Neutralization Escape Variants Research Articles

ACE2-Fc and DPP4-Fc decoy receptors against SARS-CoV-2 and MERS-CoV variants: a quick therapeutic option for current and future coronaviruses outbreaks.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are highly pathogenic human coronaviruses (CoVs). Anti-CoVs mAbs and vaccines may be effective, but the emergence of neutralization escape variants is inevitable. Angiotensin-converting enzyme 2 and dipeptidyl peptidase 4 enzyme are the getaway receptors for SARS-CoV-2 and MERS-CoV, respectively. Thus, we reformatted these receptors as Fc-fusion decoy receptors. Then, we tested them in parallel with anti-SARS-CoV (ab1-IgG) and anti-MERS-CoV (M336-IgG) mAbs against several variants using pseudovirus neutralization assay. The generated Fc-based decoy receptors exhibited a strong inhibitory effect against all pseudotyped CoVs. Results showed that although mAbs can be effective antiviral drugs, they might rapidly lose their efficacy against highly mutated viruses. We suggest that receptor traps can be engineered as Fc-fusion proteins for highly mutating viruses with known entry receptors, for a faster and effective therapeutic response even against virus harboring antibodies escape mutations.

Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

SARS-CoV-2 Variant B.1.1.7 is Susceptible to Neutralizing Antibodies Elicited by Ancestral Spike Vaccines

The SARS-CoV-2 Spike glycoprotein mediates virus entry and is a major target for neutralizing antibodies. All current vaccines are based on the ancestral Spike with the goal of generating a protective neutralizing antibody response. Several novel SARS-CoV-2 variants with multiple Spike mutations have emerged, and their rapid spread and potential for immune escape have raised concerns. One of these variants, first identified in the United Kingdom, B.1.1.7 (also called VUI202012/01), contains eight Spike mutations with potential to impact antibody therapy, vaccine efficacy and risk of reinfection. Here we employed a lentivirus-based pseudovirus assay to show that variant B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (~2-fold), by serum samples from convalescent individuals and recipients of two different vaccines based on ancestral Spike: mRNA-1273 (Moderna) and protein nanoparticle NVX-CoV2373 (Novavax). Some monoclonal antibodies to the receptor binding domain (RBD) of Spike were less effective against the variant while others were largely unaffected. These findings indicate that B.1.1.7 is not a neutralization escape variant that would be a major concern for current vaccines, or for an increased risk of reinfection.Funding: Original data and specimens for Protocol 20-0003 were supported by the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases. KW and BK were supported by LANL LDRD 20190441ER. DCM, XS, HT, and CM were supported bythe COVID-19 Prevention Network (CoVPN) and the National Institute of Health. DL, BFH, and KOS were supported by a grant from the State of North Carolina from federal CARES Act funds, and NIAID grant AI142596.Conflict of Interest: Rolando Pajon is an employee of Moderna, Inc. Filip, Dubovsky, Gale Smith and Gregory M. Glenn are employees of Novavax, Inc. The remaining authors have no competing interests.Ethical Approval: Clinical trials described in this manuscript were approved by the appropriate Institutional Review Boards (IRBs).

Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358–606 aa), S1 (1–751 aa), S2 (752–1296 aa), and SΔTM (1–1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506–509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV.

A Monoclonal Antibody Specific for Japanese Encephalitis Virus with High Neutralizing Capability for Inclusion as a Positive Control in Diagnostic Neutralization Tests.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 μg/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV.

Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Survivors Remorse: antibody-mediated protection against HIV-1.

It is clear that antibodies can play a pivotal role in preventing the transmission of HIV-1 and large efforts to identify an effective antibody-based vaccine to quell the epidemic. Shortly after HIV-1 was discovered as the cause of AIDS, the search for epitopes recognized by neutralizing antibodies became the driving strategy for an antibody-based vaccine. Neutralization escape variants were discovered shortly thereafter, and, after almost three decades of investigation, it is now known that autologous neutralizing antibody responses and their selection of neutralization resistant HIV-1 variants can lead to broadly neutralizing antibodies in some infected individuals. This observation drives an intensive effort to identify a vaccine to elicit broadly neutralizing antibodies. In contrast, there has been less systematic study of antibody specificities that must rely mainly or exclusively on other protective mechanisms, although non-human primate (NHP) studies as well as the RV144 vaccine trial indicate that non-neutralizing antibodies can contribute to protection. Here we propose a novel strategy to identify new epitope targets recognized by these antibodies for which viral escape is unlikely or impossible.

Maternal Neutralization-Resistant Virus Variants Do Not Predict Infant HIV Infection Risk.

ABSTRACTMother-to-child transmission (MTCT) of HIV provides a setting for studying immune correlates of protection. Neutralizing antibodies (NAbs) are suggested to contribute to a viral bottleneck during MTCT, but their role in blocking transmission is unclear, as studies comparing the NAb sensitivities of maternal viruses have yielded disparate results. We sought to determine whether transmitting mothers differ from nontransmitting mothers in the ability to neutralize individual autologous virus variants present at transmission. Ten transmitting and 10 nontransmitting HIV-infected mothers at high risk of MTCT were included in this study. Full-length HIV envelope genes (n = 100) were cloned from peripheral blood mononuclear cells obtained near transmission from transmitting mothers and at similar time points from nontransmitting mothers. Envelope clones were tested as pseudoviruses against contemporaneous, autologous maternal plasma in neutralization assays. The association between transmission and the log2 50% inhibitory concentration (IC50) for multiple virus variants per mother was estimated by using logistic regression with clustered standard errors. t tests were used to compare proportions of neutralization-resistant viruses. Overall, transmitting mothers had a median IC50 of 317 (interquartile range [IQR], 202 to 521), and nontransmitting mothers had a median IC50 of 243 (IQR, 95 to 594). Transmission risk was not significantly associated with autologous NAb activity (odds ratio, 1.25; P = 0.3). Compared to nontransmitting mothers, transmitting mothers had similar numbers of or fewer neutralization-resistant virus variants, depending on the IC50 neutralization resistance cutoff. In conclusion, HIV-infected mothers harbor mostly neutralization-sensitive viruses, although resistant variants were detected in both transmitting and nontransmitting mothers. These results suggest that MTCT during the breastfeeding period is not driven solely by the presence of maternal neutralization escape variants.

Chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes.

Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1(-/-) mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.

Antigenic site variation in foot-and-mouth disease virus serotype O grown under vaccinal serum antibodies in vitro

Foot-and-mouth disease virus (FMDV) is constantly evolving under neutralizing antibody pressure in either naturally infected or vaccinated animals. This study was carried out to understand the dynamics of evolution of antigenic sites. Neutralizing antibody-resistant populations of three strains of FMDV serotype O (INDR2/1975, IND120/2002 and IND271/2001) were isolated by serial propagation in BHK-21 cells in the presence of sub-neutralizing level of bovine vaccinal sera (BVS). In the partial neutralization escape variants, fixation of aa substitutions were observed at critical residues of all established antigenic sites of serotype O {144 of VP1 (site 1), 45 and 48 of VP1 (site 3), 72 and 134 of VP2 (Site 2)} except site 4 and 5. In majority of the variant populations, site 3 was found to be substituted and therefore immunodominance may not be associated with a particular site, rather it appears to be a virus strain and infected host specific affair. Substitutions were also observed in proximity to the identified residues {41 and 51 (βB-βC loop), 133, 140 and 143 (βG-βH loop), 201, 204 and 209 (C terminus) of VP1, 71 and 75 (βB-βC loop), 131 (βE-αB region), 174 and 179 (βG-βH loop) and 219 (C terminus) of VP3} within antigenic sites of serotype O or other serotypes which could be significant in terms of neutralizing antibody binding and immune escape. Presence of similar residues in the Indian field viruses as selected in the variants supports the importance of these sites in antigenic diversification of serotype O FMD virus.

Sequential Evolution and Escape from Neutralization of Simian Immunodeficiency Virus SIVsmE660 Clones in Rhesus Macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.

Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-β remained unchanged. However, IL-1β did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser → pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1β and its association with WNV pathogenesis.

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

BackgroundVenezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.MethodsWe used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.FindingsUsing 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.ConclusionsThe VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.

Escape from autologous humoral immunity of HIV-1 is not associated with a decrease in replicative capacity

Autologous HIV-1-specific neutralizing antibodies (NAbs) seem unable to inhibit viral replication as they rapidly select for neutralization escape variants. However, NAbs could potentially contribute indirectly to the control of HIV-1 if changes in the viral envelope coinciding with NAb escape would impair viral replication fitness. Here we analyzed the replication kinetics of HIV-1 isolated over the course of infection from five typical progressors, three of whom developed strong autologous neutralizing humoral immunity. Viral replication rate did not correlate with viral sensitivity to autologous serum neutralization or with envelope length or number of potential N-linked glycosylation sites in gp120, suggesting that the flexibility of the viral envelope allows escape from NAbs without the loss of viral fitness. Interestingly, the appearance of rapidly replicating viruses late in infection correlated with lower CD4(+) T-cell counts, suggesting that this viral characteristic may be positively selected when the availability of target cells becomes limiting.

Towards a Broadly Active Flavivirus Therapeutic Antibody

Evaluation of: Sultana H, Foellmer HG, Neelakanta G et al.: Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody. J. Immunol. 183(1), 650–660 (2009). Flaviviruses, such as West Nile virus, the dengue serotypes, Japanese encephalitis virus and yellow fever virus are important human pathogens and can co-circulate in parts of the world. Humoral immunity is important in the mammalian protective response to flaviviruses and passive immunization has been shown to be effective in preventing infection and even treating disease. Monoclonal antibodies (mAbs) against flaviviruses can be divided basically into potent neutralizing mAbs with restricted flavivirus cross-reactivity and broadly cross-reactive mAbs with low neutralizing potency and protective activity. Here, Sultana and colleagues extend the characterization of the cross-reactive mAb11 by defining its epitope within the fusion loop of the West Nile virus envelope protein and link the reduced neuroinvasiveness of a mAb11 neutralization escape variant to a diminished ability to activate Toll-like receptor 3, induce inflammatory cytokine release and compromise blood–brain barrier integrity. The results highlight the importance of the fusion loop in viral pathogenesis; however, challenges remain to develop mAb11 into a viable therapeutic. Instead, mixtures of potent neutralizing mAbs with different flavivirus specificities may be a more feasible approach toward a broadly active flavivirus therapeutic antibody product.

Development of Resistance to Passive Therapy with a Potently Neutralizing Humanized Monoclonal Antibody against West Nile Virus

Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus in animals. Here, we assess the potential for West Nile virus strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, the emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.

Fusion Loop Peptide of the West Nile Virus Envelope Protein Is Essential for Pathogenesis and Is Recognized by a Therapeutic Cross-Reactive Human Monoclonal Antibody

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

Epitope Determinants of a Chimpanzee Dengue Virus Type 4 (DENV-4)-Neutralizing Antibody and Protection against DENV-4 Challenge in Mice and Rhesus Monkeys by Passively Transferred Humanized Antibody

The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 deltaD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 deltaD at 20 microg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 deltaD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10(6) MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

Human Monoclonal Antibody Combination against SARS Coronavirus: Synergy and Coverage of Escape Mutants

BackgroundExperimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties.Methods and FindingsHuman mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318–510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one.ConclusionsThe combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.