Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis.

Malaria is a vector-borne disease caused by Plasmodium parasites of which Plasmodium falciparum contributed to an estimated 247 million cases worldwide in 2021 (WHO malaria report 2022). The P. falciparum Circumsporozoite protein (PfCSP) covers the surface of the sporozoite which is critical to cell invasion in the human host. PfCSP is the leading pre-erythrocytic vaccine candidate and forms the basis of the RTS'S (Mosquirix®) malaria vaccine. However, high-yield production of full-length PfCSP with proper folding has been challenging. Here, we describe expression and purification of full-length PfCSP (containing 4 NVDP and 38 NANP repeats) with proper conformation by a simple three-step procedure in the Lactococcus lactis expression system.

A randomized first-in-human phase I trial of differentially adjuvanted Pfs48/45 malaria vaccines in Burkinabé adults.

BACKGROUNDMalaria transmission-blocking vaccines aim to interrupt the transmission of malaria from one person to another.METHODSThe candidates R0.6C and ProC6C share the 6C domain of the Plasmodium falciparum sexual-stage antigen Pfs48/45. R0.6C utilizes the glutamate-rich protein (GLURP) as a carrier, and ProC6C includes a second domain (Pfs230-Pro) and a short 36-amino acid circumsporozoite protein (CSP) sequence. Healthy adults (n = 125) from a malaria-endemic area of Burkina Faso were immunized with 3 intramuscular injections, 4 weeks apart, of 30 µg or 100 µg R0.6C or ProC6C each adsorbed to Alhydrogel (AlOH) adjuvant alone or in combination with Matrix-M (15 µg or 50 µg, respectively). The allocation was random and double-blind for this phase I trial.RESULTSThe vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of 7 adverse events, mild to moderate in intensity and considered possibly related to the study vaccines, were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 µg ProC6C-AlOH with Matrix-M, and 13 of 20 (65%) individuals in the group showed greater than 80% transmission-reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA.CONCLUSIONAll formulations were safe and well tolerated in a malaria-endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation.TRIAL REGISTRATIONPan-African Clinical Trials Registry (https://pactr.samrc.ac.za) PACTR202201848463189.FUNDINGThe study was funded by the European and Developing Countries Clinical Trials Partnership (grant RIA2018SV-2311).

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

Background: The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. Methods: The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. Results: We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Conclusion: Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria.

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.

Immunodominant T cell peptides from four candidate malarial antigens as biomarkers of protective immunity against malaria.

A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.

Towards large-scale identification of HLA-restricted T cell epitopes from four vaccine candidate antigens in a malaria endemic community in Ghana.

Sterile protection against clinical malaria has been achieved in animal models and experimental human challenge studies involving immunization with radiation attenuated Plasmodium falciparum sporozoite vaccines as well as by live sporozoites under chloroquine prophylaxis. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection. Although the exact parasite targets of protective CD8 + T cell responses are not fully defined, responses against a handful of vaccine candidate antigens have been associated with protection. Identifying the T cell targets in these antigens will facilitate the development of simpler, cost-effective, and efficacious next generation multi-epitope vaccines. The aim of this study was to identify immunodominant portions of four malaria vaccine candidate antigens using peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites. Cryopreserved PBMCs from 291 HLA-typed subjects were stimulated with pools of overlapping 15mer peptides spanning the entire sequences of P. falciparum circumsporozoite protein (CSP, 9 pools), apical membrane antigen 1 (AMA1, 12 pools), thrombospondin related anonymous protein (TRAP, 6 pools) and cell traversal for ookinetes and sporozoites (CelTOS, 4 pools) in FluoroSpot assays. 125 of 291 subjects made IFN-γ responses to 30 of the 31 peptide pools tested and 22 of 291 made granzyme B responses, with 20 making dual responses. The most frequent responses were to the CSP C-terminal region and the least frequent responses were to TRAP and CelTOS. There was no association between FluoroSpot responses and active malaria infection, detected by either microscopy, RDT, or PCR. In conclusion, CSP and AMA1 have relatively higher numbers of epitopes that trigger IFN-γ and granzyme B-secreting T cells in adults with life-long malaria parasite exposure compared to the other two antigens tested, and highlights the continued relevance of these two antigens as vaccine candidates.

