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Background False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa. Methods From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs. Results Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6–89.8]; specificity, 99.4% [95% CI, 97.7–99.9]). Conclusions We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

Preerythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading preerythrocytic vaccine, RTS,S/AS01E (Mosquirix), entered implementation programs in 2019 and targets the major sporozoite surface antigen, circumsporozoite protein (CSP). However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver-stage proteins that could potentially be used in combination with CSP; they are referred to as preerythrocytic vaccine antigens (PEVAs). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (protein prime-adenovirus 5 [Ad5] boost) and Plasmodium berghei (DNA prime-Ad5 boost) in mice. When combined as individual antigens with P. yoelii CSP (PyCSP), three of eight P. yoelii PEVAs significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when three P. berghei PEVAs and P. berghei CSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP plus PEVA vaccinations. Both P. yoelii and P. berghei CSP plus PEVA combination vaccines induced robust CD8(+) T cell responses, including signature gamma interferon (IFN-γ) increases. In the P. berghei model system, IFN-γ responses were significantly higher in hepatic versus splenic CD8(+) T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.

Highlights • Pfs230 is a candidate malaria transmission blocking vaccine against P. falciparum. • Pfs230 vaccine is being tested in areas where malaria and helminth infections are co-endemic. • Chronic helminth infection induces a marked increase in systemic Th2 and regulatory cytokine levels in mice. • Chronic H. polygyrus bakeri infection does not alter Pfs230 vaccine specific-antibody levels. • Functional activity of Pfs230 vaccine was not impaired by chronic helminth infection in mice., Introduction Malaria transmission blocking vaccines (TBV) are innovative approaches that aim to induce immunity in humans against Plasmodium during mosquito stage, neutralizing the capacity of the infected vectors to transmit malaria. Pfs230D1-EPA/Alhydrogel®, a promising protein-protein conjugate malaria TBV, is currently being tested in human clinical trials in areas where P. falciparum malaria is coendemic with helminth parasites. Helminths are complex metazoans that share the master capacity to downregulate the host immune response towards themselves and also to bystander antigens, including vaccines. However, it is not known whether the activity of a protein-based malaria TBV may be affected by a chronic helminth infection. Methods Using an experimental murine model for a chronic helminth infection (Heligmosomoides polygyrus bakeri - Hpb), we evaluated whether prior infection alters the activity of Pfs230D1-EPA/Alhydrogel® TBV in mice. Results After establishment of a chronic infection, characterized by a marked increase of parasite antigen-specific IgG1, IgA and IgE antibody responses, concomitant with an increase of systemic IL-10, IL-5 and IL-6 levels, the Hpb-infected mice were immunized with Pfs230D1-EPA/Alhydrogel® and the vaccine-specific immune response was compared with that in non-infected immunized mice. TBV immunizations induced an elevated vaccine specific-antibody response, however Pfs230D1 specific-IgG levels were similar between infected and uninfected mice at days 15, 25 and 35 post-vaccination. Absolute numbers of Pfs230D1-activated B cells generated in response to the vaccine were also similar among the vaccinated groups. Finally, vaccine activity assessed by reduction of oocyst number in P. falciparum infected mosquitoes was similar between Hpb-infected and immunized mice with non-infected immunized mice. Conclusion Pfs230D1-EPA/Alhydrogel® efficacy is not impaired by a chronic helminth infection in mice.

Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.

Plasmodium vivaxmalaria incidence has increased in Latin America and Asia and is responsible for nearly 74.1% of malaria cases in Latin America. Immune responses toP. vivaxare less well characterized than those toP. falciparum, partly becauseP. vivaxis more difficult to cultivate in the laboratory. While antibodies are known to play an important role inP. vivaxdisease control, few studies have evaluated responses toP. vivaxsexual stage antigens. We collected sera or plasma samples fromP. vivax-infected subjects from Brazil (n= 70) and Cambodia (n= 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, theP. falciparumortholog of Pvs230D1M. We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and CambodianP. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure toP. falciparum, or co-infection. We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses amongP. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia.