Biology subjects

Zavala, F.

Publications and source records attributed to Zavala, F..

12 recordsLinked to original sources

bioRxiv2026

The live attenuated DGAT1-knockout whole-cell Toxoplasma vaccine confers protective immunity against acute and chronic toxoplasmosis

The intravacuolar parasite Toxoplasma gondii scavenges fatty acids from host mammalian cells and stores excess in lipid droplets. To investigate the physiological relevance of neutral lipid storage in Toxoplasma, we generated a mutant lacking DGAT1, an ER-localized enzyme that synthesizes triacylglycerols, from the virulent type I RH strain of T. gondii. Compared to WT, RH {Delta}DGAT1 parasites grow poorly in mammalian cells, form few LD, suffer from lipotoxicity, and do not cause disease or lethality in immunocompetent or immunodeficient mice. Importantly, mice immunized with RH {Delta}DGAT1 parasites mount strong, long-term immune responses involving both cellular and humoral components, with higher levels of T. gondii-specific IgG antibodies, effector memory T cells, and both pro-inflammatory and anti-inflammatory cytokines, indicating a mixed Th1/Th2 response with Th1 predominance. This immunity provides complete, long-lasting protection (up to 6 months) against rechallenge from homologous type I (acute infection) and heterologous cyst-forming type II (chronic infection) T. gondii strains. Additional analyses reveal that IFN-{gamma}, CD8+ T cells, as well as B cells are crucial for defending against type I T. gondii in immunized mice. Overall, our live-attenuated RH {Delta}DGAT1 strain is a promising vaccine candidate and a model for studying immune responses that control T. gondii infections.

