Pentameric Complex Research Articles

The IFT81‐IFT74 complex acts as an unconventional RabL2 GTPase‐activating protein during intraflagellar transport

Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70‐amino‐acid‐long coiled‐coil region of IFT81/74. We present structural models for RabL2‐containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

MEK1/2 regulate normal BCR and ABL1 tumor-suppressor functions to dictate ATO response in TKI-resistant Ph+ leukemia

Resistance to tyrosine kinase inhibitors (TKIs) remains a clinical challenge in Ph-positive variants of chronic myeloid leukemia. We provide mechanistic insights into a previously undisclosed MEK1/2/BCR::ABL1/BCR/ABL1-driven signaling loop that may determine the efficacy of arsenic trioxide (ATO) in TKI-resistant leukemic patients. We find that activated MEK1/2 assemble into a pentameric complex with BCR::ABL1, BCR and ABL1 to induce phosphorylation of BCR and BCR::ABL1 at Tyr360 and Tyr177, and ABL1, at Thr735 and Tyr412 residues thus provoking loss of BCR’s tumor-suppression functions, enhanced oncogenic activity of BCR::ABL1, cytoplasmic retention of ABL1 and consequently drug resistance. Coherently, pharmacological blockade of MEK1/2 induces dissociation of the pentameric MEK1/2/BCR::ABL1/BCR/ABL1 complex and causes a concurrent BCRY360/Y177, BCR::ABL1Y360/Y177 and cytoplasmic ABL1Y412/T735 dephosphorylation thereby provoking the rescue of the BCR’s anti-oncogenic activities, nuclear accumulation of ABL1 with tumor-suppressive functions and consequently, growth inhibition of the leukemic cells and an ATO sensitization via BCR-MYC and ABL1-p73 signaling axes activation. Additionally, the allosteric activation of nuclear ABL1 was consistently found to enhance the anti-leukemic effects of the MEK1/2 inhibitor Mirdametinib, which when combined with ATO, significantly prolonged the survival of mice bearing BCR::ABL1-T315I-induced leukemia. These findings highlight the therapeutic potential of MEK1/2-inhibitors/ATO combination for the treatment of TKI-resistant leukemia.

Structural basis of mRNA binding by the human FERRY Rab5 effector complex

The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.

Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL.

In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, which synthesises peptidoglycan strands from its substrate Lipid II, and the transpeptidase FtsI that cross-links these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ-FtsB-FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities; however, the structure of this pentameric complex is unknown. Here, we present the cryogenic electron microscopy structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. As FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.

SWIP mediates retromer-independent membrane recruitment of the WASH complex.

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.

The WAVE complex drives the morphogenesis of the photoreceptor outer segment cilium

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

112. Interim Results From a Phase 2, Randomized, Observer-Blind, Placebo-Controlled, Dose-Finding Trial of an mRNA-Based Cytomegalovirus Vaccine in Healthy Adults