Corrigendum: Asymptomatic Malaria Infection Is Maintained by a Balanced Pro- and Anti-inflammatory Response.

[This corrects the article DOI: 10.3389/fmicb.2020.559255.].

Asymptomatic Malaria Infection Is Maintained by a Balanced Pro- and Anti-inflammatory Response.

BACKGROUND: Pro- and anti-inflammatory cytokines are important mediators of immunity and are associated with malaria disease outcomes. However, their role in the establishment of asymptomatic infections, which may precede the development of clinical symptoms, is not as well-understood. METHODS: We determined the association of pro and anti-inflammatory cytokines and other immune effector molecules with the development of asymptomatic malaria. We measured and compared the plasma levels of pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-6, IL-12p70, IL-17A, and granzyme B, the anti-inflammatory cytokine IL-4 and the regulatory cytokine IL-10 from children with asymptomatic malaria infections (either microscopic or submicroscopic) and uninfected controls using Luminex. RESULTS: We show that individuals with microscopic asymptomatic malaria had significantly increased levels of TNF-α and IL-6 compared to uninfected controls. Children with either microscopic or submicroscopic asymptomatic malaria exhibited higher levels of IFN-γ, IL-17A, and IL-4 compared to uninfected controls. The levels of most of the pro and anti-inflammatory cytokines were comparable between children with microscopic and submicroscopic infections. The ratio of IFN-γ/IL-10, TNF-α/IL-10, IL-6/IL-10 as well as IFN-γ/IL-4 and IL-6/IL-4 did not differ significantly between the groups. Additionally, using a principal component analysis, the cytokines measured could not distinguish amongst the three study populations. This may imply that neither microscopic nor submicroscopic asymptomatic infections were polarized toward a pro-inflammatory or anti-inflammatory response. CONCLUSION: The data show that asymptomatic malaria infections result in increased plasma levels of both pro and anti-inflammatory cytokines relative to uninfected persons. The balance between pro- and anti-inflammatory cytokines are, however, largely maintained and this may in part, explain the lack of clinical symptoms. This is consistent with the generally accepted observation that clinical symptoms develop as a result of immunopathology involving dysregulation of inflammatory mediator balance in favor of pro-inflammatory mediators.

Prevalence of sickle cell disorders and malaria infection in children aged 1-12 years in the Volta Region, Ghana: a community-based study.

BACKGROUND: Alterations in the structure of haemoglobin (Hb) are usually brought about by point mutations affecting one or, in some cases, two codons encoding amino acids of the globin chains. One in three Ghanaians are said to have sickle cell disorders, whereas malaria continues to be one of the leading causes of mortality among children. This study determined the prevalence of sickle cell disorders and malaria infection among children aged 1-12 years in the Volta Region. METHODS: This was a community-based cross-sectional survey that involved 938 children aged 1-12 years selected from three districts, one each from the 3 geographical zones of the Volta Region using a multistage sampling method. Demographic information was collected using a standard questionnaire and anthropometric indices were measured. Isoelectric focusing (IEF) electrophoresis was used to determine the Hb genotypes and sub-microscopic parasites were determined by PCR. RESULTS: The prevalence of sickling screening positive was 16.0% with an overall prevalence of sickle cell disorders being 2.0%. Among the individual genotypes making up the sickle cell disorders, genotype HbSF was the highest (0.9% as compared to 0.2%; HbSS, 0.6%; HbSC and 0.3%; HbSCF). Microscopic Plasmodium falciparum parasitaemia was detected among 5.5% of the children and 14.2% sub-microscopic prevalence by PCR. Children with sickle cell disorders were more likely to have sub-microscopic parasitaemia (AOR = 5.51 95%CI (2.15, 14.10), p < 0.001) as well as anaemia (AOR = 3.03 95% CI (1.04, 8.82), p = 0.042), compared to those with normal genotypes. There was no significant difference observed between sickle cell disorders and growth and development of the children screened. CONCLUSIONS: Sickle cell disorders were significantly associated with sub-microscopic parasitaemia as well as anaemia in this study. Establishment of sickle cell clinics in the district and regional hospitals will help in the management of children with the disorder and also generate a national database on sickle cell disorders. National neonatal screening policies must also be put in place to help in early detection and management of these disorders.