microbiology↗

bioRxiv2025

Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

The monoclonal antibody L9 mediates high-level protection against malaria in children for up to 6 months in Africa. L9 preferentially binds with high affinity to the NVDP minor repeat on the P. falciparum circumsporozoite protein (PfCSP). Here, we sought to improve the affinity of L9 to enhance protection against rare strains with two spatially separated minor repeats or a single minor repeat. Site saturation mutagenesis and yeast display-screening identified a panel of affinity-improved variants. In vivo challenge showed one variant, L9_yd19, to be modestly more potent against a chimeric transgenic Plasmodium encoding PfCSP with two widely spaced minor repeats from a Kenyan parasite strain, with no loss in potency against the benchmark 3D7 strain with its standard complement of minor repeats. L9_yd19 also had high affinity against NANP major repeats and was protective against transgenic Plasmodium with PfCSP containing only NANP major repeats (NANP12). Cryo-EM studies revealed L9_yd19 to recognize PfCSP with two distinct homotypic interfaces, which combined to yield two trimeric layers of antibodies comprising asymmetric trimers that dimerized in a head-to-head fashion. These data reveal a new antibody mechanism that utilizes interfaces involving dual homotypic symmetry elements, a 2-fold and an asymmetric 3-fold, for potentially improved malaria prevention. HIGHLIGHTSO_LIL9 is a highly protective antimalarial antibody that preferentially binds the NVDP minor repeat on Plasmodium falciparum circumsporozoite protein (PfCSP) and also binds with low affinity to the NANP major repeat; due to these targeting preferences, it has shown reduced protection against designed transgenic malaria strains with only a single NVDP motif (Fig. 1). C_LIO_LIUsing yeast display, a panel of L9 variants were generated based on higher affinity against the minor NVDP and major NANP motifs to determine if they could improve protection against strains with fewer minor repeat regions or only containing major repeats (Figs 1-4). C_LIO_LIOne L9 variant, L9_yd19 showed enhanced protection against chimeric transgenic CSP variants with a single minor repeat or two minor repeats in which the spacing was separated; L9_yd19 also showed protection against chimeric transgenic CSP variants containing only the NANP major repeat (Figs. 4-5). C_LIO_LICryo-EM analyses revealed L9_yd19 recognition of CSP to comprise two distinct homotypic interfaces: a side-to-side interface within asymmetric antibody trimer and a head-to-head interface between antibody trimers related by 2-fold symmetry that combined to yield a higher-order complex comprising two trimeric layers of antibodies (Figs. 6-7) C_LIO_LIStructure-function studies reveal a new antibody-based structural mechanism with dual homotypic interfaces mediating protection against varying numbers and spacing of minor repeats and major repeats. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/679367v1_fig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1978620org.highwire.dtl.DTLVardef@e6b84forg.highwire.dtl.DTLVardef@14e750eorg.highwire.dtl.DTLVardef@1e6ff51_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Improving affinity to target the CSP minor repeat enhances L9 antibody protective breadth. (A) The CSP minor repeat (NVDP) minor repeat is the principal binding target of the potent anti-malarial antibody L9. Genetic polymorphisms are present among P. falciparum strains in the number and spacing of NVDP motifs contained in the CSP repeat regions, and the loss of NPNV epitopes reduces the in vivo protective potency of the L9 antibody. (B) To enhance L9 affinity against the CSP antigen, multi-mutation libraries were generated from the L9 template and selectively screened against CSP peptide repeat motifs using yeast display. Libraries were first selected against Peptide22 (Pep22), which contains two minor and two major repeats, and subsequently selected against NTDS_5/3, a truncated CSP molecule that presents peptides in a more native-like conformation. Identified L9 variants were characterized for binding affinity in vitro and for protective potency in vivo against a panel of malaria antigens. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/679367v1_fig4.gif" ALT="Figure 4"> View larger version (59K): org.highwire.dtl.DTLVardef@844f00org.highwire.dtl.DTLVardef@5ebe9borg.highwire.dtl.DTLVardef@33d634org.highwire.dtl.DTLVardef@1342c8c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO In vitro and in vivo characterization reveals functional improvement for affinity-enhanced L9 variants against multiple transgenic strains, including the Kenya2-3 KI strain and a major repeat-only transgenic. (A) Biolayer interferometry (BLI) analysis against Peptide 22 (containing 2 NVDPs) and full-length CSP (fl_CSP) of P. falciparum 3D7. L9_WT affinity is marked with a vertical line on the legend bar. L9 variants marked with an asterisk (*) were selected for detailed in vivo characterization. (B) In the transgenic sporozoite intravenous (IV) challenge model, mice were administered IgG from L9 variants or control antibodies (-2 h), followed by IV challenge with P. berghei sporozoites (PbSPZ) encoding different PfCSP knock-in sequences (0 h), and subsequent luciferin-based evaluation (42 h). Each group comprised n=10 mice, with animals receiving the indicated antibody dose. (C) Transgenic PbSPZ challenge. Liver infection burden was quantified, and data are shown as mean {+/-} standard deviation; mean liver burden total flux for L9_WT at 12.5 {micro}g/mouse is indicated by a dashed line. Statistical significance between L9_WT and each L9 variant group at 12.5 {micro}g were determined using the ordinary one-way ANOVA test with a two-tailed P value calculation and a Dunnetts multiple test correction. Statistical significance was also analyzed between L9_yd19 and MAM01 using an unpaired t test with a two-tailed P value calculation; the two groups were significantly different when tested against 1 NVDP KI PbSPZ. See also Table S1 and Fig. S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/679367v1_fig5.gif" ALT="Figure 5"> View larger version (33K): org.highwire.dtl.DTLVardef@58bed2org.highwire.dtl.DTLVardef@580075org.highwire.dtl.DTLVardef@1864220org.highwire.dtl.DTLVardef@15f6582_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5.C_FLOATNO Affinity correlations reveal that improved protection breadth correlates with affinity to multiple malaria antigens. Correlation between binding affinity to PfCSP Kenya2-3 strain, NTDS_5/3 or PfCSP 3D7 strain (x-axis, linear scale) and normalized liver burden against various transgenic PbSPZs (y-axis, linear scale). Pearsons correlation coefficient (R) was calculated using the median normalized liver burden values, with corresponding two-tailed P-values and 95% confidence intervals. A linear regression was fit based on a Pearsons correlation analysis. See also Fig. S6. C_FIG O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig6.gif" ALT="Figure 6"> View larger version (46K): org.highwire.dtl.DTLVardef@14bf2org.highwire.dtl.DTLVardef@12123d9org.highwire.dtl.DTLVardef@b8b17aorg.highwire.dtl.DTLVardef@18c0195_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Structural analyses of L9_yd19 and PfCSP (3D7 or Kenya2-3) reveals a new interaction mode. (A) CryoEM structure of L9_yd19 in complex with PfCSP (3D7) at 3.4 [A] resolution. Heavy chain mutations are shown in red and light chain mutations are shown in blue. 27 residue stretch of PfCSP is ordered and the rest of the PfCSP is disordered. (B) CryoEM structure of L9_yd19 in complex with PfCSP (Kenya2-3) at 3.3 [A] resolution. 2D classes show both three L9_yd19 Fabs (orange arrow) and six L9_yd19 Fabs (red arrow) bound to PfCSP (Kenya2-3). 3D reconstruction for six L9_yd19 Fabs bound to PfCSP (Kenya2-3) is shown. C_FIG O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@bbe52corg.highwire.dtl.DTLVardef@1c2c4b2org.highwire.dtl.DTLVardef@1ffc3a5org.highwire.dtl.DTLVardef@85ac9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO L9_yd19 engineered mutations stabilize a new head-to-head homotypic interface allowing up to six Fabs to bind to PfCSP (Kenya2-3). (A) Structural comparison of L9_yd19 bound to PfCSP-3D7 and PfCSP-Kenya2-3. PfCSP is shown in red sticks with the N- and C-termini labeled. (B) L9_yd19 in complex with PfCSP-Kenya2-3. Fabs (3,4), (2,5), and (1,6) are related by 2-fold (C2) symmetry. Critical residues generating the head-to-head homotypic interface are shown in insets. C_FIG

