A Human Bi-specific Antibody against Zika Virus with High Therapeutic Potential.

Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.

Microglial reprogramming by Hv1 antagonism protects neurons from inflammatory and glutamate toxicity.

Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate. Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury. Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

Popularity and Entropy in Friendship and Enmity Networks in Classrooms.

Looking for regular statistical trends of relations in schools, we constructed 42 independent weighted directed networks of simultaneous friendship and animosity from surveys we made in the Mexico City Metropolitan area in classrooms with students of different ages and levels by asking them to nominate and order five friends and five foes. However, the data show that older students nominated fewer than the five required five foes. Although each classroom was independent of the others, we found several general trends involving students of different ages and grade levels. In all classrooms, friendship entropy was found to be higher than enmity entropy, indicating that fewer students received enmity links than received friendship nominations. Popular agents exhibited more reciprocal nominations among themselves than less popular agents, and opposite-sex friendships increased with age.

Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation.

Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

Cyclic Dinucleotide-Adjuvanted Dengue Virus Nonstructural Protein 1 Induces Protective Antibody and T Cell Responses.

Endothelial dysfunction and vascular leak, pathogenic hallmarks of severe dengue disease, are directly triggered by dengue virus (DENV) nonstructural protein 1 (NS1). Previous studies have shown that immunization with NS1, as well as passive transfer of NS1-immune serum or anti-NS1 mAb, prevent NS1-mediated lethality in vivo. In this study, we evaluated the immunogenicity and protective capacity of recombinant DENV NS1 administered with cyclic dinucleotides (CDNs), potent activators of innate immune pathways and highly immunogenic adjuvants. Using both wild-type C57BL/6 mice and IFN-α/ß receptor-deficient mice, we show that NS1-CDN immunizations elicit serotype-specific and cross-reactive Ab and T cell responses. Furthermore, NS1-CDN vaccinations conferred significant homotypic and heterotypic protection from DENV2-induced morbidity and mortality. In addition, we demonstrate that high anti-NS1 Ab titers are associated with protection, supporting the role of humoral responses against DENV NS1 as correlates of protection. These findings highlight the potential of CDN-based adjuvants for inducing Ab and T cell responses and validate NS1 as an important candidate for dengue vaccine development.

Impact of pulmonary hypertension and congenital heart disease with hemodynamic repercussion on the severity of acute respiratory infections in children under 5 years of age at a pediatric referral center in Colombia, South America.

BACKGROUND: Acute respiratory infection is one of the main causes of morbidity in children. Some studies have suggested that pulmonary hypertension and congenital heart disease with haemodynamic repercussion increase the severity of respiratory infections, but there are few publications in developing countries. METHODS: This was a prospective cohort study evaluating the impact of pulmonary hypertension and congenital heart disease (CHD) with haemodynamic repercussion as predictors of severity in children under 5 years of age hospitalised for acute respiratory infection. RESULTS: Altogether, 217 children hospitalised for a respiratory infection who underwent an echocardiogram were evaluated; 62 children were diagnosed with CHD with haemodynamic repercussion or pulmonary hypertension. Independent predictors of admission to intensive care included: pulmonary hypertension (RR 2.14; 95% CI 1.06-4.35, p = 0.034), respiratory syncytial virus (RR 2.52; 95% CI 1.29-4.92, p = 0.006), and bacterial pneumonia (RR 3.09; 95% CI 1.65-5.81, p = 0.000). A significant difference was found in average length of hospital stay in children with the cardiovascular conditions studied (p = 0.000). CONCLUSIONS: Pulmonary hypertension and CHD with haemodynamic repercussion as well as respiratory syncytial virus and bacterial pneumonia were predictors of severity in children with respiratory infections in this study. Early recognition of cardiovascular risks in paediatric populations is necessary to lessen the impact on respiratory infections.

Increased serum sialic acid is associated with morbidity and mortality in a murine model of dengue disease.

Dengue virus (DENV) causes the most prevalent arboviral infection of humans, resulting in a spectrum of outcomes, ranging from asymptomatic infection to dengue fever to severe dengue characterized by vascular leakage and shock. Previously, we determined that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability, disrupts the endothelial glycocalyx layer (EGL) in vitro and triggers shedding of structural components, including sialic acid (Sia) and heparan sulfate. Here, using a murine model of dengue disease disease, we found high levels of Sia and NS1 circulating in mice with DENV-induced morbidity and lethal DENV infection. Further, we developed a liquid chromatography/mass spectrometry-based method for quantifying free Sia in serum and determined that the levels of free N-glycolylneuraminic acid were significantly higher in DENV-infected mice than in uninfected controls. These data provide additional evidence that DENV infection disrupts EGL components in vivo and warrant further research assessing Sia as a biomarker of severe dengue disease.

Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components.

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components.

High α-tocopherol dosing increases lipid metabolism by changing redox state in damaged rat gastric mucosa and liver after ethanol treatment.

Regeneration of ethanol-injured rat gastric mucosa must undergo changes in major metabolic pathways to achieve DNA replication and cell proliferation. These events are highly dependent on glucose utilization and inhibited by vitamin E (VE) (α-tocopherol) administration. Therefore, the present study aimed at assessing lipid metabolism in the gastric mucosa and ethanol-induced gastric damage and the effect of α-tocopherol administration. For this, rates of fatty acid ß-oxidation and lipogenesis were tested in gastric mucosa samples. Through histological analysis, we found loss of the mucosa's superficial epithelium, which became gradually normalized during the recovery period. Proliferation of gastric mucosa occurred with augmented formation of ß-oxidation by-products, diminished synthesis of triacylglycerols (TGs), as well as of phospholipids, and a reduced cytoplasmic NAD/NADH ratio, whereas the mitochondrial redox NAD/NADH ratio was much less affected. In addition, α-tocopherol increased palmitic acid utilization in the gastric mucosa, which was accompanied by the induction of 'mirror image' effects on the cell redox state, reflected in an inhibited cell gastric mucosa proliferation by the vitamin administration. In conclusion, the present study shows, for the first time, the role of lipid metabolism in the adaptive cell gastric mucosa changes that drive proliferation after a chronic insult. Moreover, α-tocopherol increased gastric mucosa utilization of palmitic acid associated with energy production. These events could be associated with its antioxidant properties in co-ordination with regulation of genes and cell pathways, including changes in the cell NAD/NADH redox state.

ROS as Regulators of Mitochondrial Dynamics in Neurons.

Mitochondrial dynamics is a complex process, which involves the fission and fusion of mitochondrial outer and inner membranes. These processes organize the mitochondrial size and morphology, as well as their localization throughout the cells. In the last two decades, it has become a spotlight due to their importance in the pathophysiological processes, particularly in neurological diseases. It is known that Drp1, mitofusin 1 and 2, and Opa1 constitute the core of proteins that coordinate this intricate and dynamic process. Likewise, changes in the levels of reactive oxygen species (ROS) lead to modifications in the expression and/or activity of the proteins implicated in the mitochondrial dynamics, suggesting an involvement of these molecules in the process. In this review, we discuss the role of ROS in the regulation of fusion/fission in the nervous system, as well as the involvement of mitochondrial dynamics proteins in neurodegenerative diseases.

The Plasmodium falciparum Cell-Traversal Protein for Ookinetes and Sporozoites as a Candidate for Preerythrocytic and Transmission-Blocking Vaccines.

Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.

Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.

Malaria infection begins when a female Anopheles mosquito injects Plasmodium sporozoites into the skin of its host during blood feeding. Skin-deposited sporozoites may enter the bloodstream and infect the liver, reside and develop in the skin, or migrate to the draining lymph nodes (DLNs). Importantly, the DLN is where protective CD8(+) T cell responses against malaria liver stages are induced after a dermal route of infection. However, the significance of parasites in the skin and DLN to CD8(+) T cell activation is largely unknown. In this study, we used genetically modified parasites, as well as antibody-mediated immobilization of sporozoites, to determine that active sporozoite migration to the DLNs is required for robust CD8(+) T cell responses. Through dynamic in vivo and static imaging, we show the direct uptake of parasites by lymph-node resident DCs followed by CD8(+) T cell-DC cluster formation, a surrogate for antigen presentation, in the DLNs. A few hours after sporozoite arrival to the DLNs, CD8(+) T cells are primed by resident CD8α(+) DCs with no apparent role for skin-derived DCs. Together, these results establish a critical role for lymph node resident CD8α(+) DCs in CD8(+) T cell priming to sporozoite antigens while emphasizing a requirement for motile sporozoites in the induction of CD8(+) T cell-mediated immunity.

Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice.

