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OBJECTIVETo investigate maternal immunoglobulins’ (IgM, IgG) response to SARS-CoV-2 infection during pregnancy and IgG transplacental transfer, to characterize neonatal antibody response to SARS-CoV-2 infection, and to longitudinally follow actively- and passively-acquired SARS-CoV-2 antibodies in infants.DESIGNA prospective observational study.SETTINGA public healthcare system in Santa Clara County (CA, USA).PARTICIPANTSWomen with SARS-CoV-2 infection during pregnancy and their infants were enrolled between April 15, 2020 and March 31, 2021.OUTCOMESSARS-CoV-2 serology analyses in the cord and maternal blood at delivery and longitudinally in infant blood between birth and 28 weeks of life.RESULTSOf 145 mothers who tested positive for SARS-CoV-2 during pregnancy, 86 had symptomatic infections: 78 with mild-moderate symptoms, and eight with severe-critical symptoms. Of the 147 newborns, two infants showed seroconversion at two weeks of age with high levels of IgM and IgG, including one premature infant with confirmed intrapartum infection. The seropositivity rates of the mothers at delivery was 65% (95% CI 0.56-0.73) and the cord blood was 58% (95% CI 0.49-0.66). IgG levels significantly correlated between the maternal and cord blood (Rs= 0.93, p< 0.0001). IgG transplacental transfer ratio was significantly higher when the first maternal positive PCR was 60-180 days before delivery compared to CONCLUSIONSMaternal SARS-CoV-2 IgG is efficiently transferred across the placenta when infections occur more than two months before delivery. Maternally-derived passive immunity may protect infants up to six months of life. Neonates mount a strong antibody response to perinatal SARS-CoV-2 infection.

Vγ9Vδ2 T cells rapidly respond to phosphoantigens produced by Plasmodium falciparum in an innate-like manner, without prior antigen exposure or processing. Vδ2 T cells have been shown to inhibit parasite replication in vitro and are associated with protection from P. falciparum parasitemia in vivo. Although a marked expansion of Vδ2 T cells is seen after acute malaria infection in naïve individuals, repeated malaria causes Vδ2 T cells to decline both in frequency and in malaria-responsiveness, and to exhibit numerous transcriptional and phenotypic changes, including upregulation of the Fc receptor CD16. Here we investigate the functional role of CD16 on Vδ2 T cells in the immune response to malaria. We show that CD16+ Vδ2 T cells possess more cytolytic potential than their CD16- counterparts, and bear many of the hallmarks of mature NK cells, including KIR expression. Furthermore, we demonstrate that Vδ2 T cells from heavily malaria-exposed individuals are able to respond to opsonized P.falciparum-infected red blood cells through CD16, representing a second, distinct pathway by which Vδ2 T cells may contribute to anti-parasite effector functions. This response was independent of TCR engagement, as demonstrated by blockade of the phosphoantigen presenting molecule Butyrophilin 3A1. Together these results indicate that Vδ2 T cells in heavily malaria-exposed individuals retain the capacity for antimalarial effector function, and demonstrate their activation by opsonized parasite antigen. This represents a new role both for Vδ2 T cells and for opsonizing antibodies in parasite clearance, emphasizing cooperation between the cellular and humoral arms of the immune system., Author summary T cells that express the Vδ2 and Vγ9 TCR chains (Vδ2 T cells) have been shown to play an important role in controlling parasitemia during P.falciparum infection. A better understanding of how these cells interact with malaria parasites to control infection is necessary. We have previously shown that after multiple P. falciparum infections, Vδ2 T cells decrease in frequency, become less responsive to TCR stimulation, and upregulate the Fc receptor CD16. Here we investigate whether Vδ2 T cells from chronically malaria-exposed individuals can be activated directly through CD16 to release proinflammatory cytokines and degranulate. We show that in these individuals, TCR is downregulated on CD16+ Vδ2 T cells, and that these cells are more likely to express a variety of cytotoxic effector molecules. Importantly, we show that these CD16+ Vδ2 T cells can be activated directly through CD16, independent of TCR, by antibody bound to parasite antigen. These results are notable because they indicate many Vδ2 T cells from chronically-exposed individuals may not be exhausted but instead favor an alternative activation pathway, one that cooperates with a mature anti-malarial antibody response.

