Antibody-dependent immune responses elicited by blood stage-malaria infection contribute to protective immunity to the pre-erythrocytic stages.

Advances in transcriptomics and proteomics have revealed that different life-cycle stages of the malaria parasite, Plasmodium, share antigens, thus allowing for the possibility of eliciting immunity to a parasite life-cycle stage that has not been experienced before. Using the Plasmodium chabaudi (AS strain) model of malaria in mice, we investigated how isolated exposure to blood-stage infection, bypassing a liver-stage infection, yields significant protection to sporozoite challenge resulting in lower liver parasite burdens. Antibodies are the main immune driver of this protection. Antibodies induced by blood-stage infection recognise proteins on the surface of sporozoites and can impair sporozoite gliding motility in vitro, suggesting a possible function in vivo. Furthermore, mice lacking B cells and/or secreted antibodies are not protected against a sporozoite challenge in mice that had a previous blood-stage infection. Conversely, effector CD4+ and CD8+ T cells do not seem to play a role in protection from sporozoite challenge of mice previously exposed only to the blood stages of P. chabaudi. The protective response against pre-erythrocytic stages can be induced by infections initiated by serially passaged blood-stage parasites as well as recently mosquito transmitted parasites and is effective against a different strain of P. chabaudi (CB strain), but not against another rodent malaria species, P. yoelii. The possibility to induce protective cross-stage antibodies advocates the need to consider both stage-specific and cross-stage immune responses to malaria, as natural infection elicits exposure to all life-cycle stages. Future investigation into these cross-stage antibodies allows the opportunity for candidate antigens to contribute to malaria vaccine development.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations.

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N'Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126-54.00 µg/ml or LC95 × 3 = 0.7437-17.82 µg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Tissue macrophages and interferon-gamma signalling control blood-stage Plasmodium chabaudi infections derived from mosquito-transmitted parasites.

Natural infection with Plasmodium parasites, the causative agents of malaria, occurs via mosquito vectors. However, most of our knowledge of the immune response to the blood stages of Plasmodium is from infections initiated by injection of serially blood-passaged infected red blood cells, resulting in an incomplete life cycle in the mammalian host. Vector transmission of the rodent malaria parasite, Plasmodium chabaudi chabaudi AS has been shown to give rise to a more attenuated blood-stage infection in C57Bl/6J mice, when compared to infections initiated with serially blood-passaged P. chabaudi-infected red blood cells. In mouse models, the host immune response induced by parasites derived from natural mosquito transmission is likely to more closely resemble the immune responses to Plasmodium infections in humans. It is therefore important to determine how the host response differs between the two types of infections. As the spleen is considered to be a major contributor to the protective host response to P. chabaudi, we carried out a comparative transcriptomic analysis of the splenic response to recently mosquito-transmitted and serially blood-passaged parasites in C57Bl/6J mice. The attenuated infection arising from recently mosquito-transmitted parasites is characterised by an earlier and stronger myeloid- and IFNγ-related response. Analyses of spleen lysates from the two infections similarly showed stronger or earlier inflammatory cytokine and chemokine production in the recently mosquito-transmitted blood-stage infections. Furthermore, tissue macrophages, including red pulp macrophages, and IFNγ-signalling in myeloid cells, are required for the early control of P. chabaudi recently mosquito-transmitted parasites, thus contributing to the attenuation of mosquito-transmitted infections. The molecules responsible for this early activation response to recently-transmitted blood-stage parasites in mice would be important to identify, as they may help to elucidate the nature of the initial interactions between blood-stage parasites and the host immune system in naturally transmitted malaria.

Restoration of Endogenous Retrovirus Infectivity Impacts Mouse Cancer Models.

Mouse models have been instrumental in establishing fundamental principles of cancer initiation and progression and continue to be invaluable in the discovery and further development of cancer therapies. Nevertheless, important aspects of human disease are imperfectly approximated in mouse models, notably the involvement of endogenous retroviruses (ERVs). Replication-defective ERVs, present in both humans and mice, may affect tumor development and antitumor immunity through mechanisms not involving infection. Here, we revealed an adverse effect of murine ERVs with restored infectivity on the behavior of mouse cancer models. In contrast to human cancer, where infectious ERVs have never been detected, we found that ERV infectivity was frequently restored in transplantable, as well as genetic, mouse cancer models. Such replication-competent, ERV-derived retroviruses were responsible for unusually high expression of retroviral nucleic acids and proteins in mouse cancers. Infectious ERV-derived retroviruses produced by mouse cancer cells could directly infect tumor-infiltrating host immune cells and fundamentally modified the host's immune defenses to cancer, as well as the outcome of immunotherapy. Therefore, infectious retroviruses, variably arising in mouse cancer models, but not in human cancer, have the potential to confound many immunologic studies and should be considered as a variable, if not altogether avoided. Cancer Immunol Res; 6(11); 1292-300. ©2018 AACR.