Malaria caused by Plasmodium falciparum kills nearly one million children each year and imposes crippling economic burdens on families and nations worldwide. No licensed vaccine exists, but infection can be prevented by antibodies against the circumsporozoite protein (CSP), the major surface protein of sporozoites, the form of the parasite injected by mosquitoes. We have used vectored immunoprophylaxis (VIP), an adeno-associated virus-based technology, to introduce preformed antibody genes encoding anti-P. falciparum CSP mAb into mice. VIP vector-transduced mice exhibited long-lived mAb expression at up to 1,200 µg/mL in serum, and up to 70% were protected from both i.v. and mosquito bite challenge with transgenic Plasmodium berghei rodent sporozoites that incorporate the P. falciparum target of the mAb in their CSP. Serum antibody levels and protection from mosquito bite challenge were dependent on the dose of the VIP vector. All individual mice expressing CSP-specific mAb 2A10 at 1 mg/mL or more were completely protected, suggesting that in this model system, exceeding that threshold results in consistent sterile protection. Our results demonstrate the potential of VIP as a path toward the elusive goal of immunization against malaria.

Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.

Studies in animals and human volunteers demonstrate that antibodies against the repeat-region of the Plasmodium circumsporozoite protein (CSP) abrogate sporozoite infection. However, the realization that the N- and C- terminal regions flanking the repeats play essential roles in parasite infectivity raised the possibility that they could be targeted by protective antibodies. We characterized a monoclonal antibody (mAb5D5) specific for the N-terminus of the P. falciparum CSP, which inhibits the proteolytic cleavage of the CSP, a key requirement for parasite infection of hepatocytes. Adoptive transfer of mAb5D5 strongly inhibits the in vivo infection of sporozoites expressing the N-terminus of P. falciparum CSP, and this protection is greatly enhanced when combined with antirepeat antibodies. Our results show that antibodies interfering with molecular processes required for parasite infectivity can exert a strong in vivo protective activity and indicate that pre-erythrocytic vaccines against Plasmodium should include the CSP N-terminal region.

A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.

The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8⁺ T cells specific for infectious pathogens.

It is well established that immunization with attenuated malaria sporozoites induces CD8(+) T cells that eliminate parasite-infected hepatocytes. Liver memory CD8(+) T cells induced by immunization with parasites undergo a unique differentiation program and have enhanced expression of CXCR6. Following immunization with malaria parasites, CXCR6-deficient memory CD8(+) T cells recovered from the liver display altered cell-surface expression markers as compared to their wild-type counterparts, but they exhibit normal cytokine secretion and expression of cytotoxic mediators on a per-cell basis. Most importantly, CXCR6-deficient CD8(+) T cells migrate to the liver normally after immunization with Plasmodium sporozoites or vaccinia virus, but a few weeks later their numbers severely decrease in this organ, losing their capacity to inhibit malaria parasite development in the liver. These studies are the first to show that CXCR6 is critical for the development and maintenance of protective memory CD8(+) T cells in the liver.

Adjuvant-like effect of vaccinia virus 14K protein: a case study with malaria vaccine based on the circumsporozoite protein.

Development of subunit vaccines for malaria that elicit a strong, long-term memory response is an intensive area of research, with the focus on improving the immunogenicity of a circumsporozoite (CS) protein-based vaccine. In this study, we found that a chimeric protein, formed by fusing vaccinia virus protein 14K (A27) to the CS of Plasmodium yoelii, induces strong effector memory CD8(+) T cell responses in addition to high-affinity Abs when used as a priming agent in the absence of any adjuvant, followed by an attenuated vaccinia virus boost expressing CS in murine models. Moreover, priming with the chimeric protein improved the magnitude and polyfunctionality of cytokine-secreting CD8(+) T cells. This fusion protein formed oligomers/aggregates that led to activation of STAT-1 and IFN regulatory factor-3 in human macrophages, indicating a type I IFN response, resulting in NO, IL-12, and IL-6 induction. Furthermore, this vaccination regimen inhibited the liver stage development of the parasite, resulting in sterile protection. In summary, we propose a novel approach in designing CS based pre-erythrocytic vaccines against Plasmodium using the adjuvant-like effect of the immunogenic vaccinia virus protein 14K.

Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

Full-length Plasmodium falciparum circumsporozoite protein administered with long-chain poly(I·C) or the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion elicits potent antibody and CD4+ T cell immunity and protection in mice.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I · C) [poly(I · C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4(+) T cell responses. Moreover, E. coli-derived CS proteins in combination with poly(I · C)LC induced potent multifunctional (interleukin 2-positive [IL-2(+)], tumor necrosis factor alpha-positive [TNF-α(+)], gamma interferon-positive [IFN-γ(+)]) CD4(+) effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ~50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4(+) T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4(+) T cell immunity that was significantly less potent than that with poly(I · C)LC. Overall, these data suggest that full-length CS proteins and poly(I · C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.