Background: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8–100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3–100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7–94.8%. No consistent cross-reactivity was observed. Conclusion: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

The use of pesticides to reduce mosquito vector populations is a cornerstone of global malaria control efforts, but the biological impact of most pesticides on human populations, including pregnant women and infants, is not known. Some pesticides, including carbamates, have been shown to perturb the human immune system. We measure the systemic absorption and immunologic effects of bendiocarb, a commonly used carbamate pesticide, following household spraying in a cohort of pregnant Ugandan women and their infants. We find that bendiocarb is present at high levels in maternal, umbilical cord, and infant plasma of individuals exposed during pregnancy, indicating that it is systemically absorbed and trans-placentally transferred to the fetus. Moreover, bendiocarb exposure is associated with numerous changes in fetal immune cell homeostasis and function, including a dose-dependent decrease in regulatory CD4 T cells, increased cytokine production, and inhibition of antigen-driven proliferation. Additionally, prenatal bendiocarb exposure is associated with higher post-vaccination measles titers at one year of age, suggesting that its impact on functional immunity may persist for many months after birth. These data indicate that in utero bendiocarb exposure has multiple previously unrecognized biological effects on the fetal immune system., Control of mosquito populations using pesticides is important for malaria elimination, but effects of pesticides on humans aren’t well understood. Here, Prahl et al. show in a cohort of pregnant Ugandan women and their infants that household spraying with bendiocarb affects the fetal immune system and response to vaccination in infancy.

BackgroundSerological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data.MethodWe conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system.ResultsAmong specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed.ConclusionOur evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

Background Young children are at greatest risk for malaria-associated morbidity and mortality. The immune response of young children differs in fundamental ways from that of adults, and these differences likely contribute to the increased susceptibility of children to severe malaria and to their delayed development of immunity. Elevated levels of pro-inflammatory cytokines and chemokines in the peripheral blood during acute infection contribute to the control of parasitaemia, but are also responsible for much of the immunopathology seen during symptomatic disease. Clinical immunity to malaria may depend upon the ability to regulate these pro-inflammatory responses, possibly through mechanisms of immunologic tolerance. In order to explore the effect of age on the immune response to malaria and the development of clinical immunity, cytokines and chemokines were measured in the plasma of children at day 0 of an acute malaria episode and during convalescence. Results Younger children presenting with acute malaria exhibited much higher levels of TNF, IL2, and IL6, as well as increased Th1 associated chemokines IP10, MIG, and MCP1, compared to older children with acute malaria. Additionally, the regulatory cytokines IL10 and TNFRI were dramatically elevated in younger children compared to older children during acute infection, indicating that regulatory as well as pro-inflammatory cytokine responses are dampened in later childhood. Conclusions Together these data suggest that there is a profound blunting of the cytokine and chemokine response to malaria among older children residing in endemic settings, which may be due to repeated malaria exposure, intrinsic age-based differences in the immune response, or both.

Background. Experimental inoculation of viable Plasmodium falciparum sporozoites administered with chemoprevention targeting blood-stage parasites results in protective immunity. It is unclear whether chemoprevention similarly enhances immunity following natural exposure to malaria. Methods. We assessed P. falciparum–specific T-cell responses among Ugandan children who were randomly assigned to receive monthly dihydroartemisinin-piperaquine (DP; n = 87) or no chemoprevention (n = 90) from 6 to 24 months of age, with pharmacologic assessments for adherence, and then clinically followed for an additional year. Results. During the intervention, monthly DP reduced malaria episodes by 55% overall (P < .001) and by 97% among children who were highly adherent to DP (P < .001). In the year after the cessation of chemoprevention, children who were highly adherent to DP had a 55% reduction in malaria incidence as compared to children given no chemoprevention (P = .004). Children randomly assigned to receive DP had higher frequencies of blood-stage specific CD4+ T cells coproducing interleukin-2 and tumor necrosis factor α (P = .003), which were associated with protection from subsequent clinical malaria and parasitemia, and fewer blood-stage specific CD4+ T cells coproducing interleukin-10 and interferon γ (P = .001), which were associated with increased risk of malaria. Conclusions. In this setting, effective antimalarial chemoprevention fostered the development of CD4+ T cells that coproduced interleukin 2 and tumor necrosis factor α and were associated with prospective protection, while limiting CD4+ T-cell production of the immunoregulatory cytokine IL-10.