immunology↗

bioRxiv2025

Virus-like particle vaccines targeting a key epitope in circumsporozoite protein provide sterilizing immunity against malaria in a mouse challenge model

Vaccines that target the pre-erythrocytic stage of the malaria lifecycle have the potential to provide sterilizing immunity but must elicit sustained, high-titer antibody responses to completely prevent infection. Most pre-erythrocytic vaccines target circumsporozoite protein (CSP), the major surface antigen on Plasmodium falciparum sporozoites. Antibodies targeting distinct epitopes within the central repeat region of CSP have the potential to provide protection from infection, but we have focused on developing vaccines that target a highly vulnerable CSP epitope that is targeted by the potent monoclonal antibody L9. In a previous study, we produced a pre-erythrocytic vaccine displaying a synthetic peptide representing the L9 epitope on Q{beta} bacteriophage virus-like particles (VLPs). This vaccine elicited strong anti-CSP antibody responses that protected mice from malaria challenge. Here, we asked whether the structural context of the L9 epitope influences the quality of antibody responses. We compared the immunogenicity and protective efficacy of Q{beta} L9 VLPs to recombinant VLPs that display the L9 peptide in a structure that is hypothesized to mimic its native conformation. Recombinant MS2 bacteriophage VLPs displaying various lengths of the L9 epitope were produced and immunogenicity and protective efficacy were evaluated in mice. Our results demonstrate that MS2 L9 VLPs, particularly those displaying longer L9 peptides and in combination with a potent novel adjuvant, elicit strong and durable antibody responses that lower malaria liver burden and prevent infection. We also compared the efficacy of L9-targeted vaccines to the licensed vaccine, RTS,S/AS01E (Mosquirix, GSK). Immunization with Q{beta} L9 VLPs, MS2 L9 VLPs, and RTS,S/AS01E provided significant protection from liver-stage infection in a mouse model; immunization with Q{beta} L9 VLPs elicited sterilizing immunity in the highest percentage of mice. A combination vaccine consisting of MS2 L9 and Q{beta} L9 VLPs, each presenting the L9 epitope in distinct structural forms, provided the strongest protection, reducing liver parasite burden and promoting sterilizing immunity more effectively than the licensed RTS,S/AS01E vaccine.