Abstract Background Cytomegalovirus (CMV) is the most common congenital viral infection and can cause severe long-term health consequences, including hearing loss and neurodevelopmental delay. A safe and effective method for prevention of CMV infection is an unmet need and public health priority. An mRNA-based vaccine against CMV, mRNA-1647, is in development and consists of 6 mRNA sequences encoding 2 CMV antigens (glycoprotein B and the pentameric glycoprotein complex) in a lipid nanoparticle formulation. Methods In this Phase 2, randomized, placebo-controlled, observer-blind, dose-finding trial, safety and immunogenicity of mRNA-1647 was evaluated in healthy adults aged 18 to 40 years (NCT04232280). In Part 1, CMV-seronegative and CMV-seropositive men and women were randomized 3:1 to receive mRNA-1647 (doses of 50, 100, or 150 µg) or placebo at Months 0, 2, and 6. The 100-µg dose was chosen for Part 2. In Part 2, CMV-seronegative and CMV-seropositive women were randomized 3:1 to receive mRNA-1647 100 µg or placebo at Months 0, 2, and 6. Safety endpoints were solicited local and systemic adverse reactions, unsolicited adverse events (AEs), and medically attended AEs through 7 days, 28 days, and 6 months after vaccination, respectively, and serious AEs throughout the study. Humoral immunogenicity endpoints were antigen-specific binding antibody titers and neutralizing antibody titers against epithelial cell infection and against fibroblast infection. Results In Parts 1 and 2, 252 and 63 participants were randomized, respectively. Interim analysis (IA) of Part 1 through 1 month after Dose 3 indicated that mRNA-1647 100 µg was generally well-tolerated, induced robust antibody responses in CMV-seronegative participants, and boosted antibody titers in CMV-seropositive participants. An additional IA of Part 1 through end of study and Part 2 through 1 month after Dose 3 showed no notable differences in the safety profile compared with the previous IA (Part 1, 1 month after Dose 3); immunogenicity data for this additional IA is being generated. Conclusion Available data from this Phase 2 trial suggest that mRNA-1647 100 µg was immunogenic in CMV-seronegative and CMV-seropositive participants and was generally well-tolerated. The mRNA-1647 candidate vaccine is being evaluated in a Phase 3 trial. Disclosures Lori Panther, MD, MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Richard Leggett, DO, Crossroads Clinical Research: Contract employee James Peterson, MD, Moderna, Inc.: Received payment as a study investigator Jiang Lin, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Kai Wu, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Heather Lee, BS, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Roxane Hasselbeck, BA, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Andrew Natenshon, MA, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Jacqueline Miller, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds.

Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit.

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis-specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

PCRCR complex is essential for invasion of human erythrocytes by Plasmodium falciparum

The most severe form of malaria is caused by Plasmodium falciparum. These parasites invade human erythrocytes, and an essential step in this process involves the ligand PfRh5, which forms a complex with cysteine-rich protective antigen (CyRPA) and PfRh5-interacting protein (PfRipr) (RCR complex) and binds basigin on the host cell. We identified a heteromeric disulfide-linked complex consisting of P. falciparum Plasmodium thrombospondin-related apical merozoite protein (PfPTRAMP) and P. falciparum cysteine-rich small secreted protein (PfCSS) and have shown that it binds RCR to form a pentameric complex, PCRCR. Using P. falciparum lines with conditional knockouts, invasion inhibitory nanobodies to both PfPTRAMP and PfCSS, and lattice light-sheet microscopy, we show that they are essential for merozoite invasion. The PCRCR complex functions to anchor the contact between merozoite and erythrocyte membranes brought together by strong parasite deformations. We solved the structure of nanobody–PfCSS complexes to identify an inhibitory epitope. Our results define the function of the PCRCR complex and identify invasion neutralizing epitopes providing a roadmap for structure-guided development of these proteins for a blood stage malaria vaccine.

Neutralization Epitopes in Trimer and Pentamer Complexes Recognized by Potent Cytomegalovirus-Neutralizing Human Monoclonal Antibodies.