Vδ2+ γδ T cells are semi-innate T cells that expand markedly following P. falciparum (Pf) infection in naïve adults, but are lost and become dysfunctional among children repeatedly exposed to malaria. The role of these cells in mediating clinical immunity (i.e. protection against symptoms) to malaria remains unclear. We measured Vδ2+ T cell absolute counts at acute and convalescent malaria timepoints (n = 43), and Vδ2+ counts, cellular phenotype, and cytokine production following in vitro stimulation at asymptomatic visits (n = 377), among children aged 6 months to 10 years living in Uganda. Increasing age was associated with diminished in vivo expansion following malaria, and lower Vδ2 absolute counts overall, among children living in a high transmission setting. Microscopic parasitemia and expression of the immunoregulatory markers Tim-3 and CD57 were associated with diminished Vδ2+ T cell pro-inflammatory cytokine production. Higher Vδ2 pro-inflammatory cytokine production was associated with protection from subsequent Pf infection, but also with an increased odds of symptoms once infected. Vδ2+ T cells may play a role in preventing malaria infection in children living in endemic settings; progressive loss and dysfunction of these cells may represent a disease tolerance mechanism that contributes to the development of clinical immunity to malaria.

BackgroundIn malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses.MethodsUsing cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes.ResultsCord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12-20weeks of gestation; p=0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p=0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3-CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p=0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p&nbsp

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

Naturally acquired immunity to malaria emerges slowly in malaria-endemic populations. In areas of high transmission, adults experience relatively few clinical episodes, compared with children, despite similar exposure to parasites, indicating that the adult immune system is able to control parasite burden and/or immunopathology associated with disease. Murine models have shown that cytokine-producing CD4+ T cells are critical for protection from malaria [1, 2]. In naturally exposed humans, interferon γ (IFN-γ) production by CD4+ T cells in response to merozoite [3, 4] and sporozoite [5–7] antigens has been associated with protection from disease. The importance of Plasmodium falciparum–specific CD4+ T-cell responses was further demonstrated by a study of naive adults vaccinated with an ultra-low dose of blood-stage parasites, which found that the CD4+ T-cell response was protective from malaria, even in the absence of antibody [8]. Advances in multiparametric flow cytometry have made it clear that assessment of pathogen-specific T cells by a single parameter is often inadequate for identifying correlates of protection. Antigen-specific T cells are functionally heterogeneous and can exhibit a complex variety of effector mechanisms. Mounting evidence from many infectious models suggests that the quality, rather than the quantity, of response is critical for immunity [9]. For instance, multifunctional CD4+ T cells coproducing IFN-γ, tumor necrosis factor α (TNF-α), and interleukin 2 (IL-2) correlate with vaccine-mediated protection from Leishmania major [10] and are implicated in protection induced by vaccinia virus immunization [11]. Multifunctional CD4+ T cells that coproduce TNF-α and/or IL-2 in conjunction with IFN-γ are thought to have increased function [12] and are associated with nonprogression of human immunodeficiency virus (HIV) infection [13]. In P. falciparum malaria, sterilizing immunity induced by experimental vaccination with whole sporozoites is associated with the induction of CD4+ T cells that coproduce IFN-γ, IL-2, and/or TNF-α [14–16]. CD4+ T-cell responses of a similar phenotype have been implicated in protection from malaria conferred by the vaccine RTS,S [17–19]. Among naturally exposed children, several effector phenotypes of P. falciparum–specific CD4+ T cells have been described, including distinct subsets producing inflammatory (TNF-α and IFN-γ) and regulatory (interleukin 10 [IL-10]) cytokines [20–22]. Recent studies suggest that the effector phenotype may be dependent on parasite exposure intensity, as recent symptomatic malaria is associated with increased frequencies of IFN-γ/IL-10–coproducing cells and a reduced frequency of TNF-α–producing cells [20, 21]. Murine models of malaria and other parasitic infections indicate that IFN-γ/IL-10–coproducing cells play an essential role in protecting the host from immunopathology, although this may come at the cost of reduced or delayed parasite clearance [23–25]. The identification of CD4+ T-cell responses in children susceptible to symptomatic malaria that differ from those in immune adults may provide important information regarding the mechanisms mediating protection. In this study, we performed a detailed assessment of P. falciparum–specific CD4+ T-cell responses among children and their adult caregivers in Uganda, in areas of high or low parasite transmission. We hypothesized that the frequency and functional characteristics of P. falciparum–specific CD4+ T-cell responses among clinically immune adults may differ from those of children who remain vulnerable to symptomatic malaria.

