Biology subjects

Flynn, B. J.

Publications and source records attributed to Flynn, B. J..

10 recordsLinked to original sources

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↗

bioRxiv2023

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, theres a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

immunology↗

bioRxiv2023

Mucosal Adenoviral-vectored Vaccine Boosting Durably Prevents XBB.1.16 Infection in Nonhuman Primates

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/565765v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@d49b9dorg.highwire.dtl.DTLVardef@347455org.highwire.dtl.DTLVardef@1c1a196org.highwire.dtl.DTLVardef@1579130_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

immunology↗

bioRxiv2023

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. HighlightsO_LINo evidence of association between CH and COVID-19 clinical severity in macaques. C_LIO_LIThe presence of CH is associated with prolonged local inflammatory responses in COVID-19. C_LIO_LISARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection. C_LI

genetics↗

bioRxiv2022

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to antibody neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-specific vaccines would enhance immunity and protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing antibody titers against D614G were 4760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (pre-boost), respectively, and 320 and 110 for Omicron. Two weeks after boost, titers against D614G and Omicron increased to 5360 and 2980, respectively, for mRNA-1273 and 2670 and 1930 for mRNA-Omicron. Following either boost, 70-80% of spike-specific B cells were cross-reactive against both WA1 and Omicron. Significant and equivalent control of virus replication in lower airways was observed following either boost. Therefore, an Omicron boost may not provide greater immunity or protection compared to a boost with the current mRNA-1273 vaccine.

immunology↗

bioRxiv2021

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined [~]3-log10 compared to control animals. In nasal swabs, sgRNA declined 1-log10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

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↗

bioRxiv2021

Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3x106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

immunology↗

bioRxiv2019

Antibody feedback limits the expansion of cognate memory B cells but drives the diversification of vaccine-induced antibody responses

Generating sufficient antibody to block infection is a key challenge for vaccines against malaria. Here we show that antibody titres to a key target, the repeat region of the Plasmodium falciparum circumsporozoite protein (PfCSP), plateaued after two immunizations in a clinical trial of the radiation-attenuated sporozoite vaccine. To understand the mechanisms limiting vaccine responsiveness, we developed Ig-knockin mice with elevated numbers of PfCSP-binding B cells. We determined that recall responses were inhibited by antibody feedback via epitope masking of the immunodominant PfCSP repeat region. Importantly, the amount of antibody that prevents boosting is below the amount of antibody required for protection. Finally, while antibody feedback limited responses to the PfCSP-repeat region in vaccinated volunteers, potentially protective subdominant responses to C-terminal regions did expand with subsequent boosts. These data suggest that antibody feedback drives the diversification of immune responses and that vaccination for malaria will require the targeting of multiple antigens.

immunology↗