immunology↗

bioRxiv2025

Multi-System Dysregulation in Placental Malaria Contributes to Adverse Perinatal Outcomes in Mice

Sequestration of Plasmodium parasites in the placental vasculature causes increased morbidity and mortality in pregnant compared to non-pregnant patients in malaria- endemic regions. In this study, outbred pregnant CD1 mice with semi allogeneic fetuses were infected with transgenic Plasmodium berghei or mock-inoculated by mosquito bite at either embryonic day (E) 6 (first trimester-equivalent) or 10 (second trimester- equivalent) and compared with non-pregnant females. P. berghei-infected mosquitoes had greater biting avidity for E10 dams than uninfected mosquitoes, which was not apparent for E6 dams nor non-pregnant females. Infected E10 dams had greater numbers of parasites than E6 dams in the uterus and spleen, but not in the blood or liver. While parasites were found in placentas, no parasites were present in fetuses. Maternal infection at E6 caused greater maternal morbidity, with greater rates of fetal reabsorption and stillbirths than at E10. Infection at E10 caused adverse offspring outcomes, including growth restriction. To identify possible mechanisms of adverse offspring outcomes, E10 dams were euthanized during peak parasitemia (8 days post infection), and outcomes were compared with mock-infected dams. P. berghei caused significant systemic maternal immune activation with elevated circulating lymphocytes, eosinophils, and neutrophils and splenic cytokine concentrations. P. berghei infection at E10 increased corticosterone and decreased progesterone concentrations, which could contribute to adverse perinatal outcomes through immunomodulation. There were limited changes in the maternal fecal microbiome after P. berghei infection. Mosquito bite infection of outbred dams with P. berghei causes placental malaria and provides a novel, tractable model to investigate therapeutic treatments.

microbiology↗

bioRxiv2024

Virus-like particle-based vaccines targeting the Anopheles mosquito salivary protein, TRIO

Malaria is a highly lethal infectious disease caused by Plasmodium parasites. These parasites are transmitted to vertebrate hosts when mosquitoes of the Anopheles genus probe for a blood meal. Sporozoites, the infectious stage of Plasmodium, transit to the liver within hours of injection into the dermis. Vaccine efforts are hindered by the complexity of the parasites lifecycle and the speed at which the infection is established in the liver. In an effort to enhance immunity against Plasmodium, we produced a virus-like particle (VLP)-based vaccine displaying an epitope of TRIO, an Anopheles salivary protein which has been shown to enhance mobility and dispersal of sporozoites in the dermis. Previous work demonstrated that passive immunization with TRIO offered protection from liver infection and acted synergistically with a Plasmodium targeted vaccine. Immunization of mice with TRIO VLPs resulted in high-titer and long-lasting antibody responses that did not significantly drop for over 18 months post-immunization. TRIO VLPs were similarly immunogenic when combined with an anti-malaria vaccine targeting the L9 epitope of the Plasmodium falciparum circumsporozoite protein.However, when used in a malaria challenge mouse model, TRIO VLPs only provided modest protection from infection and did not boost the protection provided by L9 VLPs.

immunology↗

bioRxiv2024

Somatic hypermutation-mediated paratope flexibility improves the cross-reactivity of human malaria antibodies

The protective capacity of antibodies targeting circumsporozoite protein on sporozoites of the malaria parasite Plasmodium falciparum (PfCSP) is linked to high affinity and cross-reactivity with the PfCSP central repeat domain and N-terminal junction. However, the role of somatic hypermutation (SHM) in the development of such antibodies remains unclear. Here we define the contributions of SHM to the high affinity and strong repeat and N-junction cross-reactivity of the potent anti-PfCSP monoclonal antibody (mAb) 4493 and of similar antibodies with shared SHM and affinity maturation trajectories. Molecular dynamics simulations reveal that SHM reduces the flexibility of the unbound mAb 4493 but increases the flexibility of the antigen-bound complex, thereby lowering the entropic cost for antigen binding. Furthermore, we identify an inverse relation between antibody affinity and serum stability, which limits the protective capacity of these antibodies. Our study provides molecular level evidence for the different roles that the SHM process plays in increasing VH3-49+V{kappa}3-20 antibody affinity and cross-reactivity and demonstrates how antibody affinity maturation can negatively impact antibody stability and thereby function.