This chapter focuses on the study of viral immune evasion. Most viruses spread from one host to the next via secreted bodily fluids; to gain access to their target cells for replication they need to cross epithelial barriers, which can be considered the first line of host defense. Host cell apoptosis is triggered by cell stress signals responding to the virus' attempt to co-opt cellular machinery. Pattern recognition receptors (PRRs) recognize viral components and elicit cytokines and chemokines, which in turn recruit natural killer (NK) cells. Natural killer (NK) cells are an essential component of the first line of defense against viruses. NK cells have two antiviral mechanisms, cytotoxicity and cytokine secretion, and they are the main source of INF-g early in infection. Viral genes that interfere with the MHCI pathway of antigen presentation to CD8 T cells were the first viral immune evasion genes to be described. Specific deletion of viral immune evasion genes has revealed the extraordinary potency of innate immune responses compared to the modest impact of the sophisticated adaptive immune system. However, all viral immune evasion genes, whether they are absolutely required for host species infection or confer a barely perceptible benefit, have been selected by the same balancing evolutionary pressures: the ability of a virus to propagate its genome within a host, the ability to spread to a new host, and the need to maintain a supply of new hosts.

Cytomegalovirus’s (CMV’s) unique ability to drive the expansion of virus-specific T-cell populations over the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor antigens, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine cytomegaloviruses (MCMV) encoding a SIINFEKL-eGFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for non-recombinant (i.e. MCMV-derived) antigens. Moreover, when the virus encoded SIINFEKL, T cells specific for non-recombinant antigens displayed a phenotype indicative of less frequent exposure to antigen. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV antigens. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled co-inflation of T cells specific for both SIINFEKL and non-recombinant antigens. Because coinfection allows presentation of SIINFEKL and MCMV-derived antigens by different cells within the same animal, these data reveal that competition for, or availability of, antigen at the level of the antigen presenting cell determines the composition of the inflationary response to MCMV. SIINFEKL’s strong affinity for H2-Kb, and its early and abundant expression, may provide this epitope’s competitive advantage.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations., Author Summary In malaria endemic regions, immunity is slow to develop and does not provide substantial protection against reinfection. Rather, following repeated exposure, older children and adults eventually develop protection from most symptomatic manifestations of the infection. This may be due in part to the induction of immunoregulatory mechanisms by the P. falciparum parasite, such as FoxP3+ regulatory T cells (Tregs). Prior human studies have shown that Tregs are induced by malaria parasites both in vivo and in vitro, but the role of these cells in immunity in children who are chronically exposed to malaria remains unclear. In this study, we assessed the frequency and features of Tregs among children from areas of high malaria transmission in Uganda. We found that this regulatory T cell population declined markedly with increasing malaria episodes. This loss was associated with decreased expression of TNFR2, which is a protein implicated in stability of Tregs. Additionally, T cells from highly malaria exposed children demonstrated a reduced propensity to differentiate into Tregs following parasite stimulation. Together our data suggest that repeated episodes of malaria alter Treg homeostasis, which may influence the development of immunity to malaria in children.