Follicular Helper T Cells are Essential for the Elimination of Plasmodium Infection.

CD4+ follicular helper T (Tfh) cells have been shown to be critical for the activation of germinal center (GC) B-cell responses. Similar to other infections, Plasmodium infection activates both GC as well as non-GC B cell responses. Here, we sought to explore whether Tfh cells and GC B cells are required to eliminate a Plasmodium infection. A CD4 T cell-targeted deletion of the gene that encodes Bcl6, the master transcription factor for the Tfh program, resulted in complete disruption of the Tfh response to Plasmodium chabaudi in C57BL/6 mice and consequent disruption of GC responses and IgG responses and the inability to eliminate the otherwise self-resolving chronic P. chabaudi infection. On the other hand, and contrary to previous observations in immunization and viral infection models, Signaling Lymphocyte Activation Molecule (SLAM)-Associated Protein (SAP)-deficient mice were able to activate Tfh cells, GC B cells, and IgG responses to the parasite. This study demonstrates the critical role for Tfh cells in controlling this systemic infection, and highlights differences in the signals required to activate GC B cell responses to this complex parasite compared with those of protein immunizations and viral infections. Therefore, these data are highly pertinent for designing malaria vaccines able to activate broadly protective B-cell responses.

Dichotomy between T Cell and B Cell Tolerance to Neonatal Retroviral Infection Permits T Cell Therapy.

Elucidation of the immune requirements for control or elimination of retroviral infection remains an important aim. We studied the induction of adaptive immunity to neonatal infection with a murine retrovirus, under conditions leading to immunological tolerance. We found that the absence of either maternal or offspring adaptive immunity permitted efficient vertical transmission of the retrovirus. Maternal immunodeficiency allowed the retrovirus to induce central Th cell tolerance in the infected offspring. In turn, this compromised the offspring's ability to mount a protective Th cell-dependent B cell response. However, in contrast to T cells, offspring B cells were not centrally tolerized and retained their ability to respond to the infection when provided with T cell help. Thus, escape of retrovirus-specific B cells from deletional tolerance offers the opportunity to induce protective retroviral immunity by restoration of retrovirus-specific T cell help, suggesting similar T cell immunotherapies for persistent viral infections.

Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.

Vector transmission regulates immune control of Plasmodium virulence.

Defining mechanisms by which Plasmodium virulence is regulated is central to understanding the pathogenesis of human malaria. Serial blood passage of Plasmodium through rodents, primates or humans increases parasite virulence, suggesting that vector transmission regulates Plasmodium virulence within the mammalian host. In agreement, disease severity can be modified by vector transmission, which is assumed to 'reset' Plasmodium to its original character. However, direct evidence that vector transmission regulates Plasmodium virulence is lacking. Here we use mosquito transmission of serially blood passaged (SBP) Plasmodium chabaudi chabaudi to interrogate regulation of parasite virulence. Analysis of SBP P. c. chabaudi before and after mosquito transmission demonstrates that vector transmission intrinsically modifies the asexual blood-stage parasite, which in turn modifies the elicited mammalian immune response, which in turn attenuates parasite growth and associated pathology. Attenuated parasite virulence associates with modified expression of the pir multi-gene family. Vector transmission of Plasmodium therefore regulates gene expression of probable variant antigens in the erythrocytic cycle, modifies the elicited mammalian immune response, and thus regulates parasite virulence. These results place the mosquito at the centre of our efforts to dissect mechanisms of protective immunity to malaria for the development of an effective vaccine.

Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi.

BACKGROUND: Serial blood passage of Plasmodium increases virulence, whilst mosquito transmission inherently regulates parasite virulence within the mammalian host. It is, therefore, imperative that all aspects of experimental malaria research are studied in the context of the complete Plasmodium life cycle. METHODS: Plasmodium chabaudi chabaudi displays many characteristics associated with human Plasmodium infection of natural mosquito vectors and the mammalian host, and thus provides a unique opportunity to study the pathogenesis of malaria in a single infection setting. An optimized protocol that permits efficient and reproducible vector transmission of P. c. chabaudi via Anopheles stephensi was developed. RESULTS AND CONCLUSIONS: This protocol was utilized for mosquito transmission of genetically distinct P. c. chabaudi isolates, highlighting differential parasite virulence within the mosquito vector and the spectrum of host susceptibility to infection initiated via the natural route, mosquito bite. An apposite experimental system in which to delineate the pathogenesis of malaria is described in detail.

Mass spectrometry detection of G3m and IGHG3 alleles and follow-up of differential mother and neonate IgG3.

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.