceeded in each EU (range = 1,767-1,795). A total of 25 (0.4%) individuals were ICT positive, with the number of positives in each EU (range = 3-11) less than the TAS cutoff of 20, meaning that LF transmission had been reduced below sustainable levels. As a result, 3.5 million annual albendazole-ivermectin treatments were halted in 2013. Combined with the previous halt of MDA for LF in other parts of Plateau and Nasarawa, these are the first Nigerian states to stop LF MDA statewide. Posttreatment surveillance is ongoing to determine if LF transmission has been interrupted.

The Plasmodium falciparum Antigen MB2 Induces Interferon-γ and Interleukin-10 Responses in Adults in Malaria Endemic Areas of Western Kenya.

Background: MB2 is a novel Plasmodium falciparum antigen of unknown function expressed in pre-erythrocytic and blood stages of infection in the human host. Interferon-gamma (IFN-γ) and interleukin (IL)-10 responses to other P. falciparum antigens have been associated with protection from clinical malaria, but these responses have not been studied for MB2. The present study was undertaken to characterize IFN-γ and IL-10 responses to P. falciparum MB2 antigen in adults living in areas of differing malaria transmission in Western Kenya. Materials and Methods: Cytokine responses to two 9-mer MB2 peptides predicted to be human leukocyte antigen (HLA) class I restricted T-cell epitopes were measured by enzyme-linked immunosorbent assay (ELISA) (IFN-γ and IL-10) and enzyme-linked immunosorbent spot (ELISPOT) (IFN-γ) in adults (n = 228) in areas of unstable and stable malaria transmission. HLA class I restriction of responses was assessed in a sub-group of the study population. Results: IFN-γ and IL-10 responses to MB2 peptides by ELISA were observed in both sites with no significant difference in prevalence (IFN-γ, unstable transmission area, 18.8%, stable transmission area, 27.5%, P = 0.33; IL-10, unstable transmission area, 22.5%, stable transmission area, 25.0%, P = 0.78). Prevalence of IFN-γ responses by ELISPOT was also similar in both areas (unstable, 10.8%, stable, 10.9%, P = 0.98). Neither IFN-γ nor IL-10 responses showed evidence of HLA class I restriction. Conclusions: MB2 induces IFN-γ and IL-10 responses in adults living in both stable and unstable malaria transmission areas. Future studies should assess if these responses are associated with protection from clinical malaria.

Interferon-γ responses to Plasmodium falciparum vaccine candidate antigens decrease in the absence of malaria transmission

Background Malaria elimination campaigns are planned or active in many countries. The effects of malaria elimination on immune responses such as antigen-specific IFN- γ responses are not well characterized. Methods IFN- γ responses to the P. falciparum antigens circumsporozoite protein, liver stage antigen-1, thrombospondin-related adhesive protein, apical membrane antigen-1, MB2, and merozoite surface protein-1 were tested by ELISA in 243 individuals in highland Kenya in April 2008, October 2008, and April 2009, after a one-year period of interrupted malaria transmission from April 2007 to March 2008. Results While one individual (0.4%) tested positive for P. falciparum by PCR inOctober 2008 and another two (0.9%) tested positive in April 2009, no clinical malaria cases were detected during weekly visits. Levels of IFN-γ to all antigens decreased significantly from April 2008 to April 2009 (all P < 0.001). Discussion Naturally acquired IFN- γ responses to P. falciparum antigensare short-lived in the absence of repeated P. falciparum infection. Even short periods of malaria interruption may significantly decrease IFN-γ responses to P. falciparum antigens.