Human cytomegalovirus (HCMV) infects 36% to almost 100% of adults and causes severe complications only in immunocompromised individuals. HCMV viral surface trimeric (gH/gL/gO) and pentameric (gH/gL/UL128/UL130/UL131A) complexes play important roles in HCMV infection and tropism. Here, we isolated and identified a total of four neutralizing monoclonal antibodies (MAbs) derived from HCMV-seropositive blood donors. Based on their reactivity to HCMV trimer and pentamer, these MAbs can be divided into two groups. MAbs PC0012, PC0014, and PC0035 in group 1 bind both trimer and pentamer and neutralize CMV by interfering with the postattachment steps of CMV entering into cells. These three antibodies recognize antigenic epitopes clustered in a similar area, which are overlapped by the epitope recognized by the known neutralizing antibody MSL-109. MAb PC0034 in group 2 binds only to pentamer and neutralizes CMV by blocking the binding of pentamer to cells. Epitope mapping using pentamer mutants showed that amino acid T94 of the subunit UL128 and K27 of UL131A on the pentamer are key epitope-associated residues recognized by PC0034. This study provides new evidence and insight information on the importance of the development of the CMV pentamer as a CMV vaccine. In addition, these newly identified potent CMV MAbs can be attractive candidates for development as antibody therapeutics for the prevention and treatment of HCMV infection. IMPORTANCE The majority of the global population is infected with HCMV, but severe complications occur only in immunocompromised individuals. In addition, CMV infection is a major cause of birth defects in newborns. Currently, there are still no approved prophylactic vaccines or therapeutic monoclonal antibodies (MAbs) for clinical use against HCMV infection. This study identified and characterized a panel of four neutralizing MAbs targeting the HCMV pentamer complex with specific aims to identify a key protein(s) and antigenic epitopes in the HCMV pentamer complex. The study also explored the mechanism by which these newly identified antibodies neutralize HCMV in order to design better HCMV vaccines focusing on the pentamer and to provide attractive candidates for the development of effective cocktail therapeutics for the prevention and treatment of HCMV infection.

Endothelial Cell Infection by Guinea Pig Cytomegalovirus Is a Lytic or Persistent Infection Depending on Tissue Origin but Requires Viral Pentamer Complex and pp65 Tegument Protein.

The guinea pig is the only small animal model for congenital cytomegalovirus (CMV) but requires species-specific guinea pig cytomegalovirus (GPCMV). Infection of epithelial cells and trophoblasts by GPCMV requires the viral glycoprotein pentamer complex (PC) and endocytic entry because of the absence of platelet-derived growth factor receptor alpha (PDGFRA). Endothelial cells represent an important cell type for infection, dissemination in the host, and disease but have been poorly evaluated for GPCMV. Novel endothelial cell lines were established from animal vascular systems, including aorta (EndoC) and placental umbilical cord vein (GPUVEC). Cell lines were characterized for endothelial cell protein markers (PECAM1, vWF, and FLI1) and evaluated for GPCMV infection. Only PC-positive virus was capable of infecting endothelial cells. Individual knockout mutants for unique PC components (GP129, GP131, and GP133) were unable to infect endothelial cells without impacting fibroblast infection. Ectopic expression of PDGFRA in EndoC cells enabled GPCMV(PC-) infection via direct cell entry independent of the PC. Neutralizing antibodies to the essential viral gB glycoprotein were insufficient to prevent endothelial cell infection, which also required antibodies to gH/gL and the PC. Endothelial cell infection was also dependent upon viral tegument pp65 protein (GP83) to counteract the IFI16/cGAS-STING innate immune pathway, similar to epithelial cell infection. GPCMV endothelial cells were lytically (EndoC) or persistently (GPUVEC) infected dependent on tissue origin. The ability to establish a persistent infection in the umbilical cord could potentially enable sustained and more significant infection of the fetus in utero. Overall, results demonstrate the importance of this translationally relevant model for CMV research. IMPORTANCE Congenital CMV is a leading cause of cognitive impairment and deafness in newborns, and a vaccine is a high priority. The only small animal model for congenital CMV is the guinea pig and guinea pig cytomegalovirus (GPCMV) encoding functional HCMV homolog viral glycoprotein complexes necessary for cell entry that are neutralizing-antibody vaccine targets. Endothelial cells are important in HCMV for human disease and viral dissemination. GPCMV endothelial cell infection requires the viral pentamer complex (PC), which further increases the importance of this complex as a vaccine target, as antibodies to the immunodominant and essential viral glycoprotein gB fail to prevent endothelial cell infection. GPCMV endothelial cell infection established either a fully lytic or a persistent infection, depending on tissue origin. The potential for persistent infection in the umbilical cord potentially enables sustained infection of the fetus in utero, likely increasing the severity of congenital disease.

An Attenuated Strain of Human Cytomegalovirus for the Establishment of a Subviral Particle Vaccine.

Human cytomegalovirus (HCMV) infection is associated with severe disease conditions either following congenital transmission of the virus or viral reactivation in immunosuppressed individuals. Consequently, the establishment of a protective vaccine is of high medical need. Several candidates have been tested in preclinical and clinical studies, yet no vaccine has been licensed. Subviral dense bodies (DB) are a promising vaccine candidate. We have recently provided a GMP-compliant protocol for the production of DB, based on a genetically modified version of the HCMV laboratory strain Towne, expressing the pentameric complex of envelope protein gH-gL-pUL128-131 (Towne-UL130rep). In this work, we genetically attenuated Towne-UL130rep by abrogating the expression of the tegument protein pUL25 and by fusing the destabilizing domain ddFKBP to the N-terminus of the IE1- and IE2-proteins of HCMV. The resulting strain, termed TR-VAC, produced high amounts of DB under IE1/IE2 repressive conditions and concomitant supplementation of the viral terminase inhibitor letermovir to the producer cell culture. TR-VAC DB retained the capacity to induce neutralizing antibodies. A complex pattern of host protein induction was observed by mass spectrometry following exposure of primary human monocytes with TR-VAC DB. Human monocyte-derived dendritic cells (DC) moderately increased the expression of activation markers and MHC molecules upon stimulation with TR-VAC DB. In a co-culture with autologous T cells, the TR-VAC DB-stimulated DC induced a robust HCMV-specific T cell-activation and –proliferation. Exposure of donor-derived monocytic cells to DB led to the activation of a rapid innate immune response. This comprehensive data set thus shows that TR-VAC is an optimal attenuated seed virus strain for the production of a DB vaccine to be tested in clinical studies.

Human cytomegalovirus (HCMV) long-term shedding and HCMV-specific immune response in pregnant women with primary HCMV infection.

Human cytomegalovirus (HCMV) shedding has been extensively investigated in newborns and in young children, however, much less is known about it in immunocompetent adults. Shedding of HCMV was investigated in saliva, vaginal secretions and urine of pregnant women experiencing primary infection along with the development of the HCMV-specific immune response. Thirty-three pregnant women shed HCMV DNA in peripheral biological fluids at least until one year after onset of infection, while in blood HCMV DNA was cleared earlier. Significantly higher levels of viral load were found in vaginal secretions compared to saliva and urine. All subjects examined two years after the onset of infection showed a high avidity index, with IgM persisting in 36% of women. Viral load in blood was directly correlated with levels of HCMV-specific IgM and inversely correlated with levels of IgG specific for the pentameric complex gH/gL/pUL128L; in addition, viral load in blood was inversely correlated with percentage of HCMV-specific CD4+ and CD8+ expressing IL-7R (long-term memory, LTM) while viral load in biological fluids was inversely correlated with percentage of HCMV-specific CD4+ and CD8+ effector memory RA+(TEMRA). In conclusion, viral shedding during primary infection in pregnancy persists in peripheral biological fluids for at least one year and the development of both antibodies (including those directed toward the pentameric complex) and memory T cells are associated with viral clearance.

Viral and Cellular Factors Contributing to the Hematogenous Dissemination of Human Cytomegalovirus via Polymorphonuclear Leukocytes.

Polymorphonuclear leukocytes (PMNs) presumably transmit human cytomegalovirus (HCMV) between endothelial cells in blood vessels and thereby facilitate spread to peripheral organs. We aimed to identify viral components that contribute to PMN-mediated transmission and test the hypothesis that cellular adhesion molecules shield transmission sites from entry inhibitors. Stop codons were introduced into the genome of HCMV strain Merlin to delete pUL74 of the trimeric and pUL128 of the pentameric glycoprotein complex and the tegument proteins pp65 and pp71. Mutants were analyzed regarding virus uptake by PMNs and transfer of infection to endothelial cells. Cellular adhesion molecules were evaluated for their contribution to virus transmission using function-blocking antibodies, and hits were further analyzed regarding shielding against inhibitors of virus entry. The viral proteins pUL128, pp65, and pp71 were required for efficient PMN-mediated transmission, whereas pUL74 was dispensable. On the cellular side, the blocking of the αLβ2-integrin LFA-1 reduced virus transfer by 50% and allowed entry inhibitors to reduce it further by 30%. In conclusion, these data show that PMN-mediated transmission depends on the pentameric complex and an intact tegument and supports the idea of a virological synapse that promotes this dissemination mode both directly and via immune evasion.

Divide and Conquer: A Tailored Solid-state NMR Approach to Study Large Membrane Protein Complexes.

Membrane proteins are known to exert many essential biological functions by forming complexes in cell membranes. An example refers to the β‐barrel assembly machinery (BAM), a 200 kDa pentameric complex containing BAM proteins A–E that catalyzes the essential process of protein insertion into the outer membrane of gram‐negative bacteria. While progress has been made in capturing three‐dimensional structural snapshots of the BAM complex, the role of the lipoprotein BamC in the complex assembly in functional lipid bilayers has remained unclear. We have devised a component‐selective preparation scheme to directly study BamC as part of the entire BAM complex in lipid bilayers. Combination with proton‐detected solid‐state NMR methods allowed us to probe the structure, dynamics, and supramolecular topology of full‐length BamC embedded in the entire complex in lipid bilayers. Our approach may help decipher how individual proteins contribute to the dynamic formation and functioning of membrane protein complexes in membranes.

Divide and Conquer: A Tailored Solid‐state NMR Approach to Study Large Membrane Protein Complexes

Membrane proteins are known to exert many essential biological functions by forming complexes in cell membranes. An example refers to the β-barrel assembly machinery (BAM), a 200 kDa pentameric complex containing BAM proteins A-E that catalyzes the essential process of protein insertion into the outer membrane of gram-negative bacteria. While progress has been made in capturing three-dimensional structural snapshots of the BAM complex, the role of the lipoprotein BamC in the complex assembly in functional lipid bilayers has remained unclear. We have devised a component-selective preparation scheme to directly study BamC as part of the entire BAM complex in lipid bilayers. Combination with proton-detected solid-state NMR methods allowed us to probe the structure, dynamics, and supramolecular topology of full-length BamC embedded in the entire complex in lipid bilayers. Our approach may help decipher how individual proteins contribute to the dynamic formation and functioning of membrane protein complexes in membranes.

Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH

Human cytomegalovirus (HCMV) is a β-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

Horizontal Transmission of Cytomegalovirus in a Rhesus Model Despite High-Level, Vaccine-Elicited Neutralizing Antibody and T-Cell Responses.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

Cross Strain Protection against Cytomegalovirus Reduces DISC Vaccine Efficacy against CMV in the Guinea Pig Model.

Congenital cytomegalovirus (CMV) is a leading cause of disease in newborns and a vaccine is a high priority. The guinea pig is the only small animal model for congenital CMV but requires guinea pig cytomegalovirus (GPCMV). Previously, a disabled infectious single cycle (DISC) vaccine strategy demonstrated complete protection against congenital GPCMV (22122 strain) and required neutralizing antibodies to various viral glycoprotein complexes. This included gB, essential for all cell types, and the pentamer complex (PC) for infection of non-fibroblast cells. All GPCMV research has utilized prototype strain 22122 limiting the translational impact, as numerous human CMV strains exist allowing re-infection and congenital CMV despite convalescent immunity. A novel GPCMV strain isolate (designated TAMYC) enabled vaccine cross strain protection studies. A GPCMV DISC (PC+) vaccine (22122 strain) induced a comprehensive immune response in animals, but vaccinated animals challenged with the TAMYC strain virus resulted in sustained viremia and the virus spread to target organs (liver, lung and spleen) with a significant viral load in the salivary glands. Protection was better than natural convalescent immunity, but the results fell short of previous DISC vaccine sterilizing immunity against the homologous 22122 virus challenge, despite a similarity in viral glycoprotein sequences between strains. The outcome suggests a limitation of the current DISC vaccine design against heterologous infection.