immunology↗

bioRxiv2022

Affinity-matured homotypic interactions induce spectrum of PfCSP-antibody structures that influence protection from malaria infection

The generation of high-quality antibody responses to PfCSP, the primary surface antigen of Plasmodium falciparum sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the IGHV3-33 germline gene, which is among the most prevalent and potent antibody families induced in the anti-CSP immune response and targets the NANP repeat region. Cryo-EM reveals a remarkable spectrum of helical Fab-CSP structures stabilized by homotypic interactions between tightly packed Fabs, many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to CSP and in protection from P. falciparum malaria infection. These data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3-33 antibodies, advance our understanding of the mechanism(s) of antibody-mediated protection, and inform next generation CSP vaccine design.

immunology↗

bioRxiv2021

A vaccine targeting the L9 epitope of the malaria circumsporozoite protein confers protection from blood-stage infection in a mouse challenge model

Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.

immunology↗

bioRxiv2021

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAbs ontogeny and mechanisms of binding PfCSP to neutralize sporozoites will facilitate vaccine development. Here, we isolated mAbs clonally related to L9 and showed that this B-cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9{kappa}) with clonally-related heavy chains resulted in chimeric mAbs that cross-linked two NVDP, cross-reacted with NANP, and more potently neutralized sporozoites compared to their original light chain. Structural analyses revealed that chimeric mAbs bound the minor repeat motif in a type-1 {beta}-turn seen in other repeat-specific antibodies. These data highlight the importance of L9{kappa} in binding NVDP on PfCSP to neutralize SPZ and suggest that PfCSP-based immunogens might be improved by presenting [≥]2 NVDP.

immunology↗

bioRxiv2020

An Epitope-based Malaria Vaccine Targeting the Junctional Domain of Circumsporozoite Protein

A malaria vaccine that elicits long-lasting protection and is suitable for use in endemic areas remains urgently needed. Here, we assessed the immunogenicity and prophylactic efficacy of a vaccine targeting a recently described epitope on the major surface antigen on Plasmodium falciparum sporozoites, circumsporozoite protein (CSP). Using a virus-like particle (VLP)-based vaccine platform technology, we developed a vaccine that targets the junctional region between the N-terminal and central repeat domains of CSP. This region is recognized by monoclonal antibodies, including mAb CIS43, that have been shown to potently prevent liver invasion in animal models. We show that CIS43 VLPs elicit high titer and long-lived anti-CSP antibody responses in mice and non-human primates. Immunization with CIS43 VLPs confers partial protection from malaria infection in a mouse model, and both immunogenicity and protection were enhanced when mice were immunized with CIS43 VLPs in combination with adjuvants including delta inulin polysaccharide particles and TLR9 agonists. Passive transfer of serum from immunized macaques also inhibited parasite liver invasion in the mouse infection model. Our findings demonstrate that a Q{beta} VLP-based vaccine targeting the CIS43 epitope combined with various adjuvants is highly immunogenic in mice and macaques, elicits long-lasting anti-CSP antibodies, and inhibits parasite infection in a mouse model. Thus, the CIS43 VLP vaccine is a promising pre-erythrocytic malaria vaccine candidate.

immunology↗

bioRxiv2020

Structural and biophysical correlation of anti-NANP antibodies with in vivo protection against P. falciparum

The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I {beta}- and Asn pseudo 310 turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.

microbiology↗

bioRxiv2019

A Vaccine Composed of Antigen Fused to a Ligand for a Receptor on Immature Dendritic Cells Elicits Protective Antibody Responses to Malaria Sporozoites in Infant Macaques

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3 genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized one and six month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